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PROGRAMA DE PÓS-GRADUAÇÃO EM CIENCIAS MEDICAS
Dissertação de Mestrado
Caracterização fenotípica e genotípica de
Staphylococcus epidermidis isolados de
hemoculturas do Complexo Hospitalar
Santa Casa de Misericórdia de
Porto Alegre no período de 2002 a 2004
Ana Lúcia Souza Antunes
Biblioteca Paulo Lacerda de Azevedo
2006
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PROGRAMA DE PÓS-GRADUAÇÃO EM CIENCIAS MEDICAS
Dissertação de Mestrado
Caracterização fenotípica e genotípica de
Staphylococcus epidermidis isolados de
hemoculturas do Complexo Hospitalar
Santa Casa de Misericórdia de
Porto Alegre no período de 2002 a 2004
Ana Lúcia Souza Antunes
Orientador: Pedro Alves d’Azevedo
Co-orientador: Ana Lúcia Peixoto de Freitas
Biblioteca Paulo Lacerda de Azevedo
2006
ads:
A627c Antunes, Ana Lúcia Souza
Caracterização fenotípica e genotípica de Staphylococcus
epidermidis isolados de hemoculturas do Complexo Hospitalar Santa
Casa de Misericórdia de Porto Alegre no período de 2002 a 2004 /
Ana Lúcia Souza Antunes; orient. Pedro Alves d’Azevedo ; co-
orient. Ana Lúcia Peixoto de Freitas. - Porto Alegre: FFFCMPA,
2006.
133 f.; graf. ; tab.
Dissertação (Mestrado). Fundação Faculdade Federal de Ciências
Médicas de Porto Alegre. Faculdade de Medicina. Curso de Pós-
Graduação em Ciências Médicas.
1. Staphylococcus epidermidis. 2. Métodos de identificação. 3.
Desferroxamina. 4. Fosfomicina. 5. Staphylococcus spp meticilina
resistente. I. d’Azevedo, Pedro Alves. II. Freitas, Ana Lúcia Peixoto
de. III. Título.
CDD 6l6.9297
CDU 6l6981.21
Ruth B. F. Oliveira/ Bibliotecária
CRB10/501
Ao meu marido Enrique, por todo o apoio, amor, paciência e compreensão.
Aos meus filhos Felipe e Gabriela que entenderam a minha ausência.
Aos meus pais Lucília e Eli por terem incentivado a minha caminhada na busca pelo conhecimento.
AGRADECIMENTOS________________________________________________________
Ao Prof. Dr. Pedro Alves d’Azevedo pela sua dedicada orientação na conduta dos
trabalhos científicos e também pela sua paciência e incentivo. Pelos inúmeros e extensos e-
mails científicos, sempre acompanhados de palavras de apoio descontraídas e espirituosas,
trocados durante esse último ano, período que esteve em São Paulo no Laboratório Especial de
Microbiologia Clínica (LEMC-UNIFESP) realizando seu Pós-Doutorado.
À Prof. Dra. Ana Lúcia Peixoto de Freitas pela co-orientação atenta, segura e
carinhosa que propiciou meu crescimento científico e pessoal. Pelas palavras de sabedoria e
pelo entusiasmo demonstrado durante a realização deste trabalho. Pelas inúmeras tardes de
estudo que passamos juntas.
À Carina Secchi e ao Leandro Reus Rodrigues Perez pelo carinho, amizade,
coleguismo, colaboração nos experimentos e sem dúvida, pelo grande companheirismo
durante este período que foi tão significativo em minha vida.
Às colegas da pós-graduação, em especial a Juliana Caierão e Silvana Vargas Superti
pelo incentivo carinhoso e auxílio técnico-científico.
À Keli Cristine Reiter por toda a sua dedicação, carinho, amizade e auxílio técnico
durante estes anos de convivência diária no laboratório de Análises Clínicas da Faculdade de
Farmácia.
À Lisandra Silvani Massi e Clara Lia Brandelli pela compreensão por entenderem
meus momentos de ausência da bancada de trabalho.
À Prof. Dra. Valquíria Linck Bassani ex-Diretora da Faculdade de Farmácia e atual
pró-Reitora de Pós-Graduação da UFRGS, pelo incentivo a realização desta etapa importante
de minha formação profissional.
Ao Prof. Dr. Paulo Mayorga, diretor da Faculdade de Farmácia, pelo incentivo e apoio
durante estes anos de trabalho.
Ao Prof. Dr. Afonso Luiz Barth, profissional exemplar que permitiu a realização de
uma parte de meu estudo no Laboratório do Hospital de Clínicas de Porto Alegre.
Ao Prof. Cícero Armídio Gomes Dias pelas palavras de sabedoria durante a realização
deste trabalho.
À Prof. Dra. Ana Paula Frazon pela dedicação e entusiasmo durante a realização das
PCRs.
À Prof. Dra. Kátia Regina Netto dos Santos pela doação de um controle ATCC
biofilme positivo.
À Dra. Alice Beatriz M. Pinheiro Machado pelo coleguismo demonstrado e suporte
técnico.
Às colegas de laboratório Maria Beatriz, Rosângela, Mara e Sandra pelo carinho,
amizade, dedicação e apoio técnico, fundamental para execução deste trabalho.
Aos colegas do laboratório de Análises Clínicas da Faculdade de Farmácia por me
substituírem nos momentos de ausência.
Às alunas de iniciação científica do Laboratório de Cocos Gram-positivos, Ananda
Cristine Galvão e Letícia Ganassini pelo suporte técnico durante a realização deste trabalho.
À Fundação Faculdade Federal de Ciências Médicas de Porto Alegre (FFFCMPA) por
incentivar o aprimoramento científico.
A Universidade Federal do Rio Grande do Sul (UFRGS) pela parceria e incentivo.
A todos que de uma forma ou outra auxiliaram na realização deste trabalho.
SUMÁRIO
RESUMO………………………………………………………………………………09
ABSTRACT....................................................................................................................11
INTRODUÇÃO..............................................................................................................12
REVISÃO DA LITERATURA......................................................................................15
Aspectos Gerais do Gênero Staphylococcus .................................................................15
Epidemiologia.................................................................................................................16
Patogenicidade................................................................................................................17
Resistência aos antimicrobianos.....................................................................................21
Caracterização Fenotípica...............................................................................................26
Caracterização Genotípica..............................................................................................31
REFERÊNCIAS BIBLIOGRÁFICAS...........................................................................34
JUSTIFICATIVAS DO ESTUDO.................................................................................42
OBJETIVOS DO ESTUDO...........................................................................................44
Geral...............................................................................................................................44
Específicos.....................................................................................................................44
DELINEAMENTO DO ESTUDO.................................................................................45
HIPÓTESES DO ESTUDO...........................................................................................45
ARTIGOS PRINCIPAIS...............................................................................................46
Staphylococcus epidermidis: a simple phenotypic method for identification...............47
Evaluation of oxacillin and cefoxitin disks for detection of resistance in Coagulase
Negative Staphylococci ………………………………………..….….………….…...61
Detection of biofilm production in Staphylococcus spp. isolates by the Congo Red
agar test………………………………………………………………………………..73
ANEXO 1……………..……………………………………………………………...86
Bacteremias of Coagulase-negative Staphylococci (CoNS) in Intensive Care Unit
Hospital Geral of São Paulo city, SP, Brazil………………………………………….87
ANEXO 2………………………………………………………………………….…98
Is cefoxitin a better predictor for the mecA gene than oxacillin?...............................99
ANEXO 3…………………………………………………………………………..114
Use of the D test method of detect inducible clindamycin resistance in Coagulase
Negative Staphylococci (CNS)………………………………………………….…115
ANEXO 4…………………………………..............................................................126
Tabela de resistência a meticilina em S. epidermidis................................................127
ANEXO 5..................................................................................................................132
Comitê de Ética.........................................................................................................133
RESUMO
Nas últimas décadas Staphylococcus spp coagulase negativo (SCoN) têm sido
identificados como patógenos importantes, principalmente em pacientes com
dispositivos médicos, baixa imunidade e neonatos. S. epidermidis é a espécie mais
frequentemente isolada dentre os SCoN e sua patogenicidade está relacionada com a
habilidade em formar biofilme. A emergência de SCoN como reservatório de resistência
aos antimicrobianos, torna fundamental a identificação rápida e confiável, que, no
entanto, não é realizada na rotina dos laboratórios de microbiologia clínica. Os objetivos
desse estudo foram identificar S. epidermidis, através de métodos fenotípicos de
referência e método proposto e de método genotípico. Além de estabelecer o perfil de
suscetibilidade desse microrganismo frente aos antimicrobianos usados frequentemente
no tratamento destas infecções e analisar a resistência a meticilina através das técnicas
propostas pelo CLSI. O método convencional modificado por Bannerman (2003) é
trabalhoso, e os métodos comerciais de identificação são dispendiosos e frequentemente
fornecem resultados pouco confiáveis devido à expressão variável das características
fenotípicas. O método proposto utilizando discos de desferroxanima e fosfomicina
mostrou ser uma excelente ferramenta para identificação do S. epidermidis. Em nosso
estudo identificamos 49,2% (241/490) de S. epidermidis, com uma resistência a
meticilina de 77,6% (187/241). Recentemente, o “Clinical and Laboratory Standards
Institute” (CLSI) propuseram uma modificação na metodologia para predizer a
resistência a meticilina no gênero Staphylococcus mediada pelo gene mecA, através de
teste de difusão com disco de cefoxitina. Apesar das recomendações do CLSI, a
detecção da resistência a oxacilina por métodos fenotípicos permanece um desafio para
o laboratório de microbiologia clinica tendo em vista a heteroresistência. Os resultados
desse trabalho são de grande importância para permitir aos laboratórios de
microbiologia clínica a identificação de rotina do S. epidermidis, considerando que este
dado é importante para levantamentos epidemiológicos. Além disso, o conhecimento
dos níveis de resistência à oxacilina auxilia na conduta terapêutica para o tratamento
destas infecções.
Palavras-chaves: Staphylococcus epidermidis, método de identificação, desferroxamina,
fosfomicina, Staphylococcus spp meticilina resistente.
ABSTRACT
In recent decades coagulase negative Staphylococcus spp (CoNS) have also emerged as
significant pathogens, especially in patients with medical devices, immunocompromised
pacients and neonates. S. epidermidis is the most frequent isolated among the CoNS,
and its pathogenicity is related to its ability to form a biofilm. The emergence of CoNS
as a reservoir of antimicrobial resistance makes reliable and fast identification essential,
which, however, is not performed routinely in clinical laboratories of microbiology. The
aim of this study were to identify S. epidermidis phenotypically and genotypically,
comparing a phenotypic reference method with one here proposed and with a molecular
method. We also evaluate the susceptibility profiles of this microorganism to the
antimicrobials frequently used in the treatment of these infections, and to analyze
methicillin resistance through the techniques proposed by CLSI.The conventional
method modified by Bannerman (2003) is laborious, and the commercially available
methods for identification often provide unreliable results due to the variable expression
of phenotypic characteristics. The method proposed used disks of desferrioxamine and
fosfomycin proved to be a usefull tool to identify S. epidermidis. In our study 49.2%
(241/490) of the samples were identified as S. epidermidis, with a meticillin resistance
of 77.6% (187/241). Despite the CLSI guidelines, detection of oxacillin resistance
through phenotypic methods remains a challenge for the clinical laboratory of
microbiology due heteroresistance. The results of this work are usefull for clinical
microbiology laboratories, making easier the identification of S. epidermidis, what is
helpful to epidemiologic trials. The rates os oxacillin resistance is fundamental to help
treatment of these infections.
Keywords: Staphylococcus epidermidis, identification method, desferrioxamine,
fosfomycin, methicillin-resistant Staphylococcus spp
12
INTRODUÇÃO________________________________________________________
O gênero Staphylococcus é constituído por importantes patógenos humanos,
causando um amplo espectro de doenças sistêmicas relevantes. As espécies mais
comumente associadas são o Staphylococcus aureus (membro mais virulento e mais
conhecido do gênero), Staphylococcus epidermidis, Staphylococcus haemolyticus,
Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus capitis,
Staphylococcus lugdunensis, Staphylococcus simulans e Staphylococcus warneri.
Staphylococcus spp coagulase negativos (SCoN), embora reconhecidos como
saprófitas por muitos anos, têm emergido como agentes etiológicos de um grupo grande
de infecções. Atualmente têm sido associados com septicemia em neonatos, infecções
em idosos, em pacientes imunossuprimidos e pacientes que fazem uso de dispositivos
médicos, como cateteres, próteses, lentes intra-oculares e válvulas. Estudos relacionados
com infecção hospitalar se intensificaram na década de 50, após a introdução da
penicilina no tratamento de infecções por Staphylococcus spp. Já naquela época,
isolados de S. aureus produtores de penicilinases começaram a ser relatados, indicando
que as bactérias poderiam desenvolver resistência aos antimicrobianos. Entretanto, a
partir dos anos 70, a emergência de Staphylococcus aureus resistentes a meticilina
(MRSA) em diversas regiões do mundo, assim como o aumento da incidência de
infecções causadas por bactérias resistentes, como o S. epidermidis e Enterococcus spp
mudaram definitivamente o panorama das infecções hospitalares (WEBER &
RUTALA, 1989). Apesar do surgimento de novos antimicrobianos, o ritmo do
desenvolvimento de resistência nos diferentes patógenos Gram-positivos e Gram-
negativos, representa um constante desafio terapêutico. O uso inapropriado dos
antimicrobianos pode estar relacionado a fatores como: falta de atividade da droga
13
escolhida, doses sub-terapêuticas, esquemas terapêuticos curtos, baixa penetração no
local da infecção, idade, imunidade do paciente e não adesão ao tratamento
(COURVALIN, 1999).
O aumento da resistência a meticilina, que indica resistência cruzada para toda a
classe de ß-lactâmicos incluindo cefalosporinas e carbapenêmicos, tem sido motivo de
investigação em todo o mundo. Como conseqüência desta resistência tem havido um
aumento do uso dos glicopeptídeos em terapias empíricas e até mesmo profiláticas
(GOLDSTEIN et al. 2004; NUNES et al. 2006). Na maioria dos casos, a resistência
bacteriana é mediada por genes, cuja detecção constitui método de referência na
determinação desta resistência. Entre os genes mais estudados está o gene mecA, que
determina a resistência para a meticilina e demais ß-lactâmicos. O mecanismo de
resistência à meticilina é baseado na produção de uma única proteína de ligação a
penicilina, que não está presente nos isolados suscetíveis, a PBP 2a (Penicillin Binding
Proteins). Essa proteína é codificada pelo gene mecA, presente no cromossomo
bacteriano (ARCHER & NIEMEYER, 1994) e possui baixa afinidade por agentes
antimicrobianos com anel ß-lactâmico. Recentemente, pesquisadores do CLSI
propuseram uma nova metodologia para predizer a resistência no gênero
Staphylococcus mediada pelo gene mecA. A detecção de resistência aos macrolideos e
outras classes de antimicrobianos, também são realizadas através de metodologias
estabelecidas por esse comitê. Devido ao surgimento de resistência a agentes
tradicionalmente utilizados, outras classes de antimicrobianos como estreptograminas,
oxazolidinona e lipopeptídeos podem ser opções no tratamento destas infecções. No
entanto, o uso criterioso dessas novas classes é imperativo para que possamos preservar
essas opções terapêuticas.
14
Embora os SCoN sejam os microrganismos mais isolados em hemocultura de
pacientes com implante de biomateriais, espécies e sub-espécies não são identificadas
na rotina do laboratório de microbiologia clínica (COUTO et al. 2001). O método
convencional proposto por Kloos & Sheleifer (1975) e modificado por Bannerman
(2003) é trabalhoso e moroso, tornando-o inadequado aos tempos atuais. A utilização
destes testes na rotina laboratorial se torna inviável, o que tem levado ao
desenvolvimento de esquemas de identificação com um menor número de testes,
capazes de identificar uma espécie em particular ou um maior número delas (MONSEN
et al. 1998; De PAULIS et al. 2003). Além disso, os métodos comerciais disponíveis
como alternativas de identificação são dispendiosos e frequentemente fornecem
resultados pouco confiáveis devido à variabilidade de expressão das características
fenotípicas.
Realizar uma identificação confiável através de testes simples e factíveis na
rotina do laboratório de microbiologia clínica é um grande desafio, pois os esquemas e
testes propostos não podem ser onerosos ou demorados, o que impediria sua realização
na rotina laboratorial.
15
REVISÃO DA LITERATURA____________________________________________
Aspectos Gerais do Gênero Staphylococcus
Staphylococcus spp são bactérias Gram-positivas, catalase positivas, da família
Micrococcaceae. O gênero Staphylococcus na atualidade é composto por
aproximadamente 40 espécies, 17 das quais podem ser isoladas de amostras biológicas
humanas (MONSEN et al. 1998;
BANNERMAN, 2003; CUNHA et al. 2004).
Staphylococcus são geralmente encontrados na pele e em mucosas do homem e de
outros animais. Para poderem expressar sua patogenicidade, passando de habitantes
normais da pele a agentes de infecção é necessário que haja predisposição do
hospedeiro.
O gênero Staphylococcus é constituído por importantes patógenos humanos,
causando um amplo espectro de doenças sistêmicas relevantes. Esses microrganismos
apresentam elevado risco potencial de bacteremia nosocomial entre pacientes
imunodeprimidos, idosos e recém-nascidos de baixo peso, os quais são
imunologicamente imaturos, e frequentemente requerem procedimentos invasivos para
administração de substancias nutritivas e medicamentosas (CUNHA et al. 2002;
MURRAY et al. 2004).
S. epidermidis é o membro mais freqüentemente isolado dentre os SCoN
envolvidos em bacteremia. S. epidermidis tem emergido como o mais importante
patógeno hospitalar responsável por infecções sistêmicas e associadas à implantação de
dispositivos médicos como prótese articular, válvula cardíaca, marca passo, cateter de
diálise peritoneal, lente intra-ocular (WIESER & BUSSE, 2000; De PAULIS et al.
2003). Seu sucesso como patógeno, deve-se ao fato de se aderir a superfície destes
16
dispositivos médicos, formando uma substância extracelular amorfa denominada de
biofilme ou slime, composta por produtos da bactéria e do hospedeiro.
Epidemiologia
Embora desde a década de 50 já houvesse relatos de SCoN causando infecções,
somente nos anos 80 a comunidade científica começou a estudar melhor estes
microrganismos. SCoN foram considerados não patogênicos e seu isolamento no
laboratório clínico era atribuído à contaminação pela microbiota cutânea normal. As
infecções causadas pelo S. epidermidis são freqüentemente persistentes e recidivantes, e
a emergência de resistência antimicrobiana entre os SCoN trouxe mais complicações
para o tratamento, especialmente na presença de biomateriais (O’GARA &
HUMPHREYS, 2002; BEEKMANN et al. 2003; CHRISTENSEN et al. 2003). A
importância e a incidência de infecções causadas por SCoN têm crescido
substancialmente (HUSSAIN et al. 1998). Esta mudança no panorama epidemiológico
deve-se entre outros fatores as condições de higidez de cada paciente, pois pacientes
submetidos a quimioterápicos, imunossupressores e dispositivos médicos apresentam
situações de risco para o acometimento de infecções. Na América do Norte, nas últimas
três décadas houve um aumento nas infecções causadas por estes microrganismos,
paralelamente ao aumento da resistência a meticilina nestes isolados (HUSSAIN et al.
2002).
SCoN são a principal causa de infecções no sistema circulatório em pacientes
hospitalizados, responsáveis por uma elevada morbidade e mortalidade (QIAN et al.
2001; FERREIRA et al. 2003). A freqüência com que S. epidermidis é encontrado varia
de 43% a 92% dependendo da região geográfica pesquisada (IEVEN et al. 1995;
COUTO et al. 2001; VUONG & OTTO, 2002; DE PAULIS et al. 2003; FERREIRA et
al. 2003; CUNHA et al. 2004; CAIERÃO et al. 2006).
17
Em um estudo multicêntrico realizado pelo SENTRY “Antimicribial
Surveillance Program” (JONES, 2003), foi avaliado o aumento da resistência nos SCoN
em um período de cinco anos (1997-2001), em diferentes regiões do mundo, sendo que
as taxas encontradas foram: 22,4 a 38,7% na América do Norte; 22,1 a 30,4% na Europa
e 29,2 a 36,0% na América Latina. Biedenbach et al. (2004) demonstraram que os
SCoN foram a classe de microrganismo mais isolado em bacteremias de neonatos e
crianças até um ano de idade. Em outro estudo realizado por Sader et al. (2004),
avaliando questões de resistência na América Latina, incluindo o Brasil, foi observada
uma diminuição nos níveis de suscetibilidade a teicoplanina entre os SCoN, sendo que
no Brasil, o perfil de suscetibilidade frente às cefalosporinas e meticilina também vem
se modificando, mostrando uma suscetibilidade de apenas 17,7%. Dados nacionais
mostram uma resistência de 80% a oxacilina em isolados de hemocultura já havia sido
reportada por Sader et al. (2001). Em outro estudo brasileiro, Ferreira et al. (2002)
encontraram uma freqüência de resistência de SCoN à oxacilina de 64%, em isolados de
diferentes sítios clínicos. Na Ásia, a prevalência de S. epidermidis oxacilina resistente
(MRSE) foi de 60 a 66% em bacteremia (JEONG et al. 2002).
A rápida emergência e disseminação de multiresistência entre as bactérias têm
aumentado a necessidade de controlar estes patógenos em hospitais. A caracterização
de clones dentro de um grupo fenotipicamente resistente tem um impacto direto no
método de intervenção corretiva. Em uma escala mais ampla, a identificação de clones
de microrganismos resistentes com um grande alcance geográfico, pode fornecer uma
visão da patogenicidade e virulência das espécies; podendo também resultar em
intervenções de saúde públicas mais amplas, como vacinação e restrições
antimicrobianas, objetivando a limitação da proliferação de patógeno resistentes
(JONES, 2001).
18
Patogenicidade
A aquisição de infecção por SCoN é facilitada pela presença de alguns fatores de
risco, tais como: quebra da barreira cutâneo-mucosa do hospedeiro, estado de
imunossupressão e idade (idosos e neonatos), que somados à implantação de
dispositivos médicos aumentam a suscetibilidade do individuo. Entre os SCoN,
S. epidermidis é a espécie mais comumente isolada em bacteremia e S. haemolitycus é
a segunda, ambos são associados com endocardite, septicemia, peritonite, infecções do
trato urinário, infecções de ferida operatória e infecções em articulações. Por outro lado,
S. lugdunensis tem sido associado a infecções de sítio cirúrgico, abscessos, peritonite,
osteomielite e endocardite (KLOOS & BANNERMAN, 1994). A mortalidade atribuída
a infecções no sistema circulatório causadas por S. epidermidis varia de 10 a 34%, com
aumento dos custos e acréscimo de 7 a 19 dias na duração da internação (RUPP &
ARCHER, 1994).
Os fatores de virulência presentes nos SCoN, não estão claramente estabelecidos
como em S. aureus. S. epidermidis não produz toxinas, nem exoenzimas que causam
danos severos aos tecidos, como ocorre com o S. aureus, de modo que seu sucesso
como patógeno deve-se, provavelmente, ao fato de se aderir a superfícies e conseguir
permanecer sob a cobertura de um material extracelular de proteção, passando
despercebido e assim causando infecções subagudas ou crônicas (VUONG et al. 2003).
Até mesmo instrumentos esterilizados podem ser contaminados com membros da
microbiota normal durante sua inserção no local de interesse. Se o biomaterial
permanecer no local por um período de tempo suficientemente grande, poderá haver
desenvolvimento de biofilme, que funciona como fonte de infecção duradoura e
proteção contra agentes antimicrobianos (RAAD et al. 1998).
19
O mecanismo pelo qual SCoN formam o biofilme é um processo complexo e de
múltiplos passos, onde a relevância de cada componente ainda requer mais investigação
(SILVA et al. 2002). Esta estrutura é composta por densos agregados de células
microbianas embebidas em uma matriz viscosa que se adere a uma superfície plástica.
Os agregados celulares são compostos por inúmeras micro-colônias de células e são
permeados por canais de água e áreas intersticiais vazias. O fluxo de água através dos
canais intersticiais aumenta a oferta de nutrientes para as células do biofilme (RAAD et
al. 1998).
A formação do biofilme inclui uma substância extracelular amorfa, “slime”, que
circunda o aglomerado de células composto por produtos da bactéria e das células do
hospedeiro com composição química diversa, embora seu principal componente seja o
ácido teicóico. Plasma e proteínas do tecido conectivo como fibronectina, fibrinogênio,
vitronectina, trombospondina, laminina, colágeno e o fator Von Willebrand estão
envolvidos nesse processo (EIFF et al. 1999).
O processo de formação do biofilme ocorre em dois estágios:
1º Adesão primária – onde ocorre a rápida adesão da bactéria ao material plástico ou
material protéico do hospedeiro presente no plástico, e a posterior formação da
multicamada de células. A adesão direta a superfície do material, se deve as
propriedades físico-químicas do plástico e da
superfície bacteriana
(VACHEETHASANEE et al. 1998; VACHEETHASANEE & MARCHANT, 2000).
Alguns pesquisadores demonstram a existência de moléculas bacterianas específicas
para esse tipo de interação hidrofóbica com a superfície abiótica, como a autolisina AtlE
(HEILMANN et al. 1996; VUONG et al. 2003).
2º
Adesão intercelular - a formação de aglomerados celulares no topo da monocamada
de células aderidas ao plástico ou as células do hospedeiro são o segundo estágio da
20
formação do biofilme. Muitas moléculas já foram propostas como sendo responsáveis
pela adesão célula a célula, principalmente a PIA (polysaccharide intercellular
adhesin); AAP (accumulation-associated protein) e a PNSG (poly-N-
succinylglucosamine). A PIA e a PNSG podem ser a mesma molécula, pois são
codificadas pelo operon ica que contem os genes ica ADBC (HEILMANN et al. 1996;
HUSSAIN et al. 1997). A PIA é codificada especificamente pelo gene icaA, mas sua
expressão é alterada quando ocorre uma co-expressão do icaA com o icaD, aumentando
assim significativamente sua atividade e expressão fenotípica (ARCIOLA et al. 2003).
Evidências epidemiológicas mostram que existe correlação entre patogenicidade
e presença dos genes ica nas cepas virulentas do S. epidermidis (GALDBART et al.
2000; ZIEBUHR et al. 2006). Por outro lado, Vogel et al. (2000) não encontraram uma
clara associação entre produção de biofime e virulência. Outro estudo levantou a
possibilidade de outros fatores associados contribuírem para a formação do biofilme
além do gene ica (LI et al. 2005).
O biofilme confere proteção contra os mecanismos de defesa imune do
hospedeiro e a entrada dos antimicrobianos. S. epidermidis causa menor produção de
proteínas inflamatórias que S. aureus, e consequentemente, uma menor resposta
inflamatória, incluindo a inibição de linfócitos T e monócitos periféricos por indução da
produção de prostaglandina E2. A substância extracelular, “slime”, também está
envolvida de modo prejudicial à opsonização e a fagocitose, inibindo a quimiotaxia dos
leucócitos polimorfonucleares (VUONG & OTTO, 2002). O efeito bactericida “in
vitro” de antimicrobianos tem sido analisado comparativamente em isolados clínicos de
S. epidermidis produtores ou não de biofilme (AMORENA et al. 1999: MONZÓN et al.
2001). De acordo com estes autores, eritromicina, rifampicina e tetraciclina apresentam
maior efeito que a vancomicina, clindamicina, cefalotina, teicoplanina e ofloxacina, em
21
isolados clínicos com S. epidermidis produtores de biofilme. Estes estudos salientam a
relevância do teste de suscetibilidade na presença de biofilme e o potencial dano do uso
indiscriminado da monoterapia com vancomicina, inadequada frente às infecções
envolvendo microrganismos produtores de biofilme (AMORENA et al. 1999;
MONZÓN et al. 2001).
A concentração de antimicrobiano necessária para eliminar bactérias produtoras
de biofilme é de 100-1000 vezes maior que a necessária para as mesmas espécies em
suspensão. Devido ao aumento de isolados resistentes a meticilina, estratégias
profiláticas recentes incluíram pré-cobertura dos implantes com glicopeptideos, mas o
surgimento recente de amostras de S. epidermidis com suscetibilidade reduzida a
vancomicina e teicoplanina criou a necessidade de estratégias alternativas, complicando
ainda mais o tratamento das infecções onde há presença de biomateriais (VILLARI et
al. 2000).
A detecção fenotípica da presença de biofilme em Staphylococcus spp foi
proposta por Arciola et al. (2002) através da utilização de agar Congo Red, onde a
análise direta das colônias formadas no agar mostra a produção ou não de biofilme,
segundo uma escala de cores. Este é um teste qualitativo, que pode ser confirmado
genotipicamente através da pesquisa dos genes icaA e icaD nos isolados.
Resistência aos antimicrobianos
As infecções estafilocócicas são, principalmente, de natureza hospitalar e os
isolados apresentam índices elevados de resistência (Gaynes, 1997). Por outro lado,
recentemente foram descritas infecções ocasionadas por S. aureus resistentes a
meticilina adquiridos na comunidade (CA-MRSA), que inicialmente, causam infecções
na pele e tecidos moles, furunculoses e abscessos, mas também podem causar
pneumonias necrotizantes (RIBEIRO et al. 2005; HUIJSDENS et al. 2006; MCCLURE
22
et al. 2006; D’AZEVEDO et al. 2006a). Muitos destes CA-MRSA carreiam genes
responsáveis pela produção de toxinas tais como a Panton-Valentine Leucodin (PVL) e
carregam o cassete cromossômico staphylococcal mec (SCCmec) tipo IV ou V. O
aumento das infecções causadas por CA-MRSA levou a utilização de clindamicina
como tratamento empírico, entretanto a expressão de resistência induzida pode limitar a
efetividade deste antimicrobiano (PATEL et al. 2006).
A multiresistência aos antimicrobianos é uma das principais características
observadas entre os isolados hospitalares de SCoN e têm incluído, resistência a
eritromicina, clindamicina, tetraciclina, cloranfenicol, trimetoprim/sulfametoxazol,
aminoglicosídeos, ß-lactâmicos e eventualmente, quinolonas (SANTOS et al. 1997).
Para cada classe de antimicrobianos, são observados mecanismos de resistência
específicos, com envolvimento de genes plasmidiais, ou transposons que codificam
elementos inativadores do antimicrobiano ou diminuem a afinidade de ligação,
modificando o sítio alvo. A capacidade dos SCoN em albergar vários marcadores de
resistência mostra sua importância como reservatório de genes de resistência que podem
ser transmitidos para outras espécies e gêneros de microrganismos (ARCHER et al.
1994).
Com o crescimento da caracterização de infecções por SCoN, o interesse em
estudar sua suscetibilidade também tem aumentado proporcionalmente (KLOOS &
BANNERMAN, 1994). Segundo Jones (1996), há uma associação entre o aumento da
freqüência percentual dos SCoN na etiologia de bacteremia nosocomial e a resistência
desses microrganismos aos agentes antimicrobianos. SCoN de infecções nosocomiais,
particularmente S. epidermidis e S. haemolyticus, apresentam com freqüência resistência
a meticilina maior que 80%. O aumento da resistência a meticilina, e consequentemente
a toda classe de ß-lactâmicos incluindo cefalosporinas e carbapenêmicos, tem sido
23
motivo de estudos em todo o mundo, levando a um aumento do uso dos glicopeptídeos
em terapias empíricas e até mesmo profiláticas. A emergência de resistência a
teicoplanina, antes do surgimento de resistência a vancomicina, indica a possibilidade
de que a teicoplanina possa contribuir para a seleção de isolados resistentes a
vancomicina em um momento posterior (OLIVEIRA et al. 2001; GOLDSTEIN et al.
2004; NUNES et al. 2006). A redução da suscetibilidade à teicoplanina já havia sido
relatada por Bannerman et al. (1991) em isolados de S. epidermidis e S. haemolyticus.
Os autores verificaram que 7% dos S. epidermidis e 21% dos S. haemolyticus
apresentavam suscetibilidade moderada a teicoplanina, enquanto que 11% dos
S. haemolyticus foram resistentes. Aparentemente essa resistência não é transmissível
entre estas espécies, não obstante pressões seletivas possam influenciar o
desenvolvimento de resistência entre os isolados de um paciente infectado pelo
ambiente hospitalar (CUNHA & LOPES, 2002).
Até 2004, a oxacilina foi à droga indicada para detectar “in vitro” a resistência a
meticilina nos Staphylococcus spp, por ser mais resistente à degradação e mais sensível
para detecção de heteroresistência do que a meticilina. Atualmente, novas condutas têm
sido adotadas para detectar fenotipicamente esta resistência. O CLSI atualiza
periodicamente normas relativas a testes de suscetibilidade antimicrobiana e a partir de
2005, passou a recomendar o uso do disco de cefoxitina para prever a suscetibilidade à
oxacilina pelo teste de disco difusão entre isolados de Staphylococcus spp, baseado em
um experimento realizado por Swenson & Tenover (2005). A cefoxitina é uma
cefamicina, que prediz melhor a presença do gene mecA, por ter uma maior atividade
indutora da expressão gênica do que a oxacilina (DARINI et al. 2004). Diversos
treabalhos publicados nos últimos anos apontaram a superioridade da cefoxitina na
detecção da resistência a meticilina (FELTEN et al. 2002; SKOV et al. 2003;
24
POTTUMARTHY et al. 2005; SHARP et al. 2005; SWENSON & TENOVER, 2005).
No entanto, Frigatto et al. (2005) observaram uma discrepância entre oxacilina e
cefoxitina pelo método de disco difusão, onde os cinco isolados que apresentavam o
gene mecA pela técnica da PCR, foram resistentes para a oxacilina e suscetíveis para
cefoxitina. Todos os isolados foram identificados como S. epidermidis, com padrões
distintos pela ribotipagem, descartando assim a possibilidade de um surto. Com isto os
autores ressaltaram a importância, pelo menos por enquanto, de testarmos
concomitantemente o disco de oxacilina e o de cefoxitina, e ainda utilizarmos técnicas
moleculares para confirmação dos resultados.
O mecanismo de resistência a meticilina no S. epidermidis é homólogo ao
apresentado pelo S. aureus, ou seja, pode ocorrer por uma mutação nos genes pbp e abc
(mecanismo mais freqüente) ou como resultado da aquisição de um gene exógeno que
codifica para resistência. No primeiro caso, ocorre a produção de uma proteína ligante
de penicilina adicional, a Penicilin Binding Protein (PBP 2a), de baixa afinidade para
antimicrobianos β-lactâmicos (ao contrário da proteína ligante de penicilina
constitutiva, que se liga de forma covalente aos β-lactâmicos), que é codificada pelo
gene mecA (ARCHER & NIEMEYER, 1994; CHAMBERS, 1997; FOSTER, 2004). No
segundo caso, a aquisição dos genes de resistência pode ser devido a plasmídios (DNA
extra cromossômico) ou através de elementos genéticos móveis (transposons), sendo
esta forma de aquisição a mais freqüente entre os isolados clínicos. A detecção da
proteína PBP 2a, produto da expressão do gene mecA, pode ser realizada através de teste
de aglutinação em látex. Esta técnica é uma alternativa de execução fácil e rápida, para
os laboratórios clínicos que não possuem a técnica de Reação em Cadeia da Polimerase
(PCR), padrão ouro na detecção da resistência a meticilina devido a presença do gene
25
mecA (HUSSAIN et al. 2000). Contudo o alto custo destas técnicas tem estimulado a
pesquisa de métodos fenotípicos, de menor custo, para a detecção de resistência.
Uma característica importante da resistência à oxacilina é sua heterogeneidade,
sendo que o grau de resistência varia conforme as condições do meio de cultura e do
antimicrobiano β-lactâmico em uso. Esta natureza heterogênea se observa em isolados
onde a maioria das células, aproximadamente 99% são suscetíveis a baixas
concentrações de β-lactâmicos. Sob condições de crescimento bacteriano na rotina
laboratorial, uma unidade bacteriana em 10
4
a 10
8
exibe resistência heterogênea, mas
modificando-se estas condições como: crescimento em meios de cultura hipertônicos
(suplementados com NaCl ou sacarose) ou aumento de tempo de incubação, obteremos
resultados mais confiáveis para a detecção fenotípica desta resistência (CHAMBERS,
1997; HUSSAIN et al. 2002; FERREIRA et al. 2003). Estudo realizado por Rowe et al.
(2002), utilizaram para triagem de S. epidermidis agar Muller-Hinton contendo 0,6
µg/ml de oxacilina, incubação de 24h a 35 º C. Os autores também concluíram que a
identificação genérica dos SCoN pode ser inadequada, uma vez que nem todos
apresentam a mesma resposta nos testes para detecção de resistência a meticilina.
A detecção da resistência a oxacilina nos SCoN, conforme um estudo realizado
no sul do Brasil por Caierão et al.(2004), utilizou como método de triagem, agar
Muller-Hinton contendo 4 µg/ml de oxacilina e demonstrou uma correlação de 100%
com a detecção do gene mecA em S. epidermidis, S. hominis e S. haemolyticus. Os
autores comprovaram que esta concentração de oxacilina parece ser a mais adequada
para detecção fenotípica de heteroresistência em SCoN.
O uso dos glicopeptideos em infecções causadas por Staphylococcus resistentes
a meticilina contribuiu para a emergência da resistência a vancomicina e teicoplanina
entre os SCoN. Recentemente têm sido reportados alguns casos de reduzida
26
suscetibilidade aos glicopeptideos em isolados de S. warneri. O primeiro relato de
heteroresistência aos glicopeptideos em um isolado de S. warneri foi registrado por
Nunes et al. (2006). D’Azevedo et al. (2006c) avaliaram a disseminação intra-hospitalar
de um grupo clonal predominante de S. cohnii spp urealyticum, que apresentou uma
diminuição na suscetibilidade a teicoplanina e vancomicina.
Técnicas moleculares para a detecção do gene mecA em Staphylococcus spp vêm
sendo utilizadas para confirmar a resistência detectada pelas técnicas fenotípicas
(ARCHER & NIEMEYER, 1994; SCHMITZ et al. 1998). A detecção do gene mecA
através da técnica de PCR, tem sido considerada padrão ouro para a detecção da
resistência à meticilina em amostras de Staphylococcus spp e portanto utilizada para
confirmação de métodos fenotípicos. Palazzo & Darini (2006) relataram discrepâncias,
especialmente em amostras de SCoN, onde nenhuma das técnica empregadas mostraram
100% de sensibilidade e especificidade. Os autores observaram 1,3% de falsos
resistentes, quando compararam o método de disco difusão com cefoxitina e a presença
do gene mecA. Esta falsa resistência é esperada entre 1-9% (SKOV et al. 2005).
A reação da PCR é uma técnica rápida sensível e específica, porém, ainda não é
um método viável economicamente para os laboratórios de microbiologia clínica.
Caracterização Fenotípica
Embora SCoN sejam os microrganismos mais isolados em hemocultura de
pacientes com implante de biomateriais, as espécies e sub-espécies não são identificadas
na rotina do laboratório de microbiologia clínica. O método convencional proposto por
Kloos & Sheleifer (1975) e modificado por Bannerman (2003) é trabalhoso e moroso,
tornando-o inadequado aos tempos atuais. Além disso, os métodos comerciais
disponíveis como alternativas de identificação são dispendiosos e frequentemente
fornecem resultados pouco confiáveis devido à expressão variável das características
27
fenotípicas (COUTO et al. 2001; KONTOS et al. 2003; CAIERÃO et al. 2006). Além
disso, a realização das provas bioquímicas para identificação das espécies é
demasiadamente lenta para a utilização na rotina dos laboratórios de microbiologia
clinica dos hospitais (EDWARDS et al. 2001). Outros testes, como a eletroforese
enzimática ou a análise da composição celular dos ácidos graxos, também não foram
efetivas (HEIKENS et al. 2005).
A identificação convencional das espécies de SCoN é realizada a partir da
combinação de um grupo de testes laboratoriais, tais como: morfologia colonial, o
requerimento de oxigênio, a produção de hemólise, as atividades enzimáticas, a
produção de ácido a partir da fermentação de carboidratos e a resistência a determinados
antimicrobianos (BANNERMAN, 2003). Entretanto, as limitações destes testes na
rotina labortorial têm levado ao desenvolvimento de esquemas de identificação com um
menor número de testes, capazes de identificar corretamente uma espécie em particular
ou um maior número possível destas (MONSEN et al. 1998; De PAULIS et al. 2003).
Realizar uma identificação confiável utilizando testes simples e factíveis na rotina do
laboratório de microbiologia clinica é um grande desafio, em virtude da necessidade de
procedimentos simples, rápidos e de baixo custo. Considerando que as diferentes
espécies e subespécies que compõem SCoN, apresentam perfis de suscetibilidade e
patogenicidade diferenciados, e o aumento significativo do número de infecções
causadas por estes microrganismos, as técnicas fenotípicas devem ser constantemente
reavaliadas (BANNERMAN et al. 1993).
Em 1991, pesquisadores australianos propuseram um teste para identificação de
S. epidermidis, através da suscetibilidade desta espécie a desferroxamina, um sideróforo
sintetizado pelo Streptomyces pilosus, usado clinicamente para o tratamento de
sobrecarga de ferro (LINDSAY & RIDLEY, 1991). Os autores observaram que dentre
28
95 SCoN analisados, 61 (57 S. epidermidis e 4 S. hominis) eram suscetíveis a
desferroxamina, enquanto que, os outros 34 isolados eram resistentes, apresentando um
valor preditivo positivo de 93,1% e um valor preditivo negativo de 100,0%. A este teste
foi acrescida a produção de fosfatase alcalina e a fermentação de trealose, aumentando o
número de amostras identificadas. Em experimento posterior foram analisados 161
SCoN e todas as amostras de S. epidermidis e S. hominis foram suscetíveis a
desferroxamina, enquanto os demais foram resistentes (LINDSAY et al. 1993),
demonstrando uma sensibilidade de 97,3%, uma especificidade de 91,8% e um valor
preditivo positivo de 93,1%. Para distinguir estas duas espécies foi necessária a
utilização dos cartões de identificação do sistema Vitek GPI (bioMèrieux).
Ieven et al. (1995) propuseram um esquema de identificação dos SCoN
envolvendo duas etapas. Testes de fermentação da trealose, de produção de urease e
fosfatase alcalina foram realizados em um primeiro momento. Em um segundo
momento, quando necessário para identificação da espécie, utilizou a descarboxilação
da ornitina, o crescimento em anaerobiose, a suscetibilidade a novobiocina e
fosfomicina. O esquema proposto identificou corretamente 97,7% dos isolados, sendo
que 81,3% foram identificados dentro de quatro horas com base nos três primeiros
testes.
Mulder et al. (1995) propuseram um esquema de diferenciação de S. epidermidis
e S. hominis dos demais SCoN utilizando as provas de suscetibilidade a desferroxamina;
fermentação da trealose, sacarose e manitol. O esquema se mostrou de fácil execução,
baixo custo, boa qualidade e permitiu identificar satisfatoriamente S. epidermidis e
S. hominis.
Monsen et al. (1998) realizaram um estudo propondo um esquema tido como
simples, de baixo custo e factível, utilizando um painel de testes com: produção de
29
urease e de ß-galactosidade; fermentação da maltose, manose, trealose, manitol,
sacarose e ribose. Também realizaram testes de suscetibilidade com furazolidona,
desferroxamina, polimixina B, novobiocina e bacitracina. Este esquema foi comparado
com o proposto por Kloos & Schleifer (1975) e com os testes comerciais do sistema ID
32 Staph. O esquema de Monsen e colaboradores permitiram a identificação de 96,6%
das espécies, incluindo a identificação de alguns isolados que não haviam sido
corretamente identificados pelo esquema convencional ou pelo sistema comercial
testado. Os achados deste estudo enfatizaram a necessidade de redefinir os pontos de
corte para os discos de identificação.
Em um laboratório da Coréia do Sul foi desenvolvido um método para
identificação rápida e econômica usando um meio para detecção de MRSA e MRSE
chamado “Staphy Agar” (Yoshida’s MRSA detecting medium), bem como, a
identificação de S. epidermidis usando as seguintes provas: catalase, coagulase,
suscetibilidade a novobiocina e resistência a polimixina B. A diferenciação do
S. epidermidis dos outros SCoN pelo “Staphy Agar” e as demais provas foi consistente,
quando comparada com a identificação pelo Sistema VITEK (bioMèrieux), havendo
uma concordância de 94,7% nos resultados (JEONG et al. 2002).
Por outro lado, na Argentina, foi proposto um esquema para a identificação de
S. epidermidis utilizando as seguintes provas: fermentação da trealose e do manitol,
prova da ornitina descarboxilase e suscetibilidade a desferroxamina. Este esquema,
simples, mas com testes não utilizados de rotina, permitiu a identificação presuntiva do
S. epidermidis (De PAULIS et al. 2003).
Cunha et al. (2004) propuseram um método chamado de “simplificado”
envolvendo duas etapas, a primeira constando de sete provas e a segunda etapa de até 13
provas. Para a correta identificação do S. epidermidis foi necessário um número
30
reduzido de provas (sete testes), envolvendo fermentação da xilose, sacarose, trealose,
maltose e manitol, produção de hemolisina, crescimento anaeróbico em tioglicolato. As
demais provas só foram realizadas quando houve necessidade de assegurar a correta
identificação de uma espécie. Este método foi comparado com o método de referência
proposto por Kloos & Schleifer (1975) e Bannerman (2003) e apresentou 100% de
concordância na identificação correta das espécies, sendo então considerado eficiente,
embora a fermentação de carboidratos não seja realizada rotineiramente em laboratório
de microbiologia clínica. Também foi comparado com a identificação realizada pelo
sistema Staph API que apresentou uma baixa sensibilidade na identificação de alguns
SCoN.
Os sistemas automatizados comerciais utilizados como alternativa de
identificação são dispendiosos e freqüentemente apresentam resultados pouco
confiáveis, em função da variação no tempo de incubação, na concentração dos
substratos e ou marcadores de sensibilidade e na expressão da atividade metabólica que
são variáveis consistentes entre as espécies (CUNHA et al. 2004; LAYER et al. 2006).
Weinstein et al. (1998) demonstraram que o sistema automatizado MicroScan
Rapid (Dade Behring) usando três tipos de painéis, teve problemas na identificação de
S. hominis e S. haemolyticus em dois destes. Um dos painéis, após modificação,
apresentou melhor desempenho no sistema MicroScan (Dade Behring).
Em outro estudo, Spanu et al. (2003), foi realizada a identificação de 405
isolados de Staphylococcus spp pelo sistema automatizado VITEK 2 (bioMèrieux) e
pelo ID-Cocos Gram-positivos Cocci cards (ID-GPC, bioMèrieux). O sistema VITEK 2
(bioMèrieux) mostrou acurácia e confiabilidade em isolados de Staphylococcus spp,
embora para alguns SCoN não tenha sido efetivo. Também, Caierão et al. (2006)
demonstraram a superioridade do sistema MicroScan PC-13 (Dade Behring) sobre o
31
sistema VITEK GPS-105 (bioMèrieux) para identificação dos SCoN, tendo sido
observada uma acurácia de 96,8% e 78,8% respectivamente. Os testes que apresentaram
maior discrepância foram: fermentação da manose, da trealose e da sacarose; e também
na produção da urease.
Caracterização Genotípica
Algumas técnicas genotípicas vêm sendo utilizadas para a caracterização
epidemiológica das amostras hospitalares de S. epidermidis. A variabilidade genética
destas amostras pode ser avaliada através da técnica de eletroforese de gel em campo
pulsado (JORGENSEN et al. 1993; KAUFMANN et al. 1994).
Métodos moleculares também podem ser usados para detectar genes de
resistência antimicrobiana específicos em muitos microrganismos e quando associados
com métodos fenotípicos têm feito contribuições substanciais para o entendimento da
genética de resistência antimicrobiana e da disseminação dos determinantes de
resistência (BIEDENBACH et al. 2004). A detecção do gene mecA é considerado
padrão ouro para determinar a resistência à oxacilina e por isto tem sido utilizado em
vários estudos que avaliam a sensibilidade e especificidade dos métodos fenotípicos
(LOUIE et al. 2001; FERREIRA et al. 2003; CAIERÃO et al. 2004; FRIGATTO et al,
2005; SWENSON & TENOVER, 2005). A técnica da PCR consiste na detecção do
gene mecA pela amplificação de um fragmento desse gene a partir de seqüências de
oligonucleotídeos complementares. Considerando a variabilidade fenotípica na
expressão da resistência, métodos genotípicos são utilizados para a caracterização
segura da resistência à meticilina.
PCR em tempo real, ainda está um pouco distante de nossa realidade, tendo em
vista o elevado custo inicial do equipamento. Edwards et al. (2001) demonstraram as
vantagens de se utilizar esta técnica para identificação de SCoN. Esta PCR é um sistema
32
de detecção de quantificação em tempo real envolvendo análise direta do material.
Neste estudo regiões variadas do gene 16S rRNA de Staphylococcus spp foram
observadas nos diferentes graus de semelhança. A reação em tempo real apresentou
como vantagem a rapidez de execução e a não contaminação durante o processo.
Os estudos a seguir irão comparar técnicas fenotípicas com genotípicas para
obter informações sobre a patogenicidade e a epidemiologia dos SCoN. Atualmente, a
identificação da espécie nos SCoN pode ser confirmada por técnicas moleculares, o que
permite reavaliar continuamente alguns testes fenotípicos e garantir a sua acurácia.
Martineau et al. (1996 e 2001) desenvolveram uma PCR para identificação de
SCoN, tendo como alvo o gene tuf , que codifica o fator de elongação Tu. Este gene está
envolvido na formação da cadeia peptídica, sendo um constituinte essencial do genoma
bacteriano. O estudo realizado em 2001 desenvolveu também, uma combinação de
ensaios de PCR, tendo como alvo genes de identificação e de resistência, sendo neste
caso utilizado um multiplex com gene mecA.
Sivadon et al. (2004) avaliaram as vantagens de um método genotípico sobre
sistemas bioquímicos comerciais para identificação de SCoN. Houve o seqüenciamento
parcial do gene sodA em 168 isolados clínicos identificados previamente pelo sistema
ID 32 Staph (bioMèrieux). A seqüência de sodA foi útil para solucionar todas as
ambigüidades ou identificações inconclusivas gerenciadas pelo sistema comercial ID 32
Staph. O sistema comercial apresentou dificuldades de identificar determinadas espécies
de SCoN, tendo sido realmente efetivo para S. epidermidis. Os mesmos pesquisadores
em 2005 utilizaram o gene sodA para identificar genotipicamente os SCoN responsáveis
pelas infecções em pacientes com próteses ósseas e articulares. Neste, observaram a
prevalência das espécies, bem como o grau de patogenicidade de algumas delas segundo
os critérios recomendados pelo grupo OSIRIS (Oxford Skeletal Infection Research and
33
Intervention Service) (SIVADON et al. 2005). Fujita et al. (2005) reportaram o uso do
gene sodA para confirmação na identificação dos SCoN com ambigüidade ou
erroneamente identificados pelos métodos fenotípicos.
Heikens et al. (2005) compararam métodos fenotípicos comerciais para
identificação de SCoN com métodos genotípicos baseados no sequenciamento dos
genes 16S rRNA, tuf e sodA. A identificação da espécie do SCoN é altamente desejável
para permitir uma caracterização mais precisa da relação patógeno-hospedeiro. O gene
16S rRNA está presente no cromossomo de todas as bactérias, sendo o gene mais bem
conservado. O gene sodA codifica a superóxido dismutase manganês-dependente, esta
metaloproteína inativa os radicais superóxidos. O gene tuf codifica o fator de elongação
Tu e está envolvido na formação da cadeia peptídica. Esses genes são essenciais no
genoma bacteriano e por isso, são preferidos para propósitos de diagnóstico. Este estudo
comparou métodos de identificação fenotípica utilizando o sistema API Staph ID
(bioMèrieux) e o sistema automatizado BD Phoenix (Becton Dickinson-versão 4.01)
com métodos de identificação genotípica para isolados de SCoN. Os resultados obtidos
mostraram que o sequenciamento do gene 16S rRNA tem poder discriminatório
limitado, e que o gene tuf tem um excelente poder discriminatório além de elevada
especificidade. Também concluíram que o API Staph é uma alternativa na identificação
fenotípica confiável apresentando melhor desempenho que o sistema BD Phoenix.
Layer et al. (2006) avaliaram o desempenho do sistema VITEK 2 (bioMèrieux)
em combinação com VITEK 2 ID-GP (bioMèrieux) e o sistema BD Phoenix (Becton
Dickinson Diagnostic Systems) em relação a um método molecular de referência (gap
T-RFLP). Os resultados obtidos no estudo demonstraram um bom desempenho do
VITEK 2 ID-GP e um resultado menos favorável no sistema BD Phoenix. Foi também
34
observada a necessidade do uso de técnicas moleculares, nos isolados onde foi
encontrada ambigüidade.
35
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43
JUSTIFICATIVA DO ESTUDO___________________________________________
A bacteremia é uma das mais importantes situações na doença infecciosa e a
oportuna detecção e identificação do agente etiológico está diretamente relacionada com
a morbi-mortalidade do paciente.
Os custos para tratar estas infecções podem ser altos, e a crescente freqüência de
surtos de bactérias oxacilina resistentes pode causar transtornos significativos nas
rotinas hospitalares. Isolados de ScoN resistentes a oxacilina são a segunda principal
causa de infecções hospitalares e tem sido isolada com crescente freqüência em todo o
mundo, assim como em hospitais brasileiros. Os perfis de multiresistência aos
antimicrobianos obtidos entre amostras SCoN resistentes a oxacilina refletem a
problemática da pressão seletiva ocasionada pelo uso indiscriminado de antimicrobianos
e confirmam o papel dos SCoN como reservatórios de genes de resistência que poderão
ser transferidos para outras espécies.
Na maioria dos laboratórios clínicos, a identificação do S. epidermidis não é
realizada na rotina, devido à ausência de um esquema simples, exeqüível, rápido e
acurado, que possibilite investigar seus mecanismos de patogenicidade bem como seu
perfil de suscetibilidade frente aos antimicrobianos. Somente realizando a correta
identificação de gênero e espécie, seremos capazes de entender melhor estes
mecanismos e contribuir de maneira decisiva para levantamentos epidemiológicos
seguros. O método de referência é muito trabalhoso e consome tempo excessivo para ser
usado na rotina do laboratório de microbiologia clínica.
Os estudos genômicos são necessários para estabelecer conclusões a respeito da
identificação e da resistência aos antimicrobianos entre os S. epidermidis. Segundo
alguns autores, há uma associação entre o aumento da freqüência dos SCoN na
bacteremia hospitalar e a resistência aos antimicrobianos, justificando assim uma
44
análise detalhada da bactéria, para que sejam propostas novas estratégias de intervenção
para prevenção de resistência. Os principais objetivos de qualquer programa de controle
da resistência aos antimicrobianos devem ser os de melhorar a utilização, reduzir a
resistência, melhorar a evolução do paciente e reduzir tempo de internação.
Conhecer o perfil de resistência aos antimicrobianos das amostras advindas do
Complexo Hospitalar Santa Casa de Misericórdia de Porto Alegre no período de 2002 a
2004 e realizar a identificação fenotípica de um dos mais freqüentes agentes etiológicos
em infecções sistêmicas foram contemplados com a realização deste estudo.
45
OBJETIVOS DO ESTUDO_______________________________________________
Geral
Identificar Staphylococcus epidermidis isolados de hemoculturas de pacientes do
Complexo Hospitalar Irmandade Santa Casa de Misericórdia de Porto Alegre, no
período de 2002 a 2004.
Específicos
• Propor um esquema fenotípico simplificado, rápido e acurado para a
identificação de Staphylococcus epidermidis no Laboratório de Microbiologia Clínica;
• Validar o esquema proposto, comparando-o com método de referência de
identificação convencional e com o método molecular de detecção do gene tuf espécie
específico para Staphylococcus epidermidis;
• Determinar o perfil de suscetibilidade do Staphylococcus epidermidis
frente aos vários antimicrobianos utilizados na prática clínica;
• Analisar e comparar a resistência a meticilina através das técnicas de
disco difusão, Concentração Inibitória Mínima segundo o CLSI; tendo como “padrão
ouro” a detecção do gene mecA;
• Avaliar a formação de biofilme através de método fenotípico.
46
DELINEAMENTO DO ESTUDO_________________________________________
Estudo transversal (teste diagnóstico)
HIPÓTESE DO ESTUDO________________________________________________
O método simplificado com discos de desferroxamina e fosfomicina tem melhor
correlação com o gene tuf espécie específico para identificação do
Staphylococcus epidermidis do que o método fenotípico de referência.
47
ARTIGOS PRINCIPAIS______________________________________________________
48
Staphylococcus epidermidis: a simple phenotypic method for identification
Ana Lúcia Souza Antunes¹ ³
Carina Secchi¹
Keli Cristine Reiter³
Leandro Reus Rodrigues Perez¹
Ana Lúcia Peixoto de Freitas³
Pedro Alves d’Azevedo¹ ²
¹ Pós-Graduação em Ciências Médicas, Fundação Faculdade Federal de Ciências
Médicas de Porto Alegre, RS, Brasil.
² Departamento de Microbiologia e Parasitologia, Fundação Faculdade Federal de
Ciências Médicas de Porto Alegre, RS, Brasil.
³ Departamento de Análises, Faculdade de Farmácia, Universidade Federal do Rio
Grande do Sul, Porto Alegre, RS, Brasil.
Submetido para publicação no “Journal of Medical Microbiology”.
Corresponding author:
Ana Lúcia Souza Antunes
Faculdade de Farmácia – UFRGS
Av. Ipiranga 2752, sala 302, Porto Alegre, RS, Brasil, 90610-000
Telefone: (+55) 51 33165412 Fax: (+55) 51 33165437
E-mail: analucia2112@gmail.com
49
Abstract
The emergence of coagulase negative Staphylococcus spp (CoNS) as human pathogens
as well as reservoirs of antimicrobial resistance, increases the necessity of developing
reliable methods for identification of the most frequent species among them, to establish
the pathogen-host relationship. The conventional method proposed by Bannerman
(2003) is laborious and thus not applicable to use in clinical laboratory. Moreover, the
available commercial techniques of automation are expensive and most often provide
unreliable results. The aim of this study was to propose a method easy to perform,
associated with low costs and time-consuming for identification of
Staphylococcus epidermidis. At first, 490 CoNS isolates from blood culture were
identified by Bannerman's technique. Distinct approaches resulted in the use of two
disks containing each desferrioxamine and fosfomycin. Considering susceptibility to
desferrioxamine, as a major marker, 320 isolates had been selected for further
investigation in this study. Bannerman's technique identified 238 isolates as
S. epidermidis, 73 as S. hominis and 9 as others SCoN, while the method proposed in
this study identified 239 S. epidermidis and 76 S. hominis. Both techniques were
compared with the tuf gene species-specific method for S. epidermidis, using
Polymerase Chain Reaction (PCR). Considering the presence of the tuf gene as a gold
standard, the sensitivity presented by Bannerman's and the method here described were
96.3% and 98.3%, respectively. The positive predictor value of the latter was 99.2%.
This method proposed has proved to be a useful tool for identifying S. epidermidis, the
most frequent CoNS isolated from blood cultures in laboratories of clinical
microbiology.
Keywords: Staphylococcus epidermidis, identification techniques, desferrioxamine,
fosfomycin, coagulase negative Staphylococcus spp.
50
Introduction
The genus Staphylococcus currently include about 40 different species (Monsen
al., 1998; Bannerman, 2003; Cunha et al., 2004). The identification of species among
the coagulase negative Staphylococcus spp (CoNS), is not regularly made in clinical
laboratories of microbiology, because it is expensive, even with automation, and is
time-consuming when carried out manually (Edwards et al., 2001). Historically, only
S. aureus was considered pathogenic, but in the last two decades, CoNS have also
emerged as significant pathogens, especially in patients with medical devices,
immunocompromised patients, and premature newborns (Wieser & Busse, 2000).
S. epidermidis appears to be the most frequent isolated species among the CoNS and is
responsible for infections associated with temporary or permanent medical devices (De
Paulis et al., 2003). The most important mechanism of pathogenicity in S. epidermidis is
their ability of to form a biofilm (Ziebuhr et al., 2006).
The emergence of CoNS as pathogens and reservoirs of antimicrobial resistance
require fast and reliable methods for their identification, and to establish the pathogen-
host relationship, leading to a better understanding of the mechanisms of pathogenicity
and susceptibility profiles and more reliable epidemiological surveys (Couto et al.,
2001; Kontos et al., 2003).
The conventional method proposed by Kloos & Bannerman (1994) and modified
by Bannerman (2003) appears to be unreliable, and irreproducible in laboratory routine.
Furthermore, commercial identification systems and automated systems are not able to
make a reliable distinction between the different species of CoNS, because of the
variable expression of the phenotypic characters (Couto et al., 2001; Kontos et al., 2003;
Caierão et al., 2006). Other tests, such as enzyme electrophoresis or analysis of cellular
fatty acid composition, have also failed to make a reliable identification (Heikens et al.,
2005). Therefore, methods based on sequencing of Polymerase Chain Reaction (PCR)
amplicons were valuated and compared to phenotypic identifications of CoNS. A
number of PCR amplicon-sequencing-based methods for identification of CoNS have
been reported, i.e., targeting the 16S region of the ribosomal RNA, the sodA and tuf
genes. Multiple copies of the 16S rRNA gene are present on the chromosome of most
bacteria and are better conserved than the tuf gene. But the latter is a target with a better
discriminatory power to differentiate related species. Both the sodA and tuf genes are
essentially constituents of the bacterial genome and thus are preferential for
identification of Staphylococcus spp (Martineau et al., 2001; Heikens et al., 2005).
51
The difficulties to identification of Staphylococcus spp using tests that evaluate
acid production has previously been reported in several works, which evaluated manual
and automated systems (Ieven et al., 1995; Cunha et al., 2004; Caierão et al., 2006).
S. epidermidis and S. hominis presents variable results in traditional biochemical tests
and similar phenotypic identification. Susceptibility to desferrioxamine, besides being
of easily perfomance, has been described as capable of identification of S. epidermidis
(Lindsay & Riley, 1991; Lindsay et al., 1993; Mulder, 1995).
The aim of this study was to propose a method for identification of
S. epidermidis combining accuracy, rapidity and easy to perform, to allow their use in
routine laboratories of clinical microbiology. We used the phenotypic method according
to Bannerman (2003) and the tuf gene assay through PCR to evaluate the accuracy of
the approach.
Materials and Methods
A total of 490 isolates, from consecutive blood cultures obtained from Jan 2002
to Jul 2004, were used. The samples were stored in skim-milk at temperature of – 20 ºC
at the Laboratory of Gram-positive Cocci of the FFFCMPA. The quality control of the
tests was done using the Staphylococcus aureus 25923, S. epidermidis 12228, and
S. hominis 27844 of the American Type Culture Collection (ATCC).
1- Identification of S. epidermidis: The isolates were cultured in agar
supplemented with 5% sheep blood (Tryptic Soy Agar- Oxoid, Basingstoke, England)
for 24 h at 35 ºC, where colony morphology, hemolysis production and purity were
evaluated. Subsequently coagulase and catalase tests were performed. The method
proposed by Bannerman (2003) consist of a set of biochemical tests that determine the
utilization of carbohydrates such as sucrose, trehalose, maltose, mannose, mannitol,
lactose, cellobiose; and production of hemolysis; the activity of pyrrolidonyl
arylamidase; the presence of urease and ornithine decarboxylase; the resistance to
novobiocin 5µg (Oxoid Basingstoke, England); alkaline phosphatase; and anaerobic
growth in thioglycolate. Test readings were obtained after 24h, 48h, and 72h and in up
to 7 days of incubation at 35ºC. The novobiocin susceptibility test was performed by
disk diffusion in Mueller-Hinton Agar (Oxoid Basingstoke, England).
2- Genotypic method through PCR: The detection of the tuf gene was according
to Martineau et al. (1996), using S. epidermidis species-specific primers 5’- ATC AAA
AAG TTG GCG AAC CTT TTC A-3’ and 5’- CAA AAG AGC GTG GAG AAA AGT
ATC A-3’. Following amplification, 10µl of DNA were electrophoresed. The primer
52
sequence was selected because it presents 100% ubiquity among S. epidermidis
(Martineau et al., 1996).
3- Proposed method for identification of S. epidermidis: The following tests
were performed: susceptibility desferrioxamine 100µg (Desferal Novartis, Ciba-Geigy-
Sandoz Basileia, Switzerland), to fosfomycin 200µg (Oxoid Basingstoke, England) and
resistance to polymyxin B (300 UI Oxoid Basingstoke, England). Using disk diffusion,
resistance to polymyxin B, according to Bannerman (2003), was of inhibition zone <10
mm, and according to Monsen et al. (1998) ≤16 mm. Susceptibility to fosfomycin
according to Ieven et al. (1995) and Rosco diatabs diagnostic (2000) was established as
an inhibition zone >30 mm. The disks were prepared using 0.5 g desferrioxamine
diluted in 5 ml of sterile distilled water at a final concentration of 100 mg/ml, and 3 µl
of this suspension were used to impregnate the paper disk (Lyndsay & Riley, 1991;
Lindsay et al., 1993). The disks were maintained at –10º C for up to 12 months, and
consistence of results was confirmed by duplicate tests performed throughout the period
of study. Susceptibility to desferrioxamine was defined as zone ≥ 20 mm to
S. epidermidis. Those isolates with borderline inhibition values for desferrioxamine
were submitted to identification through automated system MicroScan (Dade Behring,
USA).
Results and Discussion
The 490 samples were identified through the technique proposed by
Bannerman (2003). The use of susceptibility to desferrioxamine, which included all
isolates identified as S. epidermidis, allowed the selection of 320 isolates for the
continuation of the study.
Using the tests proposed by Bannerman (2003) in those 320 isolates, 238
S. epidermidis and 73 S. hominis were identified, and nine isolates were classified as
other CoNS (Table 1). The same isolates were tested using the method proposed by this
study and evaluating the presence of the tuf gene for S. epidermidis through PCR. In the
genotypic technique, 241 isolates presented the tuf gene, while the proposed method
identified 239 S. epidermidis (Table 2).
Among the 320 CoNS, the tuf gene for S. epidermidis was found in 241 cases,
and the proposed method identified 239 of then. In two isolates (1 S. hominis and 1
S. warneri), fosfomycin resistance was observed despite the presence of the tuf gene. In
239 isolates identified as S. epidermidis by the proposed method, 237 presented the tuf
gene. On the other hand, four isolates with the tuf gene were identified as S. hominis.
53
In the 238 isolates identified as S. epidermidis with Bannerman's method (2003),
only 232 presented the tuf gene; therefore, six samples were misidentified. On the other
hand, among the 82 isolates identified as S. hominis, nine were also misidentified, since
they presented the tuf gene.
Evaluating polymyxin B resistance using two cut-points (Bannerman, 2003 and
Monsen, 1998), resistance was detected in 100 and 275 isolates, respectively. Together
with the other biochemical and enzymatic tests, 98 isolates were identified as
S. epidermidis according to Bannerman (2003); among those, 96 isolates presented the
tuf gene. It is of worth that 11 isolates not S. epidermidis by phenotypic tests also
presented tuf gene, all of them identified as S. hominis. On the other hand, the zones
proposed by Monsen et al. (1998), in conjunction with other tests identified 235
S. epidermidis, and the tuf gene were present in 227 of them. All tests for polymyxin B
were done in duplicate, and results were discrepant in 60% of the cases.
Fosfomycin, together with the other tests, showed to be highly sensitive (99.2%)
and specific (96.2%) for the identification of these two CoNS. Susceptibility to
fosfomycin was observed in 99.2% (239/241) isolates with the tuf gene and a positive
predictor value of 98.8%.
The measure of the susceptibility zone to desferrioxamine showed variations
even within the same species (Table 3). Most isolates (89.7%) presented zones between
25 and 35 mm of wide. Only three isolates presented a zone of 19 mm, which were
identified by automated system MicroScan as S. warneri (two cases) and S. hominis
(one case). In the 320 isolates susceptible to desferrioxamine, 317 presented a zone >20
mm, this value being set as cut-off point for susceptibility to desferrioxamine. The three
isolates with smaller halos presented a very close inhibition zone (19 mm). These
samples did not present the tuf gene and were submitted to identification by the
automated system MicroScan (Dade Behring, USA), with two being identified as
S. warneri and one as S. hominis. The same isolates had also presented discrepancy in
the biochemical identification and in the tests of urea and mannitol.
Considering the presence of the tuf gene as standard, the values for sensitivity
and predictive positive value were of 98.3% and 99.2% for the method here proposed
while for Bannerman's method (2003) we observed values of 96.3%. The accuracy of
the method presented was 98.1%. The method using desferrioxamine and fosfomycin
disk to identify S. epidermidis among the CoNS showed specificity of 97.5% (table 4).
54
CoNS are microorganisms frequently isolated from blood cultures. Since CoNS
are the etiological agents of a series of infectious processes, identification of these
microorganisms is important for the determination of their physiopathological
characteristics. Their clinical and epidemiological importance, have led to the
publication of various studies analyzing identification methods for these bacteria (Ieven
et al., 1995; Monsen et al., 1998; Bannerman 2003; De Paulis et al., 2003). The
frequency with which S. epidermidis was found ranges from 43% to 92%, depending on
the geographical region where the study was conducted (Ieven et al., 1995; Couto et
al., 2001; Vuong & Otto, 2002; De Paulis et al., 2003; Ferreira et al., 2003; Spanu et al.,
2003; Cunha et al., 2004; Caierão et al., 2006). In our study, we identified 241 samples
with the tuf gene for S. epidermidis, which corresponds to a prevalence of 49.2 % of all
490 CoNS studied.
Due to the difficulty of identification of non-coagulase positive Staphylococcus
many clinical laboratories don’t distinguing between S. epidermidis and other CoNS.
Although the interpretation of the susceptibility tests independs of this identification,
the choise of the antimicrobial agent may be influenced. For instance, even with
susceptibility in vitro, the use of vancomicin is not appropriate in treating infections due
to S. epidermidis; since biofilm formation may protect the microganism with failure of
treatment. Our proposed method to S. epidermidis identification proved to be a usefull
tool.This approach using desferrioxamine and fosfomycin disks is easier, cheap and
showed the same accuracy than the conventional phenotypic method. Therefore, we can
propose our method to the routine of clinical microbiology laboratory.
In the analysis of carbohydrate fermentation we evaluated the relevant data for
identification of S. epidermidis. Fermentation of mannose and threalose have being
pointed as important to identification, but we find that, mannose did not contribute
consistently to the identification of S. epidermidis, since the response was smaller than
expected. In the case of threalose, in almost half of isolates without tuf gene gave
positive results. It must be emphasized that Freney (1999) was one that refers to the
possibility of fermentation of threalose by S. epidermidis. Also, in a recent study
Caierão et al. (2006) demonstrated the failure of automated systems to evaluate the
fermentation of mannose and threalose among the CoNS.
We defined the use of Christensen's Urea Agar more appropriate than Rustigian
& Stuart's broth (data not shown). This choice was based on a sharper and faster results
55
and the routine use of this medium. The totality of S. epidermidis isolates (tuf gene) was
positive for urease production when Christensen's Urea Agar was used.
As regards to the tests of alkaline phosphatase and growth in anaerobiosis
(Bannerman's), we observed that while alkaline phosphatase was simple and easily
executed, the visualization of growth in anaerobiosis is not. The positive predictor and
negative predictor values for alkaline phosphatase were 96.2% and 85.4%, while to
growth in anaerobiosis the values were 96.6% and 96.3% respectively. The final method
of this work did not elected those two testes, although the conventional method of
Bannerman includes both of them.
Desferrioxamine is a siderophore, unavailable in disks and thus must be
prepared (Lindsay & Riley, 1991). In Lindsay & Riley's study (1991), several inhibition
zones were considered to evaluate the susceptibility to desferrioxamine. Using disks of
100 µg, these authors observed zones >16mm, but they concluded that any halo was
meaningful. We decided that a zone >20 mm was more exact. Desferrioxamine is an
excellent marker for identification of S. epidermidis when we use the proposed
breakpoint.
About 6% of the cases (20/320) presented a double zone of inhibition,
particularly in S. epidermidis rather than in S. hominis, as previously reported by
Monsen et al. (1998). Since the other proofs indicated S. epidermidis, it was considered
that the formation of internal zone should not be considered at the moment of reading,
even when the internal zone was < 20 mm. It was not in the scope of this study to
investigate the causes of this double zone.
Polymyxin B disks were also used for phenotypic identification of
S. epidermidis, since this species is resistant to this antimicrobial. Concording to our
results, this variability had been reported that the reproduction of the disk-diffusion
technique is unsatisfactory (Gales et al., 2001; Sejas et al., 2003). Due this great
variability the polymyxin B disks were replaced by fosfomycin ones. This test turned
out to be imperative in the phenotypic sorting of S. epidermidis and S. homins.
Two isolates selected by the desferrioxamine disk, presented resistance to
novobiocin, which led to further analysis, since S. epidermidis and S. hominis are both
susceptible to novobiocin. The subspecies S. hominis subsp. novobiosepticus is an
exception, and outbreaks have already been reported in neonates in Spain (Chaves et al.,
2005) and, more recently, in a Hospital Geral de São Paulo (d’Azevedo et al., 2006). In
this case the use of susceptibility testes such as desferrioxamine (100µg), fosfomycin
56
(200µg) and novobiocin (5µg) disks were finding to be the best way to identify
S. epidermidis.
Based on our results, we proposed a simplified method for identification of
S. epidermidis among the SCoN. The proposed method comprises susceptibility tests to
desferrioxamine (>20 mm) and fosfomycin (30 mm). So, we propose that both disks
should be included in the routine susceptibility tests in laboratories of clinical
microbiology (Figure 1).
Heikens et al. (2005) demonstrated the superiority of the tuf gene for
identification of CoNS species because of its excellent discriminatory power and its
high specificity, concerning the ATCC CoNS samples and the sequences stored in the
GenBank. The authors also reported the limited discriminating power of 16S rRNA
sequences for identification of CoNS species.
The use of PCR for the identification of the tuf gene supports the evidence that
the proposed phenotypic characterization can solve the problem of S. epidermidis
identification. Until the moment, all phenotypic techniques were laborious,
unsatisfactory and irreproducible. The sequencing of the tuf gene showed that
S. warneri is closely related to S. epidermidis, which may perhaps account for their
occasionally similar phenotypic behavior.
In conclusion, a method using only desferrioxamine and fosfomycin disks
showed great correlation with Bannerman's method and with gen tuf. This simple, low
cost system can be a useful tool for identifying S. epidermidis that today is the most
frequently CoNS isolated from blood cultures in laboratories of clinical microbiology.
57
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60
Table 1: Results in the biochemical tests of 320 isolates of CoNS susceptible to desferrioxamine.
species
reaction
mannitol maltose mannose sucrose trehalose lactose cellobiose urea ornithine alkaline p*
S.epidermidis pos 1 234 74 237 7 222 0 238 34 226
n= 238 neg 237 4 164 1 231 16 238 0 204 12
S.hominis pos 2 73 5 73 45 63 0 70 2 9
n= 73 neg 71 0 68 0 28 10 73 3 71 64
other CoNS pos 2 9 7 9 6 9 0 7 4 3
n= 9 neg 7 0 2 0 3 0 9 2 5 6
*alkaline phosphatase
Table 2: Prevalence of S. epidermidis
Identification Method
n %
Bannerman 238/490 48.6
Proposed 239/490 48.8
tuf
241/490 49.2
Table 3 Behavior of species regarding the desferrioxamine disk (100µg)
Inhibition zone (in mm)
species 19-24 25-29 30-35 36-41
S.epidermidis 3
63 158 17
S.hominis
2 21 48 6
S.warneri
2 0 0 0
Total = 320 7 84 206 23
Table 4: Comparison of the two phenotypic methods with the genotypic one
sensitivity specificity PPV
Bannerman's method 96.3% 92.4% 97.5%
Proposed method 98.3% 97.5% 99.2%
61
Figure 1.
Susceptibility test with both disks for identification of S. epidermidis
62
Evaluation of oxacillin and cefoxitin disks for detection of resistance in
Coagulase Negative Staphylococci
Ana Lúcia Souza Antunes¹ ³
Carina Secchi¹
Keli Cristine Reiter³
Leandro Reus Rodrigues Perez¹
Ana Lúcia Peixoto de Freitas³
Pedro Alves d’Azevedo¹ ²
¹ Programa de Pós-Graduação Ciências Médicas, Fundação Faculdade Federal de
Ciências Médicas de Porto Alegre, RS, Brasil.
² Departmento de Microbiologia e Parasitologia, Fundação Faculdade Federal de
Ciências Médicas de Porto Alegre, RS, Brasil.
³ Departmento de Análises, Faculdade de Farmácia, Universidade Federal do Rio
Grande do Sul, RS, Brasil.
Submetido para publicação no “Memórias do Instituto Oswaldo Cruz”.
Corresponding author:
Ana Lúcia Souza Antunes
Faculdade de Farmácia – UFRGS
Av. Ipiranga 2752, sala 302, Porto Alegre, RS, Brasil, 90610-000
Telefone: (+55) 51 33165412 Fax: (+55) 51 33165437
E-mail: analucia2112@gmail.com
63
Abstract
Coagulase-negative Staphylococcus spp was considered nonpathogenic until the
emergence of its multiresistance and the demonstration of their participation as
infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates,
thus increasing the use of vancomycin. Recently the Clinical and Laboratory Standards
Institute (CLSI) standardized a new method to predict oxacillin resistance in the
Staphylococcus genus. The aim of this study was to evaluate the variability among
commercial disks of oxacillin (1µg) and cefoxitin (30µg) widely used in clinical
laboratories of microbiology, using oxacillin MIC as the reference standard. Polymerase
Chain Reaction (PCR) assays for the mecA gene were performed in isolates with
discrepant results. The use of oxacillin and cefoxitin disks simultaneously allowed the
detection of important differences, particularly, in less frequent species such as
S. cohnii, S. warneri and S. sciuri, where false resistance was observed. The cefoxitin
disk of brand 2 showed good correlation with the mecA gene and oxacillin MIC
(97.8%). One of the critical points in the diffusion disk test is the quality of the disks.
The use of better quality disks, according to CLSI guidelines, associated with molecular
methods lead to results that can define the best antibiotic therapy.
Keywords: CoNS, mecA gene, methicillin resistance, cefoxitin, susceptibility tests
64
Introduction
Coagulase-negative Staphylococcus spp (CoNS) were considered nonpathogenic
until recently, when the implication on nosocomial infections and the emergence of
multiresistance changed this picture (Beekmann et al. 2003). Hospital infections caused
by these microorganisms are responsible for high morbidity and mortality rates
worldwide. The use of empirical treatment has not contributed to reduce these infections
(Frigatto et al. 2005). The emergence of CoNS populations with heterogeneous
resistance to oxacillin led to a great difficulty to detect then in clinical laboratories
(Cauwelier et al. 2004). In Brazil, oxacillin resistance may be present in over 80% of
isolates in some health institutions (Sader et al. 2001). On this account, vancomycin has
been widely used in treating these infections and it is a major cause for the emergence
of glycopeptide-resistant isolates (Oliveira et al. 2001, Nunes et al. 2006).
Recently, CLSI (2006) standardized a new method to predict resistance in
Staphylococcus spp mediated by the mecA gene, through diffusion test with cefoxitin
disk (30µg). Others studies indicate that this is the best phenotypic test to predict
resistance to beta-lactam agents among CoNS (Felten et al. 2002, Pottumarthy et al.
2005). Despite the CLSI guidelines, the detection of oxacillin resistance through
phenotypic methods remains a challenge for clinical laboratories of microbiology (Sejas
et al. 2003). Several errors was observed among the laboratories that participated in the
Antimicrobial Surveillance Program (SENTRY), suggesting non-observance of the
interpretation criteria currently recommended (Mendes et al. 2003). The quality of the
antimicrobial disks affected the results, particularly failing in detect heteroresistance,
what is especially importat in the case of oxacillin and cefoxitin, which predict
susceptibility to a large group of antimicrobial agents (Sejas et al. 2003).
The aim of this study was to evaluate two brands of oxacillin (1µg) and cefoxitin
(30µg) disks, commonly used in clinical laboratories of microbiology.
Materials and methods
1-Isolates - CoNS isolates from blood cultures, from the collection of the
Laboratory of Gram-positive Cocci of the FFFCMPA, where were maintained in
“Skim-Milk” (Difco, Detrot) at -20°C. Strains of Staphylococcus aureus ATCC 25923
(susceptible to oxacillin and penicillin) and ATCC 33591 (resistant to oxacillin and
penicillin) were used as quality control.
2-Bacterial identification - The species identification was performed with the
combination of a group of laboratory tests: colony morphology; oxygen requirement;
65
susceptibility to novobiocin (Oxoid, UK), fosfomycin (Oxoid, UK), and
desferrioxamine (Desferal, Ciba Geigy, Switzerland); enzymatic activity of coagulase
(Laborclin, Brazil), catalase, alkaline phosphatase (phenolphthalein diphosphate,
Sigma-Aldrich, Germany), ornithine decarboxilase (Oxoid,UK), urease (Oxoid,UK) and
PYR (pyrrolidinyl arylamidase, Probac, Brazil); hemolysis production in agar
supplemented with sheep blood at 5% (Trypticasein agar, Oxoid,UK); and acid
production from carbohydrates: trehalose (Sigma-Aldrich, Germany), mannitol
(Nuclear, Brazil), mannose (Vetec, Brasil), sucrose (Vetec, Brasil), maltose (Sigma-
Aldrich, Germany), lactose (Vetec, Brasil), and cellobiose (Sigma-Aldrich, Germany).
3-Susceptibility test - Oxacillin (1µg) and cefoxitin (30µg) disks from two
different brands widely used in clinical microbiology laboratories were tested. Strains
were cultivated in agar supplemented with sheep blood at 5% (Trypticasein agar -
Oxoid, UK) for 24h and a 0.5 Mcfarland standard suspension was prepared for each
sample. The disks diffusion tests were performed by using Mueller Hinton (Oxoid, UK)
agar plates. The disks were distributed maintaining a distance of 30 mm from one to
another and of 15 mm from the plate border. The diameters of the inhibition zones were
interpreted according to the criteria recommended by the CLSI. Mueller-Hinton agar
plates (Oxoid, UK) supplemented wiyh 2% NaCl and concentrations of 0.125 to 4 µg of
oxacillin (Sigma Chemical Company, St. Louis, MO) was used for determination of
MIC for oxacillin. Steers replicator was used to inoculate the plates that were incubated
at 35ºC and screened after 24h. The presence of mecA gene was checked through the
technique of Polymerase Chain Reaction (PCR), according to Bignardi (1996). MRSA-
slidex Latex test (bioMèrieux, Marcy-I’Étoile, France) according to Chambers (1997)
was also performed to verify discrepant results between the disk diffusion and the MIC.
Results
A total of 302 coagulase-negative Staphylococcus spp. isolates were studied.
Table 1 shows the distribution and resistance in each species and the table 2 shows the
percentages of oxacillin and cefoxitin resistance observed with the disks of different
brands and the percentage of oxacillin resistance MIC.
The determination of oxacillin MIC showed results that are consistent with
resistance in 91.7% (277/302) isolates. Such resistance was detected by oxacillin disk of
brand 1 in 239 cases (79.1%) and of brand 2 in 273 cases (90.4%). Using cefoxitin
disks, we observed that brand 1 showed similar results in only 149 cases (49.3%) and
brand 2 in 264 cases (87.4%). The disks of brand 2 showed good correspondence
66
between oxacillin MIC and disk diffusion tests with both substrates (98.5% for the
oxacillin disk and 97.8% for the cefoxitin disk). However, brand 1 showed
correspondence for oxacillin in 86.3% of the isolates and in only 55.0% for cefoxitin.
Disks of oxacillin and/or cefoxitin of brand 2 failed in 13 situations, 11 cases
with MIC resistant e two susceptibles. To elucidate these results, we performed MRSA
Latex test and search the presence of mecA gene by PCR (Figure 1). Both tests were
positive in 4 cases and negative in the other 9. Almost half of the cases (6/13) were
identified as S. sciuri (Table 3).
Among isolates with oxacillin MIC and mecA gene not concordant, we observed
concordance with gene and disks of oxacillin in three cases and disks of cefoxitin in
eigth out of nine cases (Table 3).
Discussion
Detection of oxacillin resitance among CoNS isolates is dificult, mainly because
it is often heterogeneous (Chambers 1997). To overcome this problem, different
methods have been used. Several studies have demonstrated that PCR is a sensitive
method; however, most laboratories are not in a position to perform the test.
Phenotypically, none of the individual tests or combinations were able to detect all
methicillin-resistant strains.
Due to the emergence of vancomycin resitance, used in oxacillin resistant
isolates, it is essencial that clinical laboratories can distinguish between oxacillin-
susceptible and oxacillin-resistant CoNS strain. Recently the CLSI advises the use of
disks of cefoxitin as a better predictor of mecA gene-mediated resistance in
staphylococci species. Cefoxitin is a stronger inducer of the expression of penicillin-
binding protein 2a (PBP2a). Several investigators reported that cefoxitin disk diffusion
tests correlate better with the presence of mecA than do the results of disk diffusion
tests using oxacillin. Our results highlight the importance of testing the oxacillin disks
concomitant with cefoxitin disks of excellent quality, and to make PCR when there is
discrepancy.
In disk diffusion tests, the main variable not directly controled by the laboratory
is the quality of the disk. Since disk diffusion test with cefoxitin in considered to be a
good test to discriminate between resistant and susceptible isolates, laboratory is
dependent of the quality of the disk, and so, it is essencial to recopgnize that not ever
they are confiable.
67
The most frequent CoNS species found was S. epidermidis, with 157 isolates
(52%), followed by S. hominis (56 isolates; 18.5%), S. haemolyticus (52 isolates;
17.2%), S. sciuri (10 isolates; 3.3%) and S. warneri (9 isolates; 3%); other species were
present at a lower rate. Concerning to oxacillin MIC, the percentage of resistance in the
3 prevalent species was very similar, about 90%.
It is important to point out that oxacillin tests were similar with both commercial
disks, while cefoxitin results showed a great difference between the two brands.
Resistance results of oxacillin MIC and diffusion were concordant in 86.3% with
oxacillin disks, and 54.2% with cefoxitin, showing a great variability among cefoxitin
commercial disks. Considering that the treatment of infections caused by
Staphylococcus genus depends primarily on determining whether the bacteria is a
methicillin resistant Staphylococcus spp (MRS) or not (Chandran & Rennie 2005), and
that this is determined by the result of susceptibility to oxacillin and cefoxitin, the high
variability of the tests may lead to an inadequate therapy. A study in the United States
reported a mortality rate of 17.5% in adult patients with bacteraemia, while patients
receiving adequate antimicrobial therapy had a lower mortality rate (Weinstein et al.
1997). False-positive resistance results account for increased costs regarding additional
clinical work, request of more cultures, and unnecessary use of antimicrobials like
vancomycin (Chandran & Rennie 2005).
The brands of the disks used for the susceptibility tests in this study are among
those most frequently used in the microbiology laboratories. It was already pointed that
brand 1 is cheaper but less reproductible (Sejas et al. 2003), while brand 2 is
significantly more expensive but with a better performance (Skov et al. 2005).
Most of the isolates (85.7%) in which there were similar results among all
methods, presented a high oxacillin MIC (≥ 4 µg/ml), making its detection easier than
for isolates with MIC values close to susceptibility levels. However, even in isolates
with a high MIC of 2 µg/ml, there was discrepancy: among 253 isolates, the oxacillin
disk of brand 1 displayed less sensitivity (86.6%), than brand 2 (98.8%).The discrepant
results were much greater with cefoxitin disks, as brand 1 failed to detect 96 (34.7%)
cases of resistance detected by brand 2.
In cases of resistance with MIC ≤ 0.5 µg/ml (18 cases), oxacillin disk of brand 1
detected 55.5% of the cases, while brand 2 detected all cases. Cefoxitin disks showed a
greater discrepancy between the brands; even considering the superior results of brand 2
had in comparison to brand 1 (38% greater).This percentage is quite smaller than
68
expected.
There was a total agreement between MIC results and disk test of brand 2 in
susceptible isolates. On the other hand, with brand 1 there were major discrepancies of
false resistance: 40% with oxacillin disks and 20% with cefoxitin disks. Even if these
results do not lead to a treatment failure, the unnecessary use of drugs other than the
beta-lactam antimicrobials, may lead to a increase of resistance.
In concordance to CLSI guidelines and the results of other studies (Swenson &
Tenover 2005, CLSI 2006), the mecA gene was not detected in seven isolates resistant
to oxacillin by the disk diffusion test. The cefoxitin disk showed greater sensitivity in
these isolates, an evidence of a greater correlation of this substrate and the presence of
the gene. Most of these isolates were of S. sciuri.
As the mecA gene is detected in most of the MRS, this is considered the best
method of resistance detection, although discrepancies have already been reported,
particularly in CoNS isolates (Swenson & Tenover 2005, Darini & Palazzo 2006).
Isolates with absence of the mecA gene and MIC for oxacillin >0.5 ug/ml had
controversial results in disk diffusion tests. For such uncommon staphylococcal species
as S. cohnii cohnii and S. warneri, this is not a surprise (Darini & Palazzo 2006,
Hussain et al. 2000). Oxacillin resistance in the absence of the mecA gene may be due to
overproduction or overexpression of penicillinase, or alteration of other PBPs (Caierão
et al. 2004).
The disk diffusion test proposed by Kirby-Bauer in 1966 is one of the most
popular methods used in brazilian clinical laboratories. In this test the quality of the
disks is crucial (Sejas et al. 2003). Our study indicated that the use disks of good quality
(brand 2) and antimicrobial susceptibility test according to CLSI led to a reliable result,
although in a few cases only one of the disks had provided the adequate response.
In our study, the discrepancy of MIC and oxacillin disks was detected in less
frequent CoNS, showing that these species present a certain difficulty to express
resistance to oxacillin, as yet dercribed (Hussain et al. 2000; Skov et al. 2005).
Also, Frigatto et al. (2005) obtained discrepant results between oxacillin and
cefoxitin resistance using the disk diffusion method among S. epidermidis. They
emphasized the importance of testing both disks concomitantly and using molecular
techniques for confirmation of results. In our study, cefoxitin disks (brand 2) showed a
better concordance with presence of mecA gene (PCR) than oxacillin disks, hence the
importance of performing tests using both disks.
69
Although no technique alone, shows 100% of sensitivity and specificity to detect
oxacillin resistance among CoNS, the combination of disk diffusion and oxacillin MIC
can reduce errors in the detection of such resistance and while molecular techniques are
suitable for confirmation of results (Skov et al. 2005)..
Microbiology laboratories require rapid, sensitive and specific techniques to
perform susceptibility tests. In the last decades, new molecular methods have been
developed and introduced in clinical laboratories of microbiology to improve the
credibility of these tests. However, for most clinical laboratories these techniques are
still very expensive and susceptibility by disk-diffusion tests is the most common
method used. To ensure the phenotypic results that will guide the antimicrobial therapy
we must use antibiogram disks of excellent quality.
Acknowledgments
The authors wish to thank the technical team of the Laboratory of Gram-positive
Cocci of the FFFCMPA. This study was supported by the following agencies:
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), Faculdade de
Farmácia da Universidade Federal do Rio Grande do Sul (UFRGS), and Fundação
Faculdade Federal de Ciências Médicas de Porto Alegre (FFFCMPA).
70
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72
Table 1: Prevalence and oxacillin MIC for each Staphylococcus spp
Total % resistance
Species n % (MIC)
S.epidermidis
157 52 94.2
S.hominis
56 18.5 94.3
S.haemolyticus
52 17.2 94.1
S.saprophyticus
6 2.0 83.3
S.sciuri
10 3.3 90.0
S.simulans
3 1.0 66.6
S.warneri
9 3.0 75.0
S.xylosus
2 0.7 100
S.capitis capitis
3 1.0 66.6
S.capitis ureolyticus
1 0.3 0.0
S.cohnii cohnii
2 0.7 50.0
S.cohnii urealyticum
1 0.3 100
Table 2: Resistance of coagulase-negative Staphylococcus spp
Resistance
Tested disk n / total %
Oxacillin, brand 1 239 / 302 79.1
Oxacillin, brand 2 273 / 302 90.4
Cefoxitin, brand 1 149 / 302 49.3
Cefoxitin, brand 2 264 / 302 87.4
Oxacillin MIC 277 / 302 91.7
Table 3: Results of susceptibility tests with brand 2 disks, discordant from oxacillin MIC
Isolates Gene MIC Oxacillin Cefoxitin
Species
mecA
µg/ml
708 positive >4 S R S.epidermidis
328 positive 0.5 S R
S.haemolyticus
605 positive 1.0 R S
S.haemolyticus
244 positive 0.25 R S
S.saprophyticus
322 negative >4 S S
S.epidermidis
226 negative 0.5 R S
S.cohnii cohnii
327 negative 0.5 R S
S.warneri
400 negative 0.5 S S
S.sciuri
201 negative 0.5 R S
S.sciuri
216 negative 0.5 R S
S.sciuri
202 negative 1.0 R S
S.sciuri
235 negative 1.0 R S
S.sciuri
229 negative 0.25 R S
S.sciuri
R = resistant; S= susceptible
73
Figure 1: PCR detection of mecA gene of 4 isolates with discrepancy regarding oxacillin
disk. Lane 1-(MW) = molecular size markers (λ 100 bp); lane 2-(CP) = positive
controls: 310-bp band obtained with DNA from S. aureus reference strain ATCC
33591; isolates: 322 – absence of band with DNA from S. epidermidis; 328 - 310-bp
band obtained with DNA from S. haemolyticus; 400- absence of band with DNA from
S. sciuri; 708 - 310-bp band obtained with DNA from S. epidermidis.
M
M
W
W
C
C
P
P
3
3
2
2
2
2
3
3
2
2
8
8
4
4
0
0
0
0
7
7
0
0
8
8
74
Detection of biofilm production in Staphylococcus spp. isolates by the Congo red
agar test
Ana Lúcia Souza Antunes *’ **
Leandro Reus Rodrigues Perez *
Carina Secchi *
Keli Cristine Reiter **
Letícia Filippon **
Paula Eidt Fornari **
Ana Lúcia Peixoto de Freitas **
Pedro Alves d’Azevedo *
* Programa de Pos Graduação em Ciências Médicas e Departamento de
Microbiologia e Parasitologia, Fundação Faculdade Federal de Ciências Médicas de
Porto Alegre, RS, Brasil.
** Departmento de Análises, Faculdade de Farmácia, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brasil.
Financial support: CAPES, CNPQ, UFRGS, FFFCMPA
Submetido para publicação no “Memórias do Instituto Oswaldo Cruz”.
Corresponding author:
Ana Lúcia Souza Antunes
Faculdade de Farmácia – UFRGS
Av. Ipiranga, 2752,sala 302, Porto Alegre, RS, Brasil, 90610-000
Telefone:(+55) 51 33165412 Fax: (+55) 51 33165437
E-mail: [email protected]
75
Abstract
Staphylococci are an increasing cause of bloodstream infections associated with medical
devices. The formation of biofilm, or slime, on the surface of biomaterials, required for
the establishment of infection, has also led to an increase in antimicrobial resistance.
This study evaluated the formation of biofilm in 28 isolates obtained from catheters and
blood cultures using evaluation of the phenotypic appearance of colonies in Congo red
agar (CRA). Overall, sixteen isolates (57.2%) were considered as biofilm producers.
Among S. aureus isolates, 43.8% were considered as biofilm producers (7/16), while 12
S. epidermidis, 8 (66.6%) were positive. Greater antimicrobial resistance was observed
among biofilm-producing S. epidermidis isolates, differently from S. aureus, in which
producers of this virulence factor showed less resistance than nonproducers. The use of
CRA proved to be practical and easily to perform in the Laboratory of Clinical
Microbiology. However, a larger number of isolates must be studied to prove its
effectiveness and thus be used in laboratory routines.
Keywords: biofilm production, Staphylococcus spp, implant materials, antimicrobial
resistance.
76
Introduction
Staphylococcus aureus and Staphylococcus epidermidis, are important causes of
infections associated with catheters and other medical devices, such as articular
prosthesis, cardiac valves, pacemakers, contact lenses and intrauterine devices (O'Gara
& Humphreys 2001,Arciola et al. 2002, Curtin et al. 2003).
S. epidermidis is one of the most frequently agent isolated among the group of
coagulase-negative Staphylococcus (CoNS). This group is distinguished from S. aureus
by its inability to produce coagulase. Together with other staphylococci, S. epidermidis
are normal inhabitants of human skin and mucous membranes (O'Gara & Humphreys
2001, Arciola et al. 2002, Donlan & Costerton 2002, Vuong & Otto 2002). The port of
entry of these microorganisms into the human body usually is an intravascular catheter
(Vuong & Otto 2002). S. epidermidis become one of the major infectious agents in
immunocompromised patients.
Microorganism adherence and fixation to the surface of biomaterials are the first
step for the development of device-related infections. Several mechanisms are involved
in bacterial adhesion, and the production of a natural polysaccharide extracellular
substance, the so-called biofilm (or slime), seems to play an important role. This
substance mediates adhesion to the surface acting as a matrix and allowing the bacterial
cells to agglomerate into biofilm multilayer, thus rendering the bacteria less accessible
to the defense system and to antimicrobials (Arciola et al. 2002, Hume et al. 2004).
Biofilm production is frequently observed in clinical isolates of S. aureus and
coagulase-negative staphylococci, especially S. epidermidis, isolated from catheter-
associated infections (Arciola et al. 2001, 2002) .
Molecular techniques, as polymerase chain reaction (PCR), provide direct
evidence for the genetic basis of biofilm production. The synthesis of the capsular
polysaccharide is mediated by the ica operon. On activation of this operon, a
polysaccharide intercellular adhesin (PIA) is synthesized, and this supports cell-to-cell
bacterial contacts by means of a multilayered biofilm (Arciola et al. 2001, 2002).
Treatment of nosocomial infections associated to S. aureus and S. epidermidis is
usually complex, due to the increased resistance against antimicrobials and the
impermeable barrier created by biofilm formation. In addition to the low diffusion or
failure of the antimicrobial to penetrate, the resistance to antimicrobials may be
associated with physiological alterations and low bacterial growth within the biofilm
(Donlan & Costerton 2002, Vuong & Otto 2002, Curtin et al. 2003). The prevalence of
77
methicillin-resistant S. aureus (MRSA) and the emergence of vancomycin-resistant
isolates make treatment of infections related to medical devices even more difficult.
Oxacillin resistance encoded by the mecA gene is disseminated in the Staphylococcus
spp. As the S. aureus and S. epidermidis resistance profile are similar, so it is assumed
that resistance occurring in one species can rapidly be transferred to another (Vuong &
Otto 2002). Failure of treatment usually results in the need of device replacement,
increasing risk to the patient’s health, and also development of bacterial resistance
(Monzon et al. 2002).
The most frequently test used to observe biofilm production in the laboratory of
clinical microbiology is the Congo red agar (CRA), as described by Freeman et al.
(1989). The direct analysis of the colonies in the surface of the CRA allow the
recognition of biofilm-producing (characterized by black colonies on the red agar) and
non-producing (rose/red colonies) (Freeman et al. 1989). A comparative study between
the CRA test and presence of gene ica by PCR technique, demonstrated concordance
between both methods, showing that the phenotypic analysis of colonies may be a
reliable test that can be used routinely in microbiology laboratories (Arciola et al. 2002).
Considering the importance of Staphylococcus species in hospital infections and
the importance to determine the ability of produce biofilm, this study evaluated a
practical method using the CRA, testing isolates from catheters and blood cultures. In
addition, resistance rate of these isolates were determined through diffusion disk testing,
as well as their relationship to biofilm production.
Materials and methods
1- Bacterial isolates - A total of 28 isolates were analyzed, 16 of S. aureus and
12 of S. epidermidis, isolated from catheter and blood cultures obtained from patients
hospitalized in two institutions: Hospital Mãe de Deus and Hospital de Clínicas of Porto
Alegre, south of Brazil, from August to September 2005. The identification was made
by acid production from carbohydrates, and susceptibility to desferrioxamine along with
classical tests (Monsen et al. 1998, Kloos & Bannerman 1999).
2- Characterization of the ability to produce biofilm - Production of biofilm from
all strains was studied by culturing each isolate in CRA (Oxoid, Basingstoke, UK).
CRA plates were prepared adding 0.8 g of CRA and 36 g of sucrose (both from Sigma,
Missouri, US) to 1l of agar trypticaseina (Oxoid, Basingstoke, UK) according by
Arciola et al. (2002). After the inoculation of a pure culture, the plates were incubated
for 24h at 35ºC, and then overnight at room temperature. A four-color scale ranging
78
from black to red was used. Black (B) colonies were considered as biofilm-producing,
while almost black (AB) colors were considered as indicative of a weak biofilm
production activity. Conversely, red (R) to bordeaux (BR) colonies were taken as
unable to produce this virulence factor. S. epidermidis ATCC 35984 was used as
positive control of CRA and S. epidermidis ATCC 12228 was used as negative control.
3- Antimicrobial susceptibility test - The susceptibility to 5 antimicrobial agents,
including vancomycin 30 µg, oxacillin 1 µg, linezolide 30 µg, gentamicin 10 µg, and
rifampicin 5 µg disks (Oxoid), was determined by the disk diffusion method according
to the guidelines of the Clinical and Laboratory Standards Institute (CLSI - 2005),
S. aureus ATCC25923 was used as a control.
Results
Phenotypic detection of biofilm production by the CRA test - The phenotypic
production of biofilm in all isolates of the study was analyzed through observation of
colonial morphology in CRA. Definition of biofilm production was considered by
observation of the color of the colony at the end of the period of incubation (Table I).
Overall, 16 isolates (16/28) were considered as biofilm producers: 12 strong (B) and
4weak (AB). Analyzing staphylococcal species alone, we find that among the 16
S. aureus isolates 8 were considered as producers, and among the S. epidermidis 8 were
considered producers. Among the patients with S. aureus biofilm producers, five (5/8)
were using catheter in the moment of the culture, while in the 8 patients with
S. epidermidis and catheter we observed 6 positive cases.
Concerning to length of the incubation, we detect change in the category only in
3 cases, from nonproducer to producer. It is important to outstand, however, that the
color became more characteristic in other cases. We also observed that more clear
results were obtained when small amount of bacteria was used.
Antimicrobial resistance pattern - As shown in table II, the frequency of
antimicrobial resistance tested in this study depends on the antimicrobial agent.
Gentamicin resistance was greater than to other antimicrobials (78.6%). All isolates
were susceptible to vancomycin and linezolid. For oxacillin, the rate of resistance was
83.3% for S. epidermidis isolates and 68.8% for S. aureus.
Biofilm formation and antimicrobial resistance - Generally it was observed a
greater resistance among biofilm nonproducers. Analyzing species separately, we
observed a greater resistance among biofilm-producing S. epidermidis; among S.
79
aureus, however, greater resistance was detected among nonproducers of this virulence
factor (Figure 1).
Discussion and Conclusions
Arciola et al. (2002) described a six-color scale to classify CRA colonies, using
the categories "very black" and "very red". In our study, however, only four colors were
considered because the differences were subtle and could lead to an inadequate
interpretation.
Concerning the technique, 48-h incubation was not found required, since in
some isolates there was alteration of the color. However, in only 3 cases this change
implicates in an alteration of category, while in the others there were only an
intensification of pigmentation. Another reading after additional 24h of incubation did
not affect the results of 20 samples, mostly being nonproducers. These situations are
comparable to from those reported as well by Arciola et al. (2001, 2002), where almost
all samples were classified after 24 hours, even that they had incubated until 72 h.
Most isolates (82%) were obtained from patients using catheters, and almost half
of them were biofilm producers. Several S. aureus isolates from patients using catheters
were not biofilm producers, in agreement with the reports that S. epidermidis is the most
frequently isolated pathogen in patients with infections associated with medical devices
due to biofilm production (Vuong & Otto 2002, Cafiso et al. 2004). On the other hand,
these data might change if the incubation time is extended (Arciola et al. 2001).
The oxacillin resistance rate (75%) is in agreement with the literature, since
about 80% of nosocomial infections are resistant to oxacillin, a first choice
antimicrobial against microorganisms of Staphylococcus (Vuong & Otto 2002). At the
same time, the susceptibility to vancomycin and linezolid agrees with studies reporting
that these antimicrobials as effective against almost all Staphylococcus spp, despite their
production of biofilm (Monzon et al. 2001, Curtin et al. 2003, Caierao et al. 2004,
Appelbaum & Jacobs 2005) .
In S. epidermidis, resistance to the tested antimicrobials was greater in biofilm
producers, as expected. Silva et al. (2002) found no association between phenotypic
expression of biofilm based on CRA and any of the antimicrobial resistances examined,
but they reported the existence of a significant association between a series of
antimicrobial resistances (including oxacillin, rifampicin and ciprofloxacin) and the
presence of ica genes. We found 50% of resistance to oxacillin among biofilm
producers and 33% among nonproducers in agreement to Arciola et al. (2005) that
80
found rates of oxacillin resistance in S. epidermidis of 44% among biofilm producers
and 34% among nonproducers. The same authors observed a percentage of gentamicin
resistance of 39% among biofilm producers and 27% among nonproducers, similarly to
our (figure I). Curtin et al (2003), also, reported low effectiveness of gentamicin against
biofilm in S. epidermidis isolated from catheters. Studies on rifampicin resistance
among biofilm-producing S. epidermidis have suggested that the slow growth of the
bacteria on the biofilm accounts for its protection against the antimicrobial (Zheng &
Stewart 2002). Despite the well-known tendency of rifampicin to cause emergence of
resistance, its efficacy against bacteria attached to biomaterials has been clearly
demonstrated. Nevertheless, there are many studies showing greater efficacy if it is used
together with other antimicrobial agents (Monzon et al. 2001, 2002). Authors have
suggested the existence of association could be indirect and mediated by the presence of
transposons. It is known that antimicrobial resistance genes are often associated to
transposons. Some transposons are flanked by insertion sequences such as IS256 that
are often associated with biofilm formation, the presence of the icaADBC operon, as
well as gentamicin and oxacillin resistance in the clinical strains (Arciola et al. 2005).
Surprisingly, biofilm-producing S. aureus isolates were more susceptible to the
tested antimicrobials than nonproducers, contradicting idea that microorganisms that
form biofilm would be more resistant to these drugs. Amorena et al. (1999) reported
that in S. aureus gentamicin does not affect significantly the viability of biofilm cells. In
our work, gentamicin resistance among biofilm-producing S. aureus was relatively low
(25%). In that study, like ours, rifampicin showed a good response against biofilm-
producing S. aureus isolates a result (Figure I).
There are some ideas about the mechanisms responsible for the increase of
antimicrobial resistance among biofilm producers. These include low penetration of the
antimicrobial; slow bacterial growth rate within mature biofilm because of limited
nutrients; slow growth as a general stress response initiated with the formation of
biofilm; and finally, induction of a phenotype characterized by an efflux pump and
alteration of protein membrane makeup. Specific antimicrobial characteristics, such as
hydrophobic properties, distribution, size, and water solubility could also affect their
diffusion across the biofilm matrix (Donlan & Costerton 2002, Monzon et al. 2002,
Arciola et al. 2005) .
The importance of S. epidermidis infections is well-established and is probably
related to increased virulence due to biofilm production. Simple methods of detection of
81
biofilm are thus necessary. The use of CRA proved to be practical and easily to
implement at Clinical Microbiology Laboratories. However, studies using a larger
number of isolates must be carried out to demonstrate the best time of incubation of the
plates and the effectiveness use of this medium in laboratory routine.
Acknowledgments
To Dr. Kátia Regina Netto dos Santos of the Institute of Microbiology Professor
Paulo Góes of the Universidade Federal do Rio de Janeiro, Brazil, for their
collaboration.
82
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Yago J 1999. Antibiotic susceptibility assay for Staphylococcus aureus in biofilms
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Appelbaum PC, Jacobs MR 2005. Recently approved and investigational antibiotics for
treatment of severe infections caused by Gram-positive bacteria. Curr Opin
Microbiol 8: 510-517.
Arciola CR, Baldassarri L, Montanaro L 2001. Presence of icaA and icaD genes and
slime production in a collection of staphylococcal strains from catheter-associated
infections. J Clin Microbiol 39: 2151-2156.
Arciola CR, Campoccia D, Gamberini S, Cervellati M, Donati E, Montanaro L 2002.
Detection of slime production by means of an optimised Congo red agar plate test
based on a colourimetric scale in Staphylococcus epidermidis clinical isolates
genotyped for ica locus. Biomaterials 23: 4233-4239.
Arciola CR, Campoccia D, Gamberini S, Donati ME, Pirini V, Visai L, Speziale P,
Montanaro L 2005. Antibiotic resistance in exopolysaccharide-forming
Staphylococcus epidermidis clinical isolates from orthopaedic implant infections.
Biomaterials 26: 6530-6535.
Cafiso V, Bertuccio T, Santagati M, Campanile F, Amicosante G, Perilli MG, Selan L,
Artini M, Nicoletti G, Stefani S 2004. Presence of the ica operon in clinical
isolates of Staphylococcus epidermidis and its role in biofilm production. Clin
Microbiol Infect 10: 1081-1088.
Caierão J, Musskopf M, Superti S, Roesch E, Dias CG, d’Azevedo PA 2004. Evaluation
of phenotypic methods for methicillin resistance characterization in coagulase-
negative Staphylococci (CNS). J Medical Microbiology 53: 1195-1199.
Clinical and Laboratory Standards Institute (CLSI) 2005. Performance Standards for
Antimicrobial Susceptibility Testing – Sixteenth Information Supplement M-
S16.S 8ª ed. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania,
USA, CLSI 2005.
Curtin J, Cormican M, Fleming G, Keelehan J, Colleran E 2003. Linezolid compared
with esperezolid, vancomycin, and gentamicin in an in vitro model of
antimicrobial lock therapy for Staphylococcus epidermidis central venous
catheter-related biofilms infections. Antimicrob Agents Chemother 47 (10): 3145-
3148.
Donlan RM, Costerton JW 2002. Biofilms: Survivel Mechanisms of Clinically Relevant
Microorganisms. Clin Microbiol Rev 15 (2): 167-193.
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Thissen H, Read R, Poole-Warren LA, Schindhelm K, Willcox MDP 2004. The
control of Staphylococcus epidermidis biofilm formation and in vivo infection
rates by covalently bound furanones. Biomaterials 25: 5023-5030.
Kloos WE, Bannerman TL 1999. Staphylococcus and Micrococcus, In PR Murray, EJ
Baron, MA Pfaller, FC Tenover, RH Yolken (eds),Manual of Clinical
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84
Table I. Characterization of Staphylococcus aureus and Staphylococcus epidermidis isolates
through Congo Red Agar.
CRA Use of
Isolate
Biofilm
24h 48h Catheter
S. epidermidis
P BR AB Yes
S. epidermidis
P AB B Yes
S. epidermidis
P B B Yes
S. epidermidis
P B B Yes
S. epidermidis
P B B Yes
S. epidermidis
P B B Yes
S. epidermidis
P B B No
S. epidermidis
P B B No
S. epidermidis
N BR BR Yes
S. epidermidis
N R R No
S. epidermidis
N R R Yes
S. epidermidis
N R BR Yes
S. aureus
P B B No
S. aureus
P AB AB Yes
S. aureus
P R AB No
S. aureus
P R AB Yes
S. aureus
P B B Yes
S. aureus
P AB B Yes
S. aureus
P AB B Yes
S. aureus
P R B Yes
S. aureus
N R R Yes
S. aureus
N R R Yes
S. aureus
N BR BR Yes
S. aureus
N R R Yes
S. aureus
N BR BR Yes
S. aureus
N BR BR Yes
S. aureus
N R R Yes
S. aureus
N R R Yes
R: red; B: black; BR: bordeaux; AB: almost black; P: biofilm producer; N: biofilm nonproducer
85
Table II. Percentage of antimicrobial resistance among Staphylococcus epidermidis and
Staphylococcus aureus isolates
Antimicrobial
Percentage (%)
S. aureus
Oxacillin 68.8
Rifampicin 6.2
Gentamicin 75.0
S. epidermidis
Oxacillin 83.3
Rifampicin 33.3
Gentamicin 83.3
Table III. Percentage of antimicrobial resistance among CRA-positive and CRA- negative
among S. epidermidis and S. aureus isolates
CRA-positive CRA-negative
Antimicrobial
No. % No. %
Oxacillin
10
66.7
11
84.6
Rifampicin 2 13.3 3 23.1
Gentamicin
11
73.3
12
92.3
CRA-positive: biofilm producer; CRA-negative: biofilm nonproducer; No.: number of samples
86
Figure 1. Antimicrobial resistance rates among biofilm-producing (CRA-positive) and
non-producing (CRA- negative) in Staphylococcus epidermidis and Staphylococcus
aureus isolates.
87
ANEXO 1______________________________________________________________
88
Bacteremias of Coagulase-Negative staphylococci (CoNS) in Intensive Care Unit in
hospital geral of São Paulo city, SP, Brasil.
Bacteremias por Staphylococcus spp coagulase negativos em um hospital geral na
cidade de São Paulo, SP, Brasil.
d’Azevedo, PA.
1,2
Secchi, C
2
Antunes, ALS
2
Sales, TC
3
Silva, FM
1
Tranchesi, R
3
Pignatari, ACC
1,3
1
Laboratório Especial de Microbiologia Clínica (LEMC), Disciplina de Infectologia da
Universidade Federal de São Paulo (UNIFESP), SP, Brasil.
2
Laboratório de Cocos Gram Positivos da Fundação Faculdade Federal de Ciências
Médicas de Porto Alegre (FFFCMPA), RS, Brasil.
3
Hospital 9 de Julho, São Paulo, SP, Brasil.
4
Laboratório Bioclínico, São Paulo, Brasil.
This study was presented in part at the 40
th
Congresso Brasileiro de Patologia Clínica e
Medicina Laboratorial, Curitiba, PR, 2006 (abstract).
Corresponding author:
Profº Pedro A. d’Azevedo
Laboratório Especial de Microbiologia Clínica
Rua Leandro Dupret 188 – Vila Clementino – São Paulo, SP.
Cep: 04025-010
Tel: 11 50812819
e-mail: [email protected].br
89
Abstract
Coagulase negative staphylococci (CoNS), especially S. epidermidis have become an
important cause of bloodstream infections, during the last decades. In addition, rates of
methicillin-resistance among CoNS have increased substantially, leading to difficulties
in therapy. The objective of this study was evaluate 11 consecutives cases of
bacteremias by CoNS methicillin-resistance in hospital localized in São Paulo city,
Brazil and characterized phenotypic/genotypic this isolates. Five differents species
were identified inclued S. epidermidis (5), S. haemolyticus (3), S. hominis (1),
S. warneri (1) and S. cohnii subsp urealyticus (1). Macrorestriction of DNA was
realized of PFGE and analysis of profiles second Tenover and collaborators. A variety
of PFGE profiles was observed. None have the predominant PFGE type. These data
indicated the heterogeneity of CoNS isolates included in the study, suggesting that
horizontal dissemination of these microorganisms was not frequent in the hospital
investigated.
Key words: Coagulase negative staphylococci; methicillin-resistant; bacteremia;
identification; PFGE (“Pulsed-Field Gel Electrophoresis”)
90
Introduction
Coagulase-negative staphylococci (CoNS) are major causes of nosocomial
bloodstream infection and have significantly high associates morbidity and mortality
mainly in patients hospitalized (Marshall et al. 1998). Members of the genera
Staphylococcus are catalase-positive, gram-positive cocci, coagulase-negative, aerobe
and when present in human infections can show multiresistant profiles (Kloss &
Bannermann 1999, Szewczyk & Rozalska 2000). These strains may constitute a
dangerous reservoir of resistance genes in a hospital (Szewczyk & Rozalska 2000).
Staphylococci generally has a benign or symbiotic relationship with their host,
however, they may develop into a pathogen if they gain entry into the host tissue
through breaking of cutaneous barrier, inoculation by needles or implantation of
medical devices (Heikens et al. 2005). CoNS has become increasingly important to
accurately identify these isolates to the species level in order to define the clinical
significance of these bacteria, to carry out a proper epidemiological surveillance, and to
manage patients infected with CoNS in case of relapse (Poyart et al. 2001).
A substantial increase in the frequency of oxacilin resistance (methicillin-
resistant) in CoNS isolates has occurred over the last decades (Ferreira et al. 2003).
Between 50% and 80%, depending on the species, are mecA positive or oxacilin
resistance (John et al. 2002, Caierão et al. 2006).
The SENTRY Program, in Latin American and in Brazil, realized antimicrobial
surveillance program over a five-year period from 1997 to 2001. The results
demonstrated the resistance of Staphylococcus spp, in isolates of bloodstreams
infections, in Brazil, showed oxacilin susceptibility in S. aureus of 68.2% and 19.2% in
CoNS (Sader et al. 2004).
S. epidermidis and S. haemolyticus are the most frequent species in nosocomial
infection presenting a higher frequency of oxacilin resistance among CoNS clinical
isolates (Ferreira et al. 2002). S. haemolyticus have been reported to show multiple
resistance to antimicrobials and quite frequently with reduced susceptibility or fully
resistant to teicoplanin from clinical isolates (Raponi et al. 2005).
Vancomycin is usually considered the treatment of choice for infections caused
by these microorganisms. However, due to the emergence of vancomycin-resistant
enterococci (Marshall et al, 1998) and vancomycin-resistant staphylococci
91
(Szarapinska-Kwaszewska & Farkas 2003), reduction in the use of this drug has been
recommended (Szewczyk & Rozalska 2000).
A few reports have show that the mechanism of glycopeptide resistance in
S. epidermidis, S. haemolyticus, S. hominis is similar to that describe in VISA and
hetero-VISA strains (Nunes et al. 2006). In 09 de Julho Hospital, where study was
occurred therapy of teicoplanin is recommended. The objective of this study was
evaluate 11 consecutives cases of bacteremias by CoNS methicillin-resistant in 09 de
Julho Hospital localized in São Paulo city, Brazil, occurred in 2005, and characterized
phenotypic/genotypic this isolates.
Materials and Methods
Bacterial isolates: We tested 11 CoNS clinical isolates obtained from
bloodstream, from patients at in hospital localized in São Paulo City, Brazil between
June and July 2005.
Identification: Identification of the staphylococcal to the species level was
carried out by oxidation-fermentation test, detection of enzyme production: coagulase
(Laborclin, Paraná, Brazil), catalase, alkaline phospatase (diphosphato de fenoftaleína,
Sigma Sigma-Aldrich, Germany), ornithine (Merck) and urease (Oxoid, UK), PYR
(pyrrolidinyl-β-naphthylamide hydrolysis, Probac do Brasil, São Paulo), hemolytics
properties on sheep blood agar, acid production (aerobically) from carbohydrates:
trehalose (Sigma-Aldrich, Germany), mannitol (Nuclear, São Paulo), mannose (Vetec,
Rio de Janeiro), sucrose (Reagen), maltose (Sigma-Aldrich, Germany), lactose (Difco
Detroit, Mich), cellobiose (Sigma-Aldrich, Germany) and anaerobic growth in
thioglicolate (Merck). Susceptibilities to novobiocin (Oxoid, UK), polymyxin B (Oxoid,
UK), bacitracina (CECON, São Paulo, Brazil), desferrioxamine (Desferal, Ciba Geigy,
Switzerland) and fosfomycin (Oxoid,UK) were also determined. Isolates were kept
frozen at –20°C in Skim Milk. (Difco, Detroit, Mich). Bacteria to be tested were
suspended in 0.5 ml of saline to a McFarland standard and 50ul was added to each sugar
carbohydrate tube. The acid production from carbohydrates was evaluated after 24, 48
and 72hs of incubation at 35-37°C. The final evaluation was at 7
th
day.
Antimicrobial susceptibility: The isolates were tested by the agar disk
diffusion method with Mueller-Hinton agar plates (Difco, Laboratories, Detroit, Mich)
according to Clinical Laboratory and Standards Institute (CLSI 2005; formerly NCCLS)
recommendations and confirmed by E-test (AB Biodisk, Solna, Sweden). Also
92
determined susceptibility for novobiocin, polymyxin B, bacitracina, desferrioxamine
and fosfomycin to the according Monsen et al. (1998).
PFGE typing: Chromosomal DNA from CoNS was prepared in agarose blocks
and cleaved with SmaI (New England BioLabs), as described elsewhere (Pfaller et al.
1992). The isolates were run on a 1% agarose gel (Invitrogen) in a CHEF DRIII system
(Bio-Rad) under the following conditions: run time, 23 h; temperature, 13°C; voltage,
200 V; initial forward time, 5’; final forward time, 60 s. The molecular weight markers
(New England BioLabs) were run in the first and in the last line. The gels were stained
with ethidium bromide, washed in water, and photographed under UV light by using the
Gel Doc 1000 system (Bio-Rad). The gel patterns were read by visual inspection. The
isolates were classified as identical if they shared the same
band profile, and isolates
differing by more than six bands were considered to represent distinct DNA types
(Tenover et al. 1995).
Results and discussion
A total of 11 CoNS strains belongings to five species were identified including
S. epidermidis (5), S. haemolyticus (3), S. hominis (1), S. warneri (1) and S. cohnii subsp
urealyticus (1) (Table 1). All isolates, except the number 20994, were identified to the
species level by the conventional method of Kloos and Banermann (Kloss &
Banermann 1994, 1999). The isolate number 20994 could not be identified by the
conventional method, so it was identified for Vitek-2 (bioMèrieux S/A, Paris, France) at
Hospital Albert Einstein, São Paulo, Brazil) like S. cohnii subsp urealyticus.
The two species most frequently encountered were S. epidermidis and
S. haemolyticus, all the tests of the phenotypic tests were accomplished at least twice for
confirmation of the species of CoNS. All the phenotypic tests were accomplished in
parallel with a control positive S. epidermidis ATCC 12228.
The species of S. epidermidis and S. hominis were identified through the proof of
diffusion disk for desferrioxamine, this susceptibility testing just the two species. All
the other species of CoNS presented resistance the desferrioxamine through the
diffusion disk, turning like this, this proof of easy execution, a guide in the
differentiation of species. The reading of the halos of diffusion disk for desferrioxamine
was 26 mm on average (25-30mm) and of fosfomycin it was of 43 mm (40-45mm), for
S. epidermidis.
To differentiate the two species with susceptibility of desferrioxamine,
S. epidermidis and S. hominis, other phenotypic tests were used, as fermentation of the
93
trehalose (negative for S. epidermidis), alkaline phospatase (positive for S. epidermidis)
and growth in thioglicolate (positive for S. epidermidis).
The production of the enzyme urease test allowed the differentiation of
S. haemolyticus (negative urease), of S. epidermidis, S. hominis and S. warneri that are
positive urease. Besides this proof the test of positive PYR together with the hemolytic
properties in sheep blood agar and fermentation of negative mannose also allowed the
differentiation of the second species more prevalent S. haemolyticus.
The clinical isolate 20994, S. cohnii subsp urealyticus, for being a species less
common of being isolated at the clinical laboratory, it was identified for the automated
system VITEK 2 (bioMèrieux S/A, Paris, France), and after for methods conventional,
confirming his identification. Classic proofs of resistance to the disk diffusion of
novobiocin, positive urease and fermentation negative of sucrose confirmed the
identification of the specie. There was discrepancy in the proof of the production of the
enzyme alkaline phospatase that negative stayed in the conventional test and positive in
the automated system.
All isolates were methicillin-resistant by disk diffusion test, with MICs ≥ 256
µg/ml confirmed by E-Test. In spite of S. epidermidis to be the species more prevalent
among Staphylococcus spp, other species see increasing her prevalence in the isolated
ones clinical, as well as the resistance to the used antimicrobials. In this study, two
species (S. epidermidis and S. haemolyticus) presented reduced susceptibility to
teicoplanin (Table 1), the according with has also been reported in this species (Nunes
et al. 2006). Strains with this characteristic may be associated with treatment failures or
may become precursors of glycopeptide-resistant strains (Sieradzki et al. 1998).
S. cohnii subsp urealyticus, is an unusual species that has been found in
hospital environment like pediatric ICUs, and this isolates may constitute a dangerous
reservoirs of resistance genes in a hospital (plasmids), presenting resistance to multiple
antimicrobials commonly used (Waldon et al. 2002, Szewczyk et al. 2004), as well as
strains opportunist in severe infections (Yamashita et al. 2005).
Among the five clinical isolates of S. epidermidis were found five different
patterns of PFGE, but the same not occurred between the three clinical isolates of
S. haemolyticus where two isolates presented similar profile. These results
demonstrated a clonal diversity between the clinical isolates of S. epidermidis,
evidencing that there was not clonal dissemination among the clinical isolates of this
94
same species (Figure 1). The other isolates were not classified by PFGE because they
belong to different species.
In the last years, CoNS has been winning a larger importance due to her
pathogenicity and involvement in human diseases. The importance of identifying all
species of CoNS in the clinical laboratories has been increasing; however it is not an
easy task, since phenotypic tests can present similar results, hindering the obtaining of a
result, as well as a great expense of time in the identification of the species than in
commercial kits. Many clinical laboratories use automated systems for identification of
the species of Staphylococcus spp although the reliability for certain species not always
it is satisfactory, particularly for species of CoNS no-epidermidis.
Acknowledgement
The authors tank to Dra. Marines Martino of Laboratory Microbiology of Albert
Eistein to support technique and Fundação Faculdade Federal de Ciências Médicas de
Porto Alegre (FFFCMPA) of financial support.
95
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97
Table1. Coagulase-negative staphyloccci methicillin resistant isolates of patients with
bloodstream infections hospitalized in 09 de Julho Hospital (June/July 2005),
São Paulo, SP, Brazil.
Lane Specie
Clinical
isolate
Isolation
date
Unid
MIC Vanco
(µg/ml)
MIC Teico
(µg/ml)
Profile
1
S. epidermidis
21170 17/07/05 Leito 145 2.0 16.0 “A”
2
S. epidermidis
21169 11/07/05 Leito 1005 1.5 4.0 “B”
3
S. epidermidis
21168 20/07/05 Leito 307 1.5 4.0 “C”
4
S. epidermidis
20944 29/06/05 CTC 2.0 4.0 “D”
5
S. epidermidis
21171 01/07/05 onco 2.0 4.0 “E”
6
S. haemolyticus
20995 07/07/05 Leito 532 2.0 12.0 “G”
7
S. haemolyticus
21172 13/07/05 onco 1.5 4.0 “F”
8
S. hominis
20947 20/06/05 CTI 0.75 0.75 NC
9
S. warneri
20993 08/07/05 Leito 543 1.0 2.0 NC
10
S. haemolyticus
20946 24/06/05 onco 2.0 3.0 “F”
11
S. cohnii spp urealyticus
20994 06/07/05 Leito 394 1.0 3.0 NC
98
Figure 1 – PFGE profile of SmaI-digested chromosomal DNA of CoNS isolates, obtained from
patients in 9 de Julho Hospital in São Paulo city, Brazil. λ Lamba ladder DNA markers; lanes 1-
5 S. epidermidis; lanes 6,7 and 10: S. haemolyticus; lane 8: S. hominis; lane 9: S. warneri; lane
11: S. cohnii subsp urealyticus.
99
ANEXO 2___________________________________________________________________
100
IS CEFOXITIN A BETTER PREDICTOR FOR THE mecA GENE THAN OXACILLIN?
Running title: Predictors of the mecA gene in staphylococci
Category: Original article
Leandro Reus Rodrigues Perez
1,2,
*, Ana Lúcia Souza Antunes
1,2,3
, Afonso Luís Barth
3,4
e Pedro Alves d’Azevedo
1,2
1
Pós-graduação em Ciências Médicas da Fundação Faculdade Federal de Ciências
Médicas de Porto Alegre, RS, Brasil.
2
Laboratório de Cocos Gram-positivos da Fundação Faculdade Federal de Ciências
Médicas de Porto Alegre, RS, Brasil.
3
Departamento de Análises Clínicas da Faculdade de Farmácia da Universidade Federal
do Rio Grande do Sul, Porto Alegre, RS, Brasil.
4
Unidade de Microbiologia e Biologia Molecular do Serviço de Patologia do Hospital
de Clínicas de Porto Alegre, RS, Brasil.
*Corresponding author and reprint requests to Leandro Reus Rodrigues Perez.
Fundação Faculdade Federal de Ciências Médicas de Porto Alegre.
Rua Sarmento Leite, 245/211, Porto Alegre, RS, Brasil, CEP 90050-170
Phone: +55-051-32248822 –217
E-mail: [email protected]
101
ABSTRACT
Objective: To assess the use of cefoxitin as compared to oxacillin through phenotypic
methods for detection of methicillin resistance in Staphylococcus aureus and coagulase-
negative staphylococci (CNS) isolates.
Methods: A total of 343 consecutive Staphylococcus isolates were tested through: disk
diffusion (cefoxitin 30µg and oxacillin 1µg); agar dilution with cefoxitin and oxacillin
(using concentrations of from 0.25 to 256 µg/ml); screening with oxacillin (6 µg/ml and
NaCl 4% for S. aureus; 4 µg/ml and NaCl 4% for CNS) and cefoxitin (4 µg/ml with and
without NaCl 2% for S. aureus and CNS); and polymerase chain reaction (PCR) for the
mecA gene as the gold standard.
Results: The mecA gene was detected in 41.3% of S. aureus and in 78.4% of CNS. Of
the phenotypic methods, oxacillin disk presented better correlation with the mecA gene
for S. aureus, while cefoxitin disk showed better correlation for CNS. The screening
method with cefoxitin 4 µg/mL
presented 100% sensitivity for all isolates. Of the 343
isolates, 3 S. aureus and 5 CNS isolates showed differing results between some
phenotypic method and the gold standard.
Conclusion: Although disk diffusion with cefoxitin was recently recommended by the
CLSI as a better predictor of mecA gene-related resistance in Staphylococcus, disk
diffusion with oxacillin showed great correlation with the mecA gene, particularly in
S. aureus. Also, the screening method with cefoxitin 4 µg/ml showed performance equal
or superior to the other screening methods applied and can be used to characterize mecA
gene-related resistance among staphylococci species.
Keywords: Staphylococcus aureus, coagulase-negative Staphylococcus, mecA gene,
cefoxitin, oxacillin
102
INTRODUCTION
Staphylococcus spp are one of the main agents of human infections in the world
and are responsible for high morbidity and mortality rates. Staphylococcus aureus has
predominated as one of the main agents of hospital infections as well as community-
acquired ones [1,2] while coagulase-negative Staphylococci (CNS) are among the most
common etiologic agents of hospital bacteremias, especially those relating to prosthetic
implants and intravascular catheters [3].
Methicillin resistance in these organisms arose shortly after the therapeutic
practice with semi-synthetic β-lactam agents and, since then, treatment of
staphylococcal infections has become increasingly more restricted and problematic [4].
Monitoring methicillin resistance in Staphylococcus is crucially important because some
microrganisms, or even genetic components of resistance, are eminently transmissible,
leading to dissemination of resistance and increase in the number of infections [1, 2, 4].
In a study carried out by antimicrobial surveillance program SENTRY in Latin
America, 35.0% of the S. aureus isolates and 80.4% of the CNS isolates, involved in
bacteremias, presented methicillin resistance. In blood stream infections in Brazil,
methicillin resistance was detected in 35.2% of S. aureus isolates and in 84.6% of CNS
isolates [5]. Methicillin resistance is result of the production of an additional penicillin
binding protein called PBP 2a or PBP 2’. This PBP 2a, encoded by mecA gene – a DNA
component designated as mec region – confers intrinsic resistance to virtually all β-
lactam agents and their derivatives because of its low binding affinity [1,2,4].
The variation in the expression of mecA gene/PBP 2a accounts due existence of
homogeneous and heterogeneous populations of methicillin-resistant staphylococci
(MRS), and this variability in the apparent decrease of resistance results in difficulty in
detecting methicillin resistance by phenotypic susceptibility methods used in laboratory
routine [6].
In order to obtain more accurate results in the detection of MRS, the new
standard of the Clinical and Laboratory Standards Institute/National Committee for
Clinical Laboratory Standards (CLSI/NCCLS)[7]
advises the use of cefoxitin, through
the disk diffusion method, as a better predictor of mecA gene-mediated resistance in
Staphylococcus spp. Accordingly, the aim of this study was to assess the accuracy of
103
cefoxitin as compared to oxacillin through phenotypic methods of disk diffusion, agar
dilution and agar screening tests for detection of mecA gene-related resistance in
S. aureus and CNS isolates.
MATERIAL AND METHODS
Bacterial Samples
A total of 343 consecutive Staphylococcus isolates were analyzed. They were
collected between Aug and Dec 2004 from blood of patients committed to three hospital
complexes (Hospital de Clínicas of Porto Alegre, Hospital Nossa Senhora da
Conceição, and Complexo Hospitalar Santa Casa of Porto Alegre) located in Porto
Alegre city, South Brazil. The identification of S. aureus and CNS was performed by
the conventional methods as per standard protocol [8]. All samples were frozen and
stored at -20°C in skim milk with 10% glycerol.
Preparation of the Bacterial Inoculum for the Susceptibility Tests
The isolates were cultivated on tryptic soy agar (Difco Laboratories, Detroit,
USA) with 5% defibrinated sheep blood at 35 °C for 24 h. A suspension of the sterile
saline colonies was prepared up to reaching turbidity of 0.5 McFarland's scale (~10
8
CFU/mL).
Disk Diffusion Test
Antimicrobial susceptibility was evaluated by disk diffusion test on Mueller-
Hinton agar (Difco Laboratories, Detroit, USA) according to CLSI/NCCLS 2005
guidelines. Disks of cefoxitin 30 µg (Oxoid, Hampshire, UK) and oxacillin 1 µg (Oxoid,
Hampshire, UK) were tested, and the plates inoculated with the samples were incubated
at 35 ºC for 24 h.
Determination of Minimum Inhibitory Concentrations by Agar Dilution Test
Aliquots of bacterial suspension of each sample, diluted at 1:10 in saline
solution, were inoculated using a Steers replicator, on surface of plates containing
Mueller-Hinton agar (Difco Laboratories, Detroit, USA). Minimum Inhibitory
104
Concentrations (MIC) was determined using range concentrations from 0.25 to
256 µg/ml for cefoxitin and oxacillin. The results were read after 24 h of incubation at
35 º C.
Screening Method
Using a Steers replicator, the bacterial isolates were inoculated on Mueller-
Hinton agar plates (Difco Laboratories, Detroit, USA), supplemented in the following
conditions:
● 6
µg/ml oxacilin and 4% NaCl, for S. aureus [7];
● 4
µg/ml oxacilin and 4% NaCl, for CNS [9] and
● 4 µg/ml cefoxitin (with or without addition of 2% NaCl), for S. aureus and
CNS isolates (proposed method).
The reading of the results was carried after 24 h of incubation at 35 °C. The
growth of more than one colony was taken as a positive result. Plates containing
Mueller-Hinton agar without antimicrobial were used as controls of bacterial growth.
Detection of PBP 2a
Detection of PBP 2a was performed by latex agglutination test Slidex MRSA
Detection (bioMérieux, l’Etoile, France) following the manufacturers' instructions. This
test was performed only for samples that showing discrepant results between cefoxitin
and oxacillin in disk diffusion test.
Detection of the mecA Gene by Polymerase Chain Reaction (PCR)
A polymerase chain reaction (PCR) procedure was used to verify the presence
of the mecA gene. Primers (mecA
1
: 5’-TGG CTA TCG TGT CAC AAT CG and
mecA
2
: 5’-CTG GAA CTT GTT GAG CAG AG) [10] amplified segment of 310-bp of
the gene that was visualized under ultraviolet light by the addition of ethidium bromide
(0.5 µg/ml) after electrophoresis in agarose gel at 1.5%.
105
Quality Control
For quality control of susceptibility tests and PCR we used the American Type
Culture Collection (ATCC): ATCC 25923 (methicillin susceptible S. aureus) and
ATCC 33591 (methicillin resistance S. aureus).
RESULTS
A total of 343 Staphylococcus isolates were analyzed, 167 S. aureus and 176
CNS. Among the latter, the most frequent was S. epidermidis (120 isolates), followed
by S. haemolyticus (22 isolates), S. capitis subsp. urealyticum (12 isolates), S. hominis
subsp. hominis (6 isolates), S. lugdunensis (5 isolates), S. sciuri (3 isolates), S. simulans
and S. warneri (2 isolates each), S. saprophyticus, S. auricularis, S. capitis subsp.
capitis and S. cohnni subsp. urealyticus (1 isolate each). The presence of the mecA gene
was detected in 41.3% of S. aureus and 79% of CNS isolates. Table 1 shows the
distribution of the mecA gene among staphylococci species.
The MICs for cefoxitin and oxacillin were determined for all isolates and the
results are presented in Table 2. All 98 S. aureus isolates which were mecA-negative
(mecA
-
) were inhibited by cefoxitin 8 µg/ml and by oxacillin 4 µg/ml. For these isolates,
the MIC
90
for cefoxitin and oxacillin was 2 µg/ml. For 38 mecA
-
CNS isolates, the
MIC
90
was 4 µg/ml for cefoxitin and 2 µg/ml for oxacillin. Also, one mecA
-
CNS isolate
presented MIC cefoxitin of 16
µg/ml. All 69 mecA-positive (mecA
+
) S. aureus presented
MIC 256
µg/ml for cefoxitin and oxacillin, and the MIC
90
for these isolates was also
256
µg/ml for both antimicrobials. Among mecA
+
CNS isolates, resistance to elevated
concentrations of cefoxitin and oxacillin was observed. Seven mecA
+
CNS isolates
presented MIC > 256
µg/ml for cefoxitin, while 20 isolates showed MIC >256 µg/ml for
oxacillin. The MIC
90
for these isolates was > 256 µg/ml for both antimicrobials.
The results of phenotypic methods (disk diffusion and screening agar) applied
for detection of methicillin resistance among staphylococci species are shown in
Table 3. For S. aureus, disk diffusion test using cefoxitin disk 30 µg presented 98.5%
sensitivity and 100% specificity, i.e. one mecA
+
S. aureus isolate was susceptible to
cefoxitin. Among CNS isolates, sensitivity and specificity were 100%. With use of
oxacillin disk 1µg, sensitivity and specificity for CNS were 100% and 92.1%,
106
respectively. For S. aureus isolates, however, oxacillin disk 1µg was 100% susceptible
and specific. Among screening tests applied, screening agar with cefoxitin 4 µg/ml
presented 100% sensitivity for all species and presented 99.0 and 97.4% of specificity
for S. aureus and CNS, respectively.
The isolates presenting discrepant results between cefoxitin and oxacillin disks
were submitted to the latex agglutination test for detection of PBP 2a. Four isolates
presented such difference, 3 CNS (S. saprophyticus, S. epidermidis and S. warneri) and
one S. aureus. The latex agglutination test properly characterized PBP 2a in
S. aureus isolate but showed a false-positive result for 3 CNS isolates.
Table 4 shows all isolates presenting some discrepance between phenotypic
methods and presence/absence of the mecA gene.
DISCUSSION
The transcription of the mecA gene is regulated by two chromosomal regions of
different, though homologue, regulatory genes, mecR1-mec1 and blaR1-bla1. MecR1
and blaR1 are promoters while mec1 and bla1 are repressors of the transcription of the
mecA and blaZ genes, respectively [4, 11]. The difficulty in characterizing methicillin
resistance in staphylococci is due to heterogeneity of the control of repressor-promoter
expression [12]. Cefoxitin, a cephamycin, would be a stronger inducer of PBP 2a than
oxacillin by inducing more effectively the promoting region mecR1. The activation of
the mecR1 region promotes the cleavage and inactivation of repressing region mec1 of
the mecA gene, allowing its genic expression [4, 11]. Among the phenotypic methods
used for characterization of methicillin resistance in S. aureus, three are currently
recommended by CSLI: disk diffusion test using oxacillin 1µg or cefoxitin 30 µg disks,
the latter also standardized with the same breakpoints for S. lugdunensis; agar dilution
with oxacillin; and agar screening method supplemented with oxacillin 6 µg/ml and 4%
NaCl. For CNS other than S. lugdunensis, only disk diffusion (with oxacillin and
cefoxitin) and agar dilution method (oxacillin only) has been recommended.
In our study, the performance of three different methods was evaluated – disk
diffusion, agar dilution and agar screening – using two antimicrobials (cefoxitin and
107
oxacillin) in order to predict mecA gene-mediated resistance among staphylococci
species.
Although disk diffusion with oxacillin 1µg proved to be a convenient method for
all S. aureus isolates analyzed here (100% sensitivity and specificity), it provided false-
positive results for three CNS isolates: S. epidermidis, S. warneri and S. saprophyticus,
thus reducing its specificity to 92.1%. Using cefoxitin 30µg, all mecA
-
S. aureus isolates
were adequately characterized. However, one mecA
+
isolate was characterized as
susceptible. This finding disagree with several literature studies showing that disk
cefoxitin 30µg is a better predictor of the presence of the mecA gene than oxacillin disk
1µg for S. aureus [6,13-17].
The best performance of the oxacillin disk diffusion test
could be attributed to the presence of Brazilian endemic clone MRSA, which more
frequently exhibits homogeneous resistance to oxacillin. Among the CNS isolates, all
were correctly characterized with cefoxitin disk. In a recent study of the SENTRY, disk
diffusion with cefoxitin presented an excellent performance for S. aureus, not
presenting any error of correlation with mecA, and only 3% of major error (false-
resistance) among CNS isolates [18]. In our study, however, one S. aureus isolate,
which presented differing results between cefoxitin 30µg and oxacillin 1µg disks, was
mischaracterized as susceptible by cefoxitin disk (diameter of the inhibition
halo = 22 mm), after confirmation with latex agglutination for PBP 2a and PCR for
mecA. For this isolate, all agar tests performed and MIC values characterized resistance
properly, although the CLSI did not recommend the use of agar dilution for cefoxitin in
the definition of methicillin resistance. It should be noted that the region flanked by
primers mecA
1
and mecA
2
, used in the PCR reaction, was sequenced and the result
obtained no showed nowhere alteration in its nucleotide base sequence (data no shown).
Perhaps total or partial sequencing of the mec gene, particularly at positions Ser-403,
Lys-597 and Tyr-446, which are antimicrobial anchorage regions and which were not
encompassed by flanked interval with the primers used, could show a genetic mutation
[19, 20]. This would lead to a potential structural alteration of the mec complex which
could explain the binding inability of cefoxitin and its false characterization of
susceptibility for this S. aureus isolate.
108
Among CNS isolates, our findings showed that cefoxitin 30µg disk was 100%
sensitive and specific for detection of mecA-mediated resistance, while oxacillin 1µg
disk presented 1.7% (3/176) of major error.
Among screening methods evaluated, agar screening with cefoxitin 4 µg/ml
proved to be the best predictor for methicillin resistance among analyzed isolates.
Fernandes et al [13] suggested that, with a cut off of 4 µg/ml for cefoxitin, the accuracy
of the method would be correct to detect methicillin resistance in staphylococci. In our
study, however, two mecA
-
isolates, one S. aureus and one S. epidermidis, with MICs of
8 and 16 µg/mL for cefoxitin, respectively, were not properly discriminated. Screening
with cefoxitin 4 µg/mL
supplemented with 2% NaCl presented excellent sensitivity
(100%), but results of false resistance, 3% (5/167) for S. aureus and 1.13% (2/176) for
CNS showed verified. The addition of salt to the culture medium is recommended for
the determination of oxacillin MICs. For cefoxitin, however, our results showed that
there was no improvement in the performance of the method (i.e. cefoxitin MICs
without salt characterized correctly the isolates). Also, the presence of salt in agar
screening method using cefoxitin seems to have conditioned an increase in the MIC
values of these isolates, favoring a misinterpretation of false resistance.
In a study by Caierão et al [9], agar screening with oxacillin 4 µg/ml presented
100% accuracy among S. epidermidis, S. haemolyticus and S. hominis. In our study,
however, two mecA
-
isolates, S. epidermidis and S. capitis subsp. urealyticum, were
characterized as resistant (1.13% of major error). The method of agar screening with
oxacillin 6 µg/ml for S. aureus presented 0.6% (1/167) very major error (one false-
susceptible result). Although the MIC of this isolate has been reported as 8 µg/ml, it is
known that the MIC of oxacillin for this isolate is a value between 4 to 6 µg/ml range,
for it presented growth in agar dilution containing 4 µg/ml but was unable to grow, even
with a tenfold increase inoculum, in agar screening with oxacillin 6 µg/ml.
Among discrepant results (Table 4), the samples with discrepant results between
disk diffusion for cefoxitin and oxacillin were submitted to the latex agglutination test
for detection of PBP 2a. Even with previous induction with oxacillin 1µg disk, three
CNS isolates – S. saprophyticus, S. epidermidis and S. warneri – presented weak
agglutination. The absence of the mecA gene in these isolates implies that an unspecific
109
agglutination has occurred, probably with PBPs other than PBP 2a, either through
overexpression or through alteration/mutation of constituent PBPs generating high
concentrations of free transpeptidases that can perform the synthesis of the cell wall
[21].
In conclusion, oxacillin disk showed great correlation with resistance among
staphylococci species with mecA-positive genotype, particularly in S. aureus, despite
the recent recommendations of cefoxitin as a better predictor of this resistance.
Cefoxitin disk, using the new breakpoints of the CLSI, proved to be accurate in
determining resistance among CNS. In addition, agar screening method with cefoxitin
4 µg/ml showed performance equal or superior to the other screening methods applied,
without the need of supplementing salt to the culture medium and, additionally, the
same antimicrobial concentration can be used for both S. aureus and CNS.
ACKNOWLEDGMENTS
The authors are grateful to the bacteriology laboratories of each participating
hospital for their collaboration and sample provision and to Cícero Armídio Gomes
Dias for his review of the paper. This research work was supported in part by CAPES,
CNPq and FFFCMPA.
110
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14. Sharp SE, Warren JA, Jr, RBT. Cefoxitin disk diffusion screen for confirmation
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112
Table 1. Occurrence of the mecA gene among Staphylococcus spp isolates
mecA Species No. (%)
positive negative
S. aureus
167 (48.7) 69 (41.3) 98 (58.7)
S. epidermidis
120 (35.0) 100 (83.3 ) 20 (16.7)
S. haemolyticus
22 (6.4) 19 (86.3) 3 (13.6)
S. capitis subsp. urealyticum 12 (3.5) 5 (41.6) 7 (58.4)
Other CNS* 22 (6.4) 14 (4.1) 8 (2.33)
Total
343 (100) 207 (60.3) 136 (39.7)
*Species (nº/n° mecA positive): S. hominis (6/6), S. lugdunensis (5/2), S. sciuri (3/2),
S. simulans (2/1), S. warneri (2/1), S. saprophyticus (1/0), S. auricularis (1/1), S. cohnni
(1/1), S. capitis subsp. capitis (1/0).
Table 2. MICs of cefoxitin (A) and oxacillin (B) for methicillin-resistant (mecA
+
) and
methicillin-susceptible (mecA
-
) S. aureus and CNS
(A)
No. of Isolates with MICs (µg/mL) for cefoxitin
Organisms (No.)
0.25 0.5 1 2 4 8 16 32 64 128 256 >256
S. aureus mecA
+
(69)
6 7 3 3 34 16
S. aureus mecA
-
(98)
1 23 68 5 1
CNS mecA
+
(138)
24 42 14 8 19 24 7
CNS mecA
-
(38)
2 16 15 4 1
(B)
No. of Isolates with MICs (µg/mL) for oxacillin
Organisms (No.)
0.25 0.5 1 2 4 8 16 32 64 128 256 >256
S. aureus mecA
+
(69)
7 2 1 22 19 18
S. aureus mecA
-
(98)
15 12 36 34 1
CNS mecA
+
(138)
31 15 6 26 15 25 20
CNS mecA
-
(38)
28 5 1 2 2
113
Table 3. Sensitivity and specificity of variations of approved agar screen tests for
detection of methicillin resistance using the presence of the mecA gene as the gold
standard
Species*
Cefoxitin disk diffusion Oxacillin disk diffusion Agar screening oxacillin
6µg/mL + 4% NaCl
Sens** Spec** Sens Spec Sens Spec
S.
aureus
98.5 100 100 100 98.5 100
CNS 100 100 100 92.1 NA*** NA***
Total
99.5 100 100 97.8 98.5 100
Species* Agar screening oxacillin
4µg/mL + 4% NaCl
Agar screening cefoxitin
4µg/mL
Agar screening cefoxitin
4µg/mL + 2% NaCl
Sens** Spec** Sens Spec Sens Spec
S.
aureus
NA*** NA*** 100 99 100 94.9
CNS 100 94.7 100 97.4 100 94.7
Total
100 94.7 100 98.5 100 94.8
*Number of isolates and mecA status are given in Table 1
**Sensitivity (Sens), the percentage of mecA-positive strains correctly categorized;
specificity (Spec), the percentage of mecA-negative strains correctly categorized.
***Not applied (NA), tests no were applied for this species determined.
114
Table 4. Staphylococcus isolates presenting a discrepant result between some
phenotypic method, latex agglutination for PBP 2a and PCR for the mecA gene
No.
isolates
Species
PCR Latex
agglutination
Phenotypic methods discrepants
mecA PBP 2a
73
S. saprophyticus
- w Oxacillin disk diffusion
86
S. epidermidis
- w Oxacillin disk diffusion
138
S. warneri
- w Oxacillin disk diffusion
150
S. aureus
+ + Cefoxitin disk diffusion
120
S. aureus
+ NT Agar screening oxacillin 6µg/mL
143
S. aureus
- NT Agar screening cefoxitin 4µg/mL
with and without salt
176 S. capitis subsp. urealyticum - NT Agar screening oxacillin 4µg/mL
179
S. epidermidis
- NT Agar screening cefoxitin 4µg/mL
with and without salt
oxacillin screen agar 4µg/mL
-, negative; +, positive; w, weakly positive; NT, not tested.
115
ANEXO 3______________________________________________________________
116
Use of the D Test Method to Detect Inducible Clindamycin Resistance in
Coagulase Negative Staphylococci (CoNS)
Running title: Inducible Clindamycin Resistance in CoNS
Leandro Reus Rodrigues Perez
1,*
Juliana Caierão
1
Ana Lúcia Souza Antunes
1,2
Pedro Alves d’Azevedo
1
1
Fundação Faculdade Federal de Ciências Médicas de Porto Alegre, RS, Brazil.
2
Laboratório de Análises Clínicas da Faculdade de Farmácia, Universidade Federal do
Rio Grande do Sul, Porto Alegre, RS, Brazil.
Aceito para publicação no “Brazilian Journal of Infect Disease”.
*Corresponding author: Leandro Reus Rodrigues Perez, Fundação Faculdade Federal de
Ciências Médicas de Porto Alegre, Rua Sarmento Leite 245/211. Zip code: 90050-170
Porto Alegre - RS, Brazil.
Phone: 55-051-32248822
Fax: 55-051-32269756
E-mail address: [email protected]
117
Abstract
According to National Committee for Clinical Laboratory Standards (NCCLS, 2004), a
method to evaluate the inducible clindamycin resistance in accordance with an approach
of the disks of erythromycin and clindamycin – the D test – has been reported. We
analyzed the performance of this method in two hundred Coagulase Negative
Staphylococci (CoNS) strains. This obtained from blood cultures of hospitalized
patients at a hospital general in Southern Brazil. Twenty-seven clinical isolates with
suitable profile (erythromycin-resistance and clindamycin-susceptible) were evaluated
for D test realization. Thus, only five CoNS showed D test positive. The D test method
show is simple and an important technique in the detection of inducible clindamycin
resistance.
Key words: clindamycin, resistance, D test.
118
INTRODUÇÃO
The determination of antimicrobial susceptibility of a clinical isolate is often
crucial for the optimal antimicrobial therapy of infected patients. This is particularly
important considering the increasing of resistance and the emergence of multidrug-
resistant microorganisms [1-3]. Several authors have screened clinical isolates of
erythromycin-resistant S. aureus and coagulase-negative staphylococci (CoNS) for
genes encoding resistance to macrolides, lincosamides and streptogramins type B
(MLS
B
) [4-10].
Resistance to macrolides (e.g. erythromycin) can occur by two different
mechanisms: efflux due to macrolide streptogramin resistance (msrA gene) and
ribosome alteration due to erythromycin ribosome methylase (erm gene) [11, 12].
Macrolide resistance due to efflux encoded by msrA has been more prevalent in CoNS
than in S. aureus [13].
Different mechanisms of acquired MLS resistance have been found in gram-
positive bacteria [11, 12]. The first mechanism of macrolide resistance described was
due to posttranscriptional modifications of the 23S rRNA by the adenine-N-6-
methyltransferase. Target modification alters a site in 23S rRNA common to the binding
of MLS
B
antibiotics. Modification of the ribosomal target confers cross-resistance to
MLS
B
antibiotics (MLS
B
resistant phenotype) and remains the most frequent
mechanism of resistance. In general, genes encoding these methylases have been
designated erm. Expression of MLS
B
resistance in staphylococci may be constitutive
(MLS
Bc
) or inducible (MLS
Bi
). When expression is constitutive, the strains are resistant
to all MLSB type antibiotics. When expression is inducible, the strains are resistant to
14- and 15-membered macrolides only [11, 12].
For MLS
Bi
strains, erythromycin will induce production of the methylase, which
allows clindamycin resistance to be expressed. Inducible clindamycin resistance can be
detected with a simple disk approximation test, commonly referred to as the D test [14].
For this test, an erythromycin disk is placed 15 mm to 26 mm (edge to edge) from a
clindamycin disk in a standard disk diffusion test. Following incubation, a flattening of
the zone in the area between the disks where both drugs have diffused indicates that the
organism has inducible clindamycin resistance [14, 15, 16].
119
The purpose of this study was to characterize the antimicrobial susceptibility
patterns (erythromycin, clindamycin and oxacillin) and to evaluate, according to D test
[17], all coagulase negative staphylococci from a collection of 200 clinical isolates from
blood cultures that had the necessary characteristics for this study: resistance to
erithromycin and susceptibility to clindamycin. We analyzed 200 consecutives clinical
isolates of CoNS obtained from patients admitted in a general hospital in Porto Alegre
city, in Southern Brazil, between January and June 2002. All the isolates were obtained
from blood cultures.
MATERIAL AND METHODS
The samples were identified (only for species-level identification) through
MicroScan, panel Pos-Combo 13 (Dade Behring – Deerfield, Illinois, USA). For
selection criteria, the method for determining clindamycin susceptibility (disk diffusion)
was perfomed rather separated on the plate and so it is not confused with the
performance of the D tests itself.
The susceptibility tests - disks for the following agents at the concentrations
specified: 15 µg erythromycin, 2 µg clindamycin and 1µg oxacillin (Difco Laboratories,
Detroit, Mich.) - were performed by the agar disk diffusion (Kirby-Bauer) method
according to the guidelines of the National Committee for Clinical Laboratory
Standards.
D test Method - For this test, the erythromycin disks were placed 15 mm and 26
mm (edge to edge) from clindamycin disks, as recommended (NCCLS, 2004) on
Muller-Hinton agar plate (Oxoid – Hampshire, England). Moreover, the disks also were
placed to 10 mm of distance. According to evaluation criterious of NCCLS 2004, the
flattened (positive test) or not (negative test) clindamycin zone between an
erythromycin and clindamycin disks was verified.
RESULTS
We performed the clindamycin induction test on CNS that had the following
profile: test resistant or intermediate to erythromycin and susceptible to clindamycin
using routine antimicrobial susceptibility test. Twenty seven CNS of our collection
(n=200) had this profile. S. aureus ATCC 25923 were used for quality control (QC) of
the clindamycin and erythromycin disks, according to the standard disk diffusion QC
procedure. The susceptibility patterns for CNS isolates are showed in the Table 1. One
120
handred tirty three (66.5%) of the isolates were oxacillin-resistant. The full and
intermediate resistance was 63% and 3.5%, respectively, for erithromycin and 53.5%
and 1% respectively, for clindamycin. The phenotypic pattern compatible for realization
of the D test was obtained for 27 CNS isolates (13.5% of the all). Thus, only five
(18.5%) were positive for inducible clindamycin resistance. The test was more visible
when the erythromycin disk was placed 15 mm or 10 mm from the clindamycin disk.
Positive reactions D test were showed when test intermediate to erythromycin were
included as well as test resistante to erythromycin. In total, five positive reactios were
observed, three (14.3%) for full resistance and two (33.4%) for intermediate resistance
to erithromycin. We observed distinct species in this five CNS isolates D test positive:
two S. epidermidis, two S. haemolyticus and one S. simulans. Both S. epidermidis (2)
and S. haemolyticus (2) are carrier of the mecA gene – oxacillin-resistant, whereas
S. simulans is not carrier (data no showed). The distance between disks more suitable to
detection of the induction of resistance was 15 mm (standard) and 10 mm (no standard).
The Figure 1 showed the induction of the resistance in 10 mm distance.
DISCUSSION
Resistance in Gram-positive bacteria not only increases morbidity and mortality,
but also the costs of management of hospitalized patients. Studies have indicated a great
increase in the ratio of resistance of staphylococci to MLS group and failure in the
treatment with clindamycin in infections for microorganisms with inducible resistance
to MLS group [17]. Reporting clindamycin as susceptible for Staphylococcus spp. that
test erythromycin resistant and clindamycin susceptible without checking for inducible
clindamycin resistance may result in inappropriate clindamycin therapy. How caution,
add comment of resistance based on detection of inducible clindamycin resistance has
been proposed [18]. On the other hand, negative results for inducible clindamycin
resistance, report clindamycin susceptible and to add comment that this
Staphylococcus spp. does not demonstrate inducible clindamycin resistance in vitro
[18].
The D test is acceptable for all Staphylococcus spp. including oxacillin
susceptible or oxacillin resistant S. aureus or CoNS [18]. Many of the recently
recognized methicillin-resistence Staphylococcus aureus (MRSA) that cause
community-associated infections have the msrA gene and the oral clindamycin may be a
treatment option for these patients. In this case, these S. aureus strains are susceptibles
121
to clindamycin and do not present inducible resistance to this antimicrobial agent.
Although the clindamycin can be effective in some patients, did not recommend it used
without before to realize the D test [18].
An important fact in our study, was that we incorporate clinical isolated of
CoNS that presented a profile of intermediate resistance to clindamycin. In fact, two
isolated that showed compatible profile with the realization of the D test (erythromycin-
intermediate and clindamycin-susceptible), resulted in resistance to clindamycin and
positive D test. This isolates were identified as S. epidermidis and S. haemolyticus.
Outbreaks caused by multiresistents and offensives S. epidermidis and S. haemolyticus
have been reported in various nosocomial settings, such as in individual intensive care
units (ICU) or other units within a hospital [19]. Save this results, ours
Staphylococcus spp.isolates have resistance levels less than in others countries (data no
published showed in 104th General Meeting of the American Society for Microbiology,
New Orleans, LA, 2004). Until now, haven´t studies what report this test in Brazilian
clinical isolates.
This disk approximation test proved to be a good method to detect staphylococci
strains with inducible clindamycin resistance. As demonstrated in the effectued
analyses, the method revealed to be adequate and viable for the evaluation of this
phenotype of resistance, when was used 15 mm (standard) and 10 mm (no standard) of
distance between the disks? With 26 mm of distance between the disks, macrolide
resistance not was detected in two isolates (1 S. epidermidis and 1 S. haemolyticus). In
summary, the D test method revealed be practical in the established conditions, being
able to be used in the qualitative determination (phenotyping) of the resistance in
coagulase-negative staphylococci, mainly when the lesser standardized distance
(15 mm) between disks was used. Additional advantages include decreased
managements costs of treating in resistant infections (by diagnostic confirmation), more
rapidity in the results and its easy adaptation in the laboratorial routine.
The D test can be used as an auxiliary and alternative method to inducible
clindamycin resistance detection in the routine of clinical laboratories. However, the
confirmation of the erm gene in staphylococci strain with D test positive would assist in
the standardization of the test (suitable distance between disks, sensitivity and
specificity of the test). Moreover, the present study verified only 5 positive tests and a
122
greater number would be required for validation of the interpretation of the distance
between the disks.
Acknowledgements
The authors wish to tank Tiza and Rosângela for their support, bacteriology of
the Complexo Hospitalar Santa Casa de Misericórdia de Porto Alegre (CHSCMPA),
Fundação Faculdade Federal de Ciências Médicas de Porto Alegre (FFFCMPA),
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasília,
Brazil.
123
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125
Table 1. Antimicrobial susceptibility patterns among coagulase negative staphylococci
isolates
Pattern Antimicrobials
Oxacillin (%) Erithromycin (%) Clindamycin (%)
Resistant 133 (66.5) 126 (63) 107 (53.5)
Intermediate - 07 (3.5) 02 (1)
Susceptible 67 (33.5) 67 (33.5) 91 (45.5)
Total 200 (100) 200 (100) 200 (100)
126
Figure 1.
Legend of the Figure 1.
Figure 1. D test positive result for S. epidermidis strain (distance of 10 mm between
disks).
127
ANEXO 4______________________________________________________________
128
Amostras SensifarFOX
Oxoid
FOX
Sensifar
OX
Oxoid OX MIC ug/ml MIC ug/ml mecA
205 S S S S 0,125 S N
207 S S S S 0,25 S N
208 S S S S 0,125 S N
225 R R R R >4 R P
227 R R R R >4 R P
230 S R R R 4 R P
232 R R R R >4 R P
234 S S S S 0,125 S N
236 S R R R >4 R P
240 R S R S 0,125 S N
246 S R R R 2 R P
248 R R R R >4 R P
251 R R R R 2 R P
252 R R R R >4 R P
253 R R R R 4 R P
254 R R R R >4 R P
258 S S S S 0,125 S N
265 S R R R 1 R P
267 S R R R 2 R P
270 R R R R 1 R P
274 S R R R >4 R P
275 S R S R >4 R P
277 R R R R >4 R P
285 S S S S 0,125 S N
289 S S S S 0,125 S N
290 S R S R 1 R P
291 S R R R >4 R P
292 S S S S 0,5 R N
294 S R R R >4 R P
296 S R R R >4 R P
300 R R R R >4 R P
304 S S S S 0,125 S N
305 S R R R >4 R P
306 S R S R >4 R P
310 S S S S 0,125 S N
317 S R R R 2 R P
318 S R R R >4 R P
319 S S S S 0,125 S N
320 S S S S 0,125 S N
321 R R R R >4 R P
322 R R S S >4 R P
326 S R R R 4 R N
329 R R R R >4 R P
335 R R R R >4 R P
339 S R S R 1 R P
340 S S S S 0,25 S N
343 S R R R >4 R P
352 S R R R >4 R P
354 R R R R >4 R P
359 R R R R >4 R P
363 S R R R >4 R P
129
Amostras SensifarFOX
Oxoid
FOX
Sensifar
OX
Oxoid OX MIC ug/ml MIC ug/ml mecA
364 S R R R >4 R P
367 R R R R >4 R P
368 R R R R >4 R P
370 S S S S 0,125 S N
372 S S S S 0,125 S N
373 R R R R >4 R P
378 S S S S 0,125 S N
379 R R R R >4 R P
382 S R R R >4 R P
384 R R R R >4 R P
385 R R R R >4 R P
389 S R S R >4 R P
393 S S R S 0,25 S N
395 S S S S 0,25 S N
399 S R R R >4 R P
402 S R R R >4 R P
403 S S S S 0,125 S N
404 S R R R >4 R P
405 S S S S 0,125 S N
406 S R R R >4 R P
410 S R R R >4 R P
413 S R R R >4 R P
414 S S S S 0,125 S N
417 R R R R >4 R P
419 R R R R >4 R P
420 S R S R >4 R P
421 R R R R >4 R P
424 S R R R 4 R P
425 R R R R >4 R P
426 S R S R 4 R P
430 R R R R >4 R P
431 R R R R >4 R P
432 S R S R 4 R P
433 S S S S 0,125 S P
437 S S S S 0,125 S N
438 S R R R >4 R P
439 R R R R >4 R P
452 R R R R >4 R N
453 S S S S 0,25 S N
454 R R R R >4 R P
458 S S S S 0,25 S N
461 R R R R >4 R P
465 R R R R 4 R P
469 R R R R >4 R P
470 R R R R >4 R P
472 R R R R 4 R P
473 S R S R 4 R P
474 R R R R >4 R N
478 R R R R >4 R P
479 R R R R >4 R P
480 R S R S 4 R P
483 R R R R >4 R P
130
Amostras SensifarFOX
Oxoid
FOX
Sensifar
OX
Oxoid OX MIC ug/ml MIC ug/ml mecA
488 S S S S 0,125 S N
493 S R R R >4 R P
496 R R R R >4 R P
497 S R R R >4 R P
505 S R R R >4 R P
507 S R R R >4 R P
509 S S S S 0,125 S N
510 R R R R >4 R P
513 R R R R >4 R P
514 R R R R >4 R P
516 R R R R >4 R P
518 S S S S 0,125 S N
521 S R R R >4 R P
524 S R R R >4 R P
526 S R S R >4 R P
527 R R R R >4 R P
530 R R R R >4 R P
531 S R R R >4 R P
532 R R R R >4 R P
533 S R R R >4 R P
538 R R R R >4 R P
539 R R R R >4 R P
542 R R R R >4 R P
543 S S S S 0,125 S N
544 S R R R >4 R P
547 R R R R >4 R P
553 S S S S 0,125 S N
554 S R R R >4 R P
555 S S S S 0,125 S N
556 R R R R >4 R P
557 R R R R >4 R P
563 S R R R >4 R P
564 S S S S 0,125 S N
566 R R R R >4 R P
572 R R R R >4 R P
575 R R R R >4 R P
577 S S S S 0,25 S N
578 S R R R 4 R P
582 S R R R >4 R P
583 R R R R >4 R P
584 S R R R >4 R P
585 S S S S 0,125 S N
588 R R R R >4 R P
594 S S S S 0,125 S N
595 S S S S 0,125 S N
598 S R R R >4 R P
602 S S S S 0,25 S N
607 R R R R >4 R P
608 R R R R >4 R P
611 S S S S 0,25 S N
612 R R R R >4 R P
614 R R R R >4 R P
131
Amostras SensifarFOX
Oxoid
FOX
Sensifar
OX
Oxoid OX MIC ug/ml MIC ug/ml mecA
618 R R R R >4 P P
623 R R R R >4 R N
625 R R R R >4 R P
627 S R R R >4 R P
628 S R R R >4 R P
630 R R R R >4 R P
632 S R R R >4 R P
634 R R R R >4 R P
636 R R R R >4 R P
637 S S S S 0,125 S N
638 R R R R 2 R P
639 R R R R 4 R P
640 S S S S 0,125 S N
641 R R R R 4 R P
642 R R R R >4 R P
645 R R R R >4 R P
647 R R R R >4 R P
650 R R R R >4 R P
651 R R R R >4 R P
652 R R R R >4 R P
655 R R R R >4 R P
659 R R R R >4 R N
660 R R R R >4 R P
663 S S S S 0,125 S N
672 S R R R >4 R P
673 S R R R >4 R P
674 R R R R >4 R P
675 R R R R >4 R P
676 S S S S 0,125 S N
677 S R S R >4 R P
680 S R R R >4 R P
681 S R R R 2 R P
682 S R R R >4 R P
685 S R S R 2 R P
686 R R R R >4 R P
687 R R R R >4 R P
689 S R R R >4 R P
690 S R R R >4 R P
692 S S S S 0,125 S N
693 S R S R >4 R P
694 R R R R >4 R P
695 R R R R >4 R P
698 R R R R >4 R P
700 R R R R >4 R P
701 R R R R 4 R P
702 S S S S 0,125 S N
703 S S S S 0,25 S P
705 R R R R >4 R P
708 S R S S >4 R P
709 S S S S 0,25 S N
710 S R R R >4 R P
711 S S S S >4 R P
132
Amostras SensifarFOX
Oxoid
FOX
Sensifar
OX
Oxoid OX MIC ug/ml MIC ug/ml mecA
713 R R R R 4 R P
714 R R R R >4 R P
715 R R R R >4 R P
716 R R R R >4 R P
717 R R R R >4 R P
718 R R R R >4 R P
722 S R S R >4 R P
723 R R R R >4 R P
725 R R R R >4 R P
727 S S S S 0,25 S N
728 S R R R >4 R P
730 S R S R 2 R P
739 R R R R >4 R P
740 R R R R >4 R P
741 R R R R >4 R P
744 R R R R >4 R P
746 R R R R >4 R P
749 R R R R >4 R P
750 R R R R >4 R P
751 R R R R >4 R P
753 R R R R >4 R P
756 R R R R >4 R P
759 R R R R >4 R P
760 R R R R >4 R P
765 R R R R >4 R P
770 S S S S 0,25 S N
772 R R R R >4 R P
773 S R R R >4 R P
778 S R R R >4 R P
779 S S S S 0,25 S P
780 S R R R >4 R P
783 R R R R >4 R P
785 S R R R >4 R P
788 S S S S 0,25 S N
133
ANEXO 5______________________________________________________________
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