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Kelly Mari Pires de Oliveira
Adesão de Salmonella Enteritidis em superfícies de
processamento de alimentos
Tese apresentada ao Curso de
Doutorado em Ciência de Alimentos da
Universidade Estadual de Londrina,
como parte dos requisitos necessários
para obtenção de título de Doutor.
Profa. Dra. Tereza Cristina Rocha Moreira de Oliveira
LONDRINA – PARANÁ
2006
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CENTRO DE CIÊNCIAS AGRÁRIAS
DEPARTAMENTO DE TECNOLOGIA DE ALIMENTOS E MEDICAMENTOS
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BANCA EXAMINADORA
____________________________________________________
Profa. Dra. Terezinha Inês Estivalet Svidzinski
Universidade Estadual de Maringá - Pr
_____________________________________________________
Prof. Dr. Nélio José de Andrade
Universidade Federal de Viçosa - MG
______________________________________________________
Profa. Dra. Regina Lúcia dos Santos
Universidade Estadual de Londrina - Pr
_____________________________________________________
Profa. Dra. Elisa Yoko Hirooka
Universidade Estadual de Londrina - Pr
____________________________________________________
Profa. Dra. Tereza Cristina Rocha Moreira de Oliveira
Universidade Estadual de Londrina - Pr
Londrina 10, março de 2006.
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Dedico
À Minha Mãe,
Que sempre me acompanhou e me apoiou e a quem
devo o percurso da minha vida, estou grata pelo amor,
confiança, estímulo e coragem que tão oportunamente
me soube transmitir.
Agradecimentos
Á Deus, que me concedeu a vida, e saúde durante a execução deste trabalho e força
nos momentos difíceis e que sempre me abençoou com as pessoas certas nas horas
em que mais precisei.
A Profa. Dra. Tereza Cristina Rocha Moreira de Oliveira, pela orientação e
profissionalismo, e pela importante contribuição para o meu crescimento científico
desde a graduação.
Aos meus Professores e amigos do Departamento de Análises Clínicas da
Universidade Estadual de Maringá, pela excelência dos conhecimentos transmitidos e
pelo incentivo e apoio na minha formação acadêmica e vida profissional.
À Doutora Rosário Oliveira, minha orientadora em Portugal, agradeço por ter me
recebido em seu laboratório, bem como pela correção dos artigos.
Ao Departamento de Tecnologia de Alimentos e Medicamentos (TAM) da
Universidade Estadual de Londrina, pelo apoio à pesquisa científica.
Ao Departamento de Engenharia Biológica da Universidade do Minho.
À CAPES, pelo apoio financeiro que foi de fundamental importância para a execução
deste trabalho.
Aos meus amigos do Laboratório da Universidade do Minho pela agradável
convivência, em especial à Mariana, por sua contribuição no início deste trabalho.
À Fátima Jacinto, pela sua generosidade e a minha querida amiga Salomé, pela
grande amizade que se construiu e se consolidou.
À Dra. Terezinha Inês Estivalet Svidzinski pela amizade e disponibilidade, pelo apoio
científico e pelas preciosas idéias que muito me auxiliaram.
A Sandra Rezende, secretária do TAM, que, com profissionalismo e competência, se
preocupa em facilitar o andamento do trabalho do pesquisador.
A toda minha família e aos meus pais Valdomiro e Lourdes pelo apoio incondicional,
vocês foram meu “porto seguro” nos momentos mais difíceis, e também
compartilharam comigo uns dos momentos mais felizes da minha vida.
À minha querida princesinha Emanuele, que só me trouxe alegria e ao Cleves pelo
apoio e compreensão.
A todos os amigos que, com palavras e gestos de incentivo e carinho, contribuíram
para a realização deste trabalho.
RESUMO
Os biofilmes são estruturas altamente organizadas nas quais microrganismos
crescem e sobrevivem a ambientes hostis. São definidos como complexos
ecossistemas microbianos embebidos em uma matriz de substâncias poliméricas
extracelulares (EPS) aderidos a uma superfície. Biofilmes apresentam uma maior
resistência aos sanitizantes que as células livres e quando em superfícies de
processamento de alimentos podem trazer problemas relacionados à contaminação
cruzada, contaminação pós-processamento e corrosão de equipamentos. A
compreensão dos fatores e processos biológicos envolvidos no estabelecimento e
desenvolvimento dos biofilmes é de fundamental importância. Os mecanismos que
governam a adesão dependem da natureza da superfície de contato e das
propriedades superficiais dos microrganismos. O objetivo deste trabalho foi estudar
os fatores determinantes da adesão de quatro isolados diferentes de Salmonella
Enteritidis, um isolado de Pseudomonas aeruginosa e um isolado de Serratia
marcescens
em diferentes materiais utilizados no processamento dos alimentos. As
características das cepas estudadas foram correlacionadas com as taxas de adesão
e hidrofobicidade. O estudo de adesão foi realizado em aço 304, polietileno,
polipropileno e granito, materiais normalmente utilizados em superfícies de
processamento de alimentos. A medida do ângulo de contato foi empregada para
determinar a hidrofobicidade e a tensão superficial das superfícies de contato e dos
microrganismos. A técnica espectroscópica de fotoelétrons excitados por raios X
(XPS) e a análise da rugosidade foram utilizadas para análise superficial das cepas
estudadas e caracterização das superfícies de contato testadas, respectivamente.
Todas as cepas estudadas mostraram valores positivos para a hidrofobicidade e
foram consideradas hidrofílicas. Os resultados encontrados com a XPS sustentam a
similaridade dos valores de hidrofobicidade obtidos pelo ângulo de contato. P.
aeruginosa foi a cepa mais hidrofóbica e apresentava a maior quantidade de N/C. O
caráter hidrofílico da S. marcescens foi relacionado com a grande quantidade de O/C.
As diferentes cepas de Salmonella mostraram similaridade na composição da parede
celular e nas propriedades físico-químicos das superfícies. A adesão das diferentes
cepas de Salmonella as diversas superfícies foram estatisticamente diferentes. A
fonte de isolamento das Salmonella spp. não parece afetar a habilidade de adesão.
Contudo, S. Enteritidis MUSC apresentou maior capacidade de adesão às superfícies
estudadas (p<0.05). A facilidade de interação desse isolado de Salmonella as
superfícies de contato poderia ser explicado por sua grande capacidade de aceitar
elétrons. Todas as superfícies apresentaram caráter hidrofóbico e não foi possível
estabelecer nenhuma correlação na capacidade de doar elétrons e receber elétrons
sobre a interação das superfícies. Não foi encontrada uma relação entre rugosidade
dos materiais e adesão bacteriana. O polietileno apresentou os maiores valores de
rugosidade média, no entanto, foi o material no qual ocorreu a menor adesão.
Considerando todas as tentativas de explicação baseadas nas propriedades físico-
químicas das bactérias e das superfícies, não é possível estabelecer nenhuma
correlação direta e deduzir uma hipótese de um modelo racional de adesão.
Palavras chaves: adesão; Salmonella Enteritidis; superfícies; hidrofobicidade; ângulo
de contato; XPS.
ABSTRACT
Biofilms are structures highly organized in which microorganisms grow and therefore
can survive to hostile environments. They are defined as a complex community of
microorganisms usually encased in an extracellular matrix of polymeric substances
(EPS) attached to a surface. Cells embedded in biofilm are more resistant to sanitizers
than free cells. Biofilms in food processing surfaces can cause problems such as
cross contamination, product contamination and equipment corrosion. A greater
understanding of biofilms formation is important for food quality and safety. The
mechanism governing microbial adhesion to surfaces depends on the nature of the
contact surface and the bacterial surface properties. The aim of this study was to
investigate the factors that are involved in the adhesion of four Salmonella Enteritidis
isolates, one Pseudomonas aeruginosa isolate and one Serratia marcescens isolate
to different materials (steel 304, polyethylene, polypropylene and granite) used as
cover surfaces or as other utensils in kitchens. The bacterial surface properties were
correlated with taxes of adhesion and hidrophobicity, which was, evaluated through
contact angle measurements using the sessile drop method. The X-ray photoelectron
spectroscopy (XPS) and roughness were used to analyze the strains surface
characteristics and the inert surfaces studied, respectively. All strains analyzed
showed positive values of hidrophobicity and were considered hydrophilic. The XPS
results corroborated with the similarity of the hidrophobicity values obtained. P.
aeruginosa was the most hydrophobic strain and had the highest amount of N/C. The
hydrophilic characteristic observed in S. marcecens was related to the high surface
quantity of O/C. All Salmonella strains analyzed showed similar wall cellular
composition and the same physical chemical surfaces properties. Statiscally, the
extent of adhesion of the different strains to the materials assayed was different
(p<0.05). The source of Salmonella spp. isolates does not seem to affect the ability of
adhesion. However, S. Enteritidis MUSC showed the highest capacity of adhesion to
all surfaces studied (p<0.05) that could be explain by its capacity to accept electrons.
All the surfaces had hydrophobic character; at least it is not possible to establish any
correlation between the electron donor and electron acceptor capabilities of the
interacting surfaces. No correlation between inert surface roughness and bacterial
adhesion was found. Polyethylene was the roughest material but it was the material
displaying less extent of bacterial colonization. Considering all the tentative
explanations based on physico-chemical properties of bacterial cells and surfaces, it is
not possible to establish any direct correlation to elicit the hypothesis of a reasonable
model of adhesion.
Keywords: Adhesion; Salmonella Enteritidis; surfaces; hydrophobicity, contact angle;
XPS.
SUMÁRIO
1
INTRODUÇÃO
.......................................................................................... 9
2 OBJETIVOS.............................................................................................. 11
3 REVISÃO BIBLIOGRÁFICA..................................................................... 12
3.1 CONSIDERAÇÕES HISTÓRICAS DE MICRORGANISMO
S
ADERIDOS................................................................................................
12
3.2 FORMAÇÃO DO BIOFILME .................................................................... 14
3.3 COMPOSIÇÃO DO BIOFILME.................................................................. 16
3.4 CONDICIONAMENTO DA SUPERFÍCIE.................................................. 17
3.5 ADESÃO.................................................................................................... 18
3.5.1
Adesão reversível
................................................................................... 19
3.5.2 Adesão irreversível ................................................................................ 20
3.6. PRODUÇÃO DE SUBSTÂNCIA EXTRACELULAR POLIMÉRICA.. ........ 21
3.7 CRESCIMENTO, DESENVOLVIMENTO E DESPRENDIMENTO D
O
BIOFILME..................................................................................................
22
3.8 ASPECTOS TERMODINÂMICOS DA ADESÃO INICIAL......................... 24
3.8.1 Teoria DLVO............................................................................................. 25
3.8.2 Forças de Curto Alcance........................................................................ 27
3.8.3.
Teoria XDLVO
.......................................................................................... 27
3.9 HIDROFOBICIDADE................................................................................. 29
3.9.1 Medida do Ângulo de Contato................................................................ 31
3.10 TÉCNICA ESPECTROSCÓPIVA DE FOTOELÉTRONS EXCITADOS
POR RAIOS X (X-ray photoelectron spectroscopy) – XPS......................
34
3.11 RUGOSIDADE DOS MATERIAIS............................................................. 38
3.12 SUPERFÍCIES ESTUDADAS................................................................... 41
3.12.1 Aço............................................................................................................ 41
3.12.2 Granito...................................................................................................... 43
3.12.3 Polietileno................................................................................................ 44
3.12.4 Polipropileno............................................................................................ 46
REFERÊNCIAS......................................................................................... 48
APÊNDICE
...…………………………………………………………….......... 56
ANEXOS...................................................................................................
ARTIGO 1 Biofilmes Microbianos e resistência aos sanitizantes:
uma revisão
ARTIGO 2
Factors involved in attachment of Salmonella Enteritidis,
Pseudomonas aeruginosa and Serratia marcescens to stainless steel
ARTIGO 3 Adhesion of Salmonella Enteritidis to materials used
in kitchen surfaces
58
Factors involved in attachment of Salmonella Enteritidis,
Pseudomonas aeruginosa and Serratia marcescens to stainless steel.
Kelly Oliveira
3
, Tereza Oliveira
1
, Pilar Teixeira
2
, Joana Azeredo and Rosário Oliveira
2
*
1Universidade Estadual de Londrina, Centro de Ciências Agrárias, Departamento de
Tecnologia de Alimentos e Medicamentos, C.P. 6001, CEP:87051-970, Londrina, PR, Brasil.
2Centro de Engenharia Biológica - CEB, Universidade do Minho, Campus de Gualtar, 4710-
057 Braga, Portugal
3Centro Universitário de Maringá – CESUMAR, Maringá-PR, Brasil
*Corresponding author:
Prof. Rosário Oliveira
Centro de Engenharia Biológica
Universidade do Minho, Campus de Gualtar
4710 - 057 Braga
Portugal
tel: 351 253604409
fax: 351 253678986
e-mail: [email protected]
Running headline: Attachment of bacteria to stainless steel
Abstract
Adhesion of microorganisms to food processing equipment surfaces and the problems it
causes are a matter of strong concern to the food industry. Contaminated food processing
surfaces may act as potential sources of transmission of pathogens in food industry, catering
and in the domestic environments. The mechanisms governing microbial adhesion to surfaces
are poorly understood; several studies have shown that adhesion of bacteria partly depends
upon the nature of the inert surfaces and partly upon the bacterial surface properties. The aim
of this study was to evaluate the effect of surface hydrophobicity in the adhesion of four
different strains of Salmonella Enteritidis, one Pseudomonas aeruginosa and one Serratia
marcescens to stainless steel 304 (SS304). Hydrophobicity was evaluated through contact
angle measurements using the sessile drop method. All the strains studied showed positive
values of the degree of hydrophobicity (
lwl
G∆ ), and so can be considered hydrophilic. P.
aeruginosa
was the less hydrophilic microorganism and stainless steel revealed a hydrophobic
character. The XPS results corroborated the similarity of the values of the degree of
hydrophobicity obtained by contact angles. The different Salmonella strains showed similar
cell wall compositions and cell surface physico-chemical properties. Nevertheless,
P.
aeruginosa
and S. Enteritidis MUSC presented higher adhesion ability to SS304 (p<0.05),
which can be explained by a facilitated interaction between their higher acceptor electron
capacity with the single donor ability of SS304.
Keywords: Adhesion; Salmonella Enteritidis; stainless steel; hydrophobicity, contact angle;
XPS.
1. Introduction
Adhesion of microorganisms to food processing equipment surfaces is of great concern
to the food industry. Adhered microorganisms to solid surfaces can have the potential to act as
a chronic source of microbial contamination, which may compromise food quality and
represent a significant health hazard (Barnes, et al., 1999). Contaminated food processing
surfaces may act as a potential source of transmission of pathogens in food industry, catering
and in the domestic environments. Several studies showed that cross-contamination can result
from hands, sponges/clothes and utensils either in domestic kitchens or in any food processing
plant (Hilton and Austin, 2000; Gorman et al., 2002; Kusumaningrum et al., 2002;
Kusumaningrum et al., 2003).
For instance, Salmonella spp. is able to colonize different inert
food contact surfaces to form biofilms (Hood and Zottola, 1997; Gough and Dodd, 1998;
Bonafonte et al., 2000; Joseph et al., 2001).
So, it has been recognized that a greater
understanding of the interaction between microorganisms and food-processing surfaces is
required to control these problems.
Salmonellosis has been one of the most commonly reported food-borne illnesses
worldwide. In many countries, including Brazil,
Salmonella Enteritidis is the most frequently
isolated serotype. Epidemiological evidence has linked the majority of outbreaks in State of
Paraná, Brazil, to contaminated poultry products.
Pseudomonas
species are often associated with spoilage of perishable foods stored at
refrigerator temperatures such as milk and meats and, therefore, have a major impact on the
quality of these foods (Hood and Zottola, 1997; Eneroth et al., 2000).
Serratia marcescens is
an opportunistic pathogen widely distributed in nature, including foods of vegetable and
animal origin. Contaminated foods can act as a vehicle of infection and in humans
Serratia
marcescens may cause diarrhea, septic arthritis, urinary tract infections, wound infections and
septicemia (Singh et al., 1997; Gran et al., 2003).
Stainless steel has been the material of choice for working surfaces and kitchen sinks
for many years because of its mechanical strength, corrosion resistance, longevity and ease of
fabrication (Holah and Thorpe, 1990). In the food processing industry most of the surfaces are
of stainless steel including, pipelines tanks (Assanta et al., 2002), machinery and working
surfaces (Hood and Zottola, 1997; Rossoni and Gaylarde, 2000; Parker et al., 2001).
Moreover, it is relatively resistant to chemical attack by oxidizing and other sanitizing agents
used in the food industry, like hypochlorite, peracetic acid and iodophors (Boulange- Peterson,
1996).
The mechanisms governing the adhesion of
Salmonella spp., Pseudomonas and
Serratia adherence to inert surfaces are poorly studied; several studies have shown that
adhesion of bacteria partly depends upon the nature of the inert surfaces and partly upon the
bacterial surface properties (Sinde and Carballo, 2000). Hydrophobicity and surface charge
are the most important surface properties in the adhesion process. Innumerous studies have
demonstrated this fact (van Loosdrecht et al., 1987; Millsap et al., 1996; Hood and Zottola,
1997; Teixeira and Oliveira, 1999; Pereira et al., 2000).
The understanding of microbial adhesion is of major importance in preventing
undesirable biofilm formation. Therefore, the aim of this study was to evaluate the effect of
surface hydrophobicity in the adhesion of
Salmonella Enteritidis, Pseudomonas aeruginosa
and Serratia marcescens to stainless steel 304 (SS304), in order to investigate the behavior of
different strains of the same species and among species.
2. Materials and methods
2.1 Media and growth conditions
The strains used in this study are presented in Table1.
Table1
Bacterial isolates used in this study.
Strains Source
Pseudomonas aeruginosa
1
Clinical human sample
Serratia marcescens
1
Clinical human sample
Salmonella Enteritidis EMB
2
Water from poultry packaging
Salmonella Enteritidis MUSC
2
Breast meat of poultry
Salmonella Enteritidis AL
3
Food sample related to food-borne outbreak
Salmonella Enteritidis PC
3
Fecal human sample
The bacterial isolates were obtained from:
1
Clinical Microbiology Laboratory, Depart. of Clinical Analysis, University of Maringá, Pr,
Brazil.
2
Food Microbiology Lab., Depart. of Food and Dry Technology, University of Londrina, Pr,
Brazil.
3
LACEN (Central Paraná Public Health Laboratory Service).
All bacterial isolates were maintained in trypticase soy agar (TSA). Every strain was
subcultured twice in trypticase soy broth (TSB) at 37°C in an orbital shaker (130 rpm),
overnight. The cells were then harvested by centrifugation at 5000 g for 10 min and washed
three times with phosphate buffered saline (PBS 0.1M pH 7). The pellets were resuspended in
PBS to an inoculum level of 10
8
CFU/ml, determined by optical density.
2.2 Material used as substratum
The test surface was stainless steel (304, finish no 4), commonly present in the food
industry and used in domestic kitchens. The coupons were cut in 0.8 x 0.8 cm, washed in a
solution of a commercial detergent (Sonasol Pril, Henkel Ibérica S.A., Portugal) in ultrapure
water for 30 min and then thoroughly rinsed in ultrapure water (to remove any remaining
detergent), followed by immersion in ethanol 90 % for 30 min to completely degrease the
surface.
2.3 Hydrophobicity and surface free energy
Hydrophobicity was evaluated through contact angle measurements and using the
approach of van Oss and co-workers (van Oss et al., 1987, 1988, 1989). Accordingly, the
degree of hydrophobicity of a given entity is expressed as the variation of the surface free
energy between two moieties of that entity when immersed in water, comprising two
components: one apolar, due to Lifshitz-van der Waals interactions (
γ
LW
) and one polar
resulting from Lewis acid-base interactions (γ
AB
). The latter comprising two parameters, γ
-
expressing the electron donor capacity and
γ
+
accounting for electron acceptance.
Contact angle measurements (at least 25 determinations with each liquid on stainless
steel and on each microbial strain) were performed automatically with the aid of an image
analysis system (G2/G40) installed in a standard contact angle apparatus (Kruss-GmbH). The
images were transmitted by a video camera to a personal computer for evaluation. All the
measurements were performed at room temperature. In the case of bacterial cells, the
measurements were performed on a cell lawn using the sessile drop method described by
Busscher et al. (1984). Briefly, bacteria were deposited on a 0.45 µm cellulose acetate
membrane filter by filtration of the suspension using negative pressure. To standardize the
moisture content, the filters were then transferred onto Petri dishes containing 1% (w/v) agar
with 10% (v/v) glycerol. Measurements of advancing water contact angles were carried out at
25°C and three liquids with different polarities were used, water (W), formamide (F) and α-
bromonaphtalene (α-B).
Hydrophobicity of the stainless steel was estimated by the same technique, with direct
measurements of contact angles on stainless steel surface, after degreasing and cleaning.
2.4 X-ray photoelectron spectroscopy
Bacterial cell surface composition was measured using X-ray photoelectron
spectroscopy (XPS). The bacterial cells were grown in 200 ml TSB at 37
o
C under 120 rpm
for 18 h and washed three times in deionized water by centrifugation (10 min at 5000 g and 4
o
C). A volume of 200 ml of a cellular suspension (10
9
cells/ml) was vacuum filtered through
an acetate cellulose membrane of 45 µm. The membrane, completely covered with cells, was
immediately frozen with liquid nitrogen and then stored at –80 ºC until the subsequent step of
lyophilization. Freeze drying was performed at 10 Pa, overnight. The samples were placed in a
dessicator, at room temperature and immediately analyzed by XPS. The XPS analysis was
performed using an apparatus ESCALAB 200A, with a VG5250 software and data analysis.
The spectrometer used monochromatized Mg Kα X-ray radiation (15.000 eV). The constant
pass energy of the analyzer was 20 eV and it was calibrated with reference to Ag 3d
5/2
(368.27
eV). The pressure during analysis was under 1x10
-6
Pa. The spectra were recorded following
the sequence C 1s, O 1s, N 1s, P 2p. The chemical composition was defined as the ratio
between oxygen and carbon (O/C), nitrogen and carbon (N/C) or phosphorous and carbon
(P/C).
2.5 Adhesion assays
The coupons of stainless steel were immersed in 2 ml of each bacterial suspension
containing 10
8
CFU/ml. After 1 h at 37
o
C with constant shaking at 100 rpm, the coupons were
rinsed twice with PBS to remove poorly adhered bacteria. An aliquot of 20 µl/ml of a 4´,6-
diamidino-2-phenylindole (DAPI) solution was added to each well containing the plates and
incubated for 30 min in the dark. After this time, the wells were rinsed with sterile distilled
water and the adherent microorganisms were quantified by automatic enumeration using
epifluorescence microscopy. Thirty fields per coupon were scanned and the fluorescent cells
were enumerated. Computerized image analysis software (Image-Pro Plus, Media
Cybernetics) was used for the quantitative estimation of the adherent cells. All experiments
were done in triplicate.
2.6 Statistical analysis
The resulting data were analysed using SPSS software (Statistical Package for the
Social Sciences). One-way ANOVA with Bonferroni test was used to compare the number of
adhered cells. All tests were performed with a confidence level of 95%.
3. Results and discussion
The contact angles formed by the three liquids (water, formamide, and α-
bromonaphtalene) on bacterial lawns are present in Table 2. The values of water contact
angles for all the strains assayed were heterogeneous ranging from 9.7
o
to 33.5
o
. P.
aeruginosa displayed the greatest water contact angle (33.5
o
). The water contact angles of all
Salmonella strains tested were quite similar (9.7
o
– 10.8
o
) and were somewhat lower then
those reported in the literature (17
o
– 35
o
) (Dickson and Koohmaraie, 1989; Sinde and
Carballo, 2000). The different serovars of Salmonella studied and the non-uniformity on
bacterial surface may explain the results obtained in this study (Donlon and Colleran, 1993).
The water contact angle value gives preliminary information about the degree of
hydrophobicity of cells. The sample is considered hydrophobic or hydrophilic if the angle is
higher or lower than 65
o
, respectively (Vogler 1998).
Table 2
Values of contact angles (in degrees) measured with water (θ
W
), formamide (θ
f
) and α -
bromonaphatelene (
θ
α-B
) on the different microorganisms assayed.
Contact angle (°)
(
±
SD)
θ
W
θ
f
θ
α-B
S. Enteritidis EMB 10.8 (±2.2) 15.6 (±1.8) 26.1 (±4.2)
S. Enteritidis MUSC 13.5 (±1.6) 15.9 (±2.3) 27.6 (±1.7)
S. Enteritidis PC 14.0 (±4.4) 17.0 (±3.2) 31.7 (±28)
S. Enteritidis AL 9.7 (±1.9) 14.8 (±2.6) 27.2 (±2.5)
Pseudomonas aeruginosa
33.5 (±3.9) 29.3 (±2.7) 32.2 (±2.5)
Serratia marcescens
19.1 (±2.8) 25.7 (±1.3) 37.2 (±1.3)
SD means standard deviation
The values of the contact angles of the three liquids were used to calculate cell surface
tension parameters and the degree of hydrophobicity (Table 3). The degree of hydrophobicity
of a given material (l) can be defined in terms of the variation of the free energy of interaction
between two entities of this material when immersed in water (w),
lwl
G∆ . If the interaction
between the two entities is stronger than the interaction of each entity with water (
lwl
G∆
<
0)
the material is considered hydrophobic. On the contrary, if
lwl
G∆> 0 the material is
hydrophilic.
As far as hydrophobicity is concerned, all strains studied showed positive values of
lwl
G∆ , and so can be considered hydrophilic, which is in accordance with the water contact
angle values. The
lwl
G∆ values were very similar, with the exception of P. aeruginosa, which
exhibited a lower degree of hydrophilicity. It has been previously shown that, generally,
microorganisms adhere in higher numbers to hydrophobic materials (Sinde and Carballo,
2000; Cunliffe et al., 1999).
Table 3
Values of the components of surface tension (
+
γ
,
−
γ
,
LW
γ
) and degree of hydrophobicity
(
lwl
G∆ ) of bacterial cells.
Surface tension (mJ/m
2
)
Cells
LW
γ
+
γ
−
γ
lwl
G∆
S. Enteritidis EMB 39.89 0.97 55.99 34.12
S. Enteritidis MUSC 26.00 4.6 51.30 31.70
S. Enteritidis PC 38.06 1.22 54.48 32.28
S. Enteritidis AL 39.50 1.05 55.84 33.79
P. aeruginosa
20.03 8.03 53.31 19.89
S. marcescens
35.87 1.02 56.30 36.18
LW
γ
represents the apolar Lifshitz-van der Waals surface free energy component;
+
γ
represents the electron acceptor parameter and
−
γ
represents the electron donor parameter
of the polar Lewis acid-base surface free energy component;
lwl
G∆
represents the degree of
hydrophobicity
From Table 3, it can be observed that all strains had surfaces that were predominantly
electron donors (higher values of
−
γ
), with a low electron acceptor parameter (
+
γ
). The
exceptions were strains
S. Enteritidis MUSC (
+
γ
= 4.61) and P. aeruginosa (
+
γ
= 8.03) that
had the greatest electron acceptor parameters, which suggest that these cells are more prone to
establish Lewis acid-base interactions by acceptance of electrons than the others.
Substrate surface physico-chemical characteristics are presented in Table 4.
Considering the values of the contact angle of water on SS 304 obtained in this study (81.2
o
)
and
lwl
G∆
<
0 (-59.8), the stainless steel assayed was hydrophobic, which is in accordance
with several authors (Sinde and Carballo, 2000; Flint et al. 2000; Teixeira et al. 2005). A point
to be noted is that stainless steel does not have an electron acceptor parameter but only an
electron-donor (
−
γ
).
Table 4
Contact angle (in degrees) measured with water (
θ
W
), formamide (
θ
f
) and α-bromonaphtalene
(θ
α-B
) and values of the surface tension components (
+
γ
,
−
γ
,
LW
γ
) and degree of
hydrophobicity (
lwl
G∆ ) of stainless steel 304.
Surface
Contact angle (°)
(±SD)
Surface tension (mJ/m
2
)
θ
W
θ
f
θ
α-B
LW
γ
+
γ
−
γ
lwl
G∆
Stainless
steel
81.2
(±0.9)
60.0
(±1.1)
23.4
(±0.46)
40.81 0.00 5.84 -59.80
SD means standard deviation
The XPS spectra of the bacterial surfaces showed that the most abundant elements on
the surface are (C) carbon, oxygen (O), and nitrogen (N) along with outline amounts of
phosphorus (P). The chemical composition of microbial cells surface is usually expressed in
terms of N/C, O/C and P/C ratios (Van der Mei et al., 2000). The corresponding values for the
microorganisms assayed are presented in Table 5. All strains used in this study exhibited high
O/C values, ranging from 0.453 (
P. aeruginosa) to 0.739 (S. marcescens), and P/C values,
ranging from 0.004 (S. marcescens) to 0.025 (P. aeruginosa). The results for P. aeruginosa
are in accordance with Bruinsma et al. (2001), while for
S. marcescens the present results are
slightly inferior, probably due to strain characteristics.
Table 5
Ratios of the major chemical elements of bacterial surface composition of Salmonella strains,
P. aeruginosa and S. marcescens obtained by XPS analysis.
Strain N/C O/C P/C
S. Enteritidis EMB 0.066 0.584 0.008
S. Enteritidis MUSC 0.118 0.465 0.009
S. Enteritidis PC 0.118 0.466 0.009
S. Enteritidis AL 0.114 0.479 0.008
S. marcescens
0.119 0.739 0.004
P. aeruginosa
0.191 0.453 0.025
Ratios of nitrogen/ carbon (N/C), oxygen/carbon (O/C) and phosphorus/carbon (P/C) of cell
walls
Microbial surface thermodynamics is a reflection of the physico-chemistry of bacterial
surfaces, which is controlled by macromolecular components, e.g., lipo-polysaccharide,
protein and exopolymers, varying in quantity with growth conditions and from strain to strain.
The amount of the macromolecular components can be represented by a variety of different
functional groups (Strevett and Chen, 2003; Vadillo-Rodríguez et al. 2004). In previous
works, cell surface hydrophobicity, assessed by the water contact angle, was directly
correlated with the concentration of nitrogen or carbon involved in hydrocarbon form and
inversely correlated with the oxygen concentration (Rouxhet et al.; 1994, Van der Mei et al.,
2000; Boonaert and Rouxhet, 2000). In this study, the higher hydrophilic character of
S.
marcescens
is in agreement with its higher value of O/C, which can also suggest the presence
of capsular polysaccharide material (Van der Mei et al., 2000). The XPS results corroborated
the similarity of the hydrophobicity values. The results obtained through XPS analysis
indicate that
P. aeruginosa shall be the most hydrophobic strain because it has the higher N/C
ratio. Cerca et al., (2005) correlated the N/C ratio with cell surface hydrophobicity, with the
less hydrophobic cells exhibiting the lower N/C ratio. The presence of proteinic appendages is
often reflected in a high nitrogen concentration at the cell surface (Rouxhet et al., 1994).
The number of cells of
Salmonella Enteritidis (EMB, MUSC, PC, AL), P. aeruginosa
and S. marcescens adhered to stainless steel are presented in Figure 1 and Figure 2. The extent
of adhesion of P. aeruginosa and Salmonella MUSC was statistically (p<0.05) higher
compared with the other cells.
0
5000
10000
15000
20000
25000
S.
Enteritidis
Embal
S.
Enteritidis
Musc
S.
Enteritidis
Pc
S.
Enteritidis
Al
P.
aeruginosa
S.
marcescens
Adhered Cells/mm
2
Figure 1. Adherence of the cells to stainless steel 304. The final concentrations of 10
8
for Salmonelas and
S. marcescens, for P. aeruginosa the concentrations were dilute 10 fold
for assays.
P. aeruginosa showed the higher degree of hydrophobicity and the higher number of
cells adhering to stainless steel. Liu et al. (2004) predicted that when both bacterial and
support surfaces are hydrophobic, microbial adhesion is highly facilitated, if both bacterial
and support surfaces are hydrophilic, microbial adhesion would proceed with difficultly, and
increased cell surface hydrophobicity would favor cell adhesion on both hydrophilic and
hydrophobic support surface. Also, Assanta et al. (2002) suggested that
Arcobacter butzzeri
could attach in higher numbers to surfaces with low surface free energy and
Aeromonas
hydrophila
cells had a tendency to attach in high numbers to hydrophobic surfaces. A recent
study by Henriques at al. (2004), reported the increase in the number of adhered yeast cells to
acrylic by an increase in the interactions between the electron-donor groups of acrylic and the
electron-acceptor groups of cells. In consonance, the greater extent of adhesion of
P.
aeruginosa
and S. Enteritidis MUSC observed in the present study could also be attributable
to the ease of interaction between the stainless steel electron-donor groups and the cells
electron-acceptor groups.
As all
Salmonella strains studied showed similar cell wall compositions, their cell
surface physico-chemical properties and the physico-chemical interaction with the adhesion
substratum were expected to be similar as well. Nevertheless,
Salmonella Enteritidis MUSC
showed a higher ability to adhere to SS304. The possible explanation can rely in its ability to
establish preferential Lewis acid-base interactions with an electron donor substratum.
Consequently, the concept of overall hydrophobicity (
lwl
G∆ ), i.e. considering its components,
is very useful for understanding the role of hydrophobic/hydrophilic interaction between cells
and support surfaces in the microbial adhesion process. A more thorough understanding of the
interactions that occur between the bacterial cells and the substratum may lead to the
development of methods to prevent bacterial adhesion, for instance by substratum surface
modification.
Acknowledgements
Kelly Oliveira fully acknowledges CAPES/ Brazil for the grant BEX 0891/01-0.
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Biofilmes Microbianos e resistência aos sanitizantes: uma revisão
Kelly Mari Pires de OLIVEIRA
1,2
,
Tereza Cristina Rocha Moreira de OLIVEIRA
2
1
Centro Universitário de Maringá – CESUMAR, Av Guedner, 1610 Jardim Aclimação. CEP 87050-390
- Maringá-PR, Brasil.
2
Universidade Estadual de Londrina - UEL, Centro de Ciências Agrárias, Departamento de Tecnologia
de Alimentos e Medicamentos, C.P. 6001, CEP:87051-970, Londrina, PR, Brasil.
Biofilmes Microbianos e resistência aos sanitizantes: uma revisão
RESUMO
Os biofilmes são estruturas altamente organizadas nas quais microrganismos
crescem e sobrevivem a ambientes hostis. São definidos como complexos
ecossistemas microbianos embebidos em uma matriz de substâncias poliméricas
extracelulares (EPS) aderidos a uma superfície. Biofilmes podem ser formados e
tornarem-se uma fonte constante de contaminação de alimentos se microrganismos
não forem completamente removidos das superfícies de contato durante a
desinfecção. Os microrganismos aderidos são mais resistentes aos sanitizantes que
as células livres. Porém, a eficácia de um sanitizante depende do tipo de
microrganismo, da natureza do sanitizante e da superfície de adesão. A
compreensão de como os biofilmes podem ser formados é importante para a
manutenção da qualidade e segurança dos alimentos. O objetivo deste trabalho foi
fazer uma revisão sobre a formação de biofilme e a sua resistência aos sanitizantes.
Palavras-chave: adesão, sanitizantes, EPS
Introdução
A vida microbiana na forma de células isoladas não é freqüente na natureza. Os
microrganismos colonizam superfícies de tecidos dos organismos vivos ou
superfícies de matéria inanimada, aderindo firmemente e formando comunidades
denominadas de biofilmes, que podem atingir alto nível de complexidade.
Biofilme é um grupo de microrganismos associados entre si por seus produtos
extracelulares, que funcionam como uma interface, aderido a uma superfície abiótica
ou biótica. O início da formação do biofilme ocorre quando bactérias livres ou
planctônicas reconhecem uma superfície e aderem-se a ela firmemente.
17
Os biofilmes formam-se sobre uma grande variedade de superfícies tais como,
dentes, epitélios, cateteres, plásticos, aço inoxidável entre outros. A acumulação em
equipamentos industriais tem sido um problema constante. A fornece de biofilme em
superfícies de industrias alimentícias pode resultar em um impacto negativo na
qualidade dos produtos finais como também à corrosão microbiana dos maquinários.
Além disso, os microrganismos quando em biofilmes na indústria de alimentos
apresenta uma maior sua resistência aos sanitizantes.
10, 27
Os microrganismos aderidos às superfícies de processamento de alimentos
oferecem problemas consideráveis em relação à contaminação cruzada e
contaminação pós-processamento.
16, 17, 32
A monitorização de biofilmes, evitando a sua formação, melhora a qualidade e
vida de prateleira dos alimentos, aumenta a vida de tubulações e equipamentos,
diminui os custos de produção com a manutenção de equipamentos e pelo uso
racional de sanitizantes, com conseqüente redução de impactos ambientais. Assim,
este trabalho teve como objetivo fazer uma revisão sobre as etapas de formação de
biofilme e à sua resistência aos sanitizantes.
Formação do Biofilme
A formação de biofilme é um processo dinâmico. Os modelos atuais,
baseados em grande parte no estudo com Pseudomonas spp., descrevem a
formação de biofilme como um processo linear, que começa quando células
bacterianas planctônicas prendem-se a uma superfície. Essa adesão é seguida de
multiplicação do microrganismo, desenvolvimento da estrutura do biofilme a
dispersão de células bacterianas na superfície. Essas várias fases de interações
microbianas com a superfície parecem requerer a produção de substância
extracelular polimérica (EPS), que ajuda na adesão inicial e na manutenção da
estrutura do biofilme.
3,11,12,15
A adesão é a primeira etapa no complexo processo de formação de um
biofilme. O mecanismo mais aceito de adesão de bactérias em superfícies sólidas,
descrito por Marshall em 1971, apresenta duas etapas, a adesão reversível e a
adesão irreversível.
15
O modelo proposto por Busscher & Weerkamp
6
considera no
processo a distância entre a bactéria e a superfície de adesão, que ocorrem em três
etapas. Em ambos, o passo final depende da habilidade do microrganismo
metabolizar e produzir material adesivo. O desenvolvimento do biofilme, descrito por
Hamilton e Characklis apud Morton et al.
25
envolve quatro etapas para o
desenvolvimento do biofilme. Primeiramente ocorre o transporte de moléculas
orgânicas e células para a superfície, em seguida a adsorção dessas moléculas
orgânicas para formar um ”condicionamento da superfície”, e somente na terceira
etapa ocorrerá a adsorção de células à superfície condicionada e o crescimento de
células com a síntese de EPS. À medida que vem sendo estudado o
desenvolvimento do biofilme novas etapas vem sendo descritas no processo de
formação.
Levando em consideração os modelos descritos na literatura, a formação do
biofilme ocorre nas seguintes etapas: (1) Transporte de moléculas orgânicas para a
superfície e adsorção para a formação do filme condicionador; (2) Transporte de
microrganismos e outras partículas para a superfície condicionada; (3) Adesão
reversível à superfície; (4) Adesão entre os microrganismos aderidos; (5) Adesão
irreversível das células, com produção de EPS; (6) Multiplicação das células e
crescimento do biofilme; (7) Transporte de subprodutos do biofilme para o exterior;
(8) Desprendimento do biofilme.
Composição do Biofilme
O biofilme é composto por aproximadamente 97% de água. Esta porcentagem varia
dependendo das características ambientais onde o biofilme se encontra. Segundo
Sutherland
30
, além da água e das células microbianas, a matriz do biofilme é
composta de um complexo de polímeros, nutrientes absorvidos e metabólitos,
produtos de lise celular e partículas de materiais do meio onde se encontra (Tabela
ｪ).
Estudos recentes mostraram que os biofilmes na natureza são heterogêneos. Os
microrganismos presentes podem exibir diferentes propriedades fisiológicas e
metabólicas. Bactérias anaeróbias e aeróbias podem ser isoladas do mesmo
biofilme. Por exemplo, P. aeruginosa tende a ser encontrada no interior do biofilme e
V. parahaemolyticus escolhe estar perto da interface do biofilme-líquido. A
interdependência metabólica pode acontecer entre espécies presentes
11, 29, 15
e
muitos patógenos humanos potenciais como Listeria pneumophila, Cryptosporidium
spp, Mycobacterium spp, Pseudomonas spp, Staphylococcus spp, Rotavirus,
Giardia, micoplasmas e protozoários podem se associar e formar biofilmes.
20, 25
Tabela ｪ. Composição dos Biofilmes
Componentes % na matriz
Água Acima de 97%
Células microbianas 2-5% (várias espécies)
Polissacarídeos (homo e hetero-polissacarideos) 1-2% (neutro e polianiônico)
Proteínas (extracelulares e resultante de lise) <1-2% (várias, incluindo enzimas)
DNA e RNA < 1-2% (de lise de células)
Íons ? (ligados ou livres)
Fonte: Sutherland
30
Condicionamento da Superfície
O primeiro estágio na formação do biofilme está relacionado com o meio onde
o microrganismo se encontra, pois serão componentes deste meio que irão adsorver
a superfície e formar o filme condicionador. Por exemplo, o filme condicionador em
laticínios será o leite, em abatedouros proteínas e gorduras da carne, nos dentes a
saliva e será em sistema de abastecimento de água a própria água.
O filme condicionador provavelmente altera as propriedades físico-químicas
da superfície. Barnes et al.
4
estudaram a adesão de Listeria monocytogenes e
Staphylococcus aureus no aço condicionado com leite e com as proteínas do leite e
observaram uma menor adesão no aço condicionado com leite comparado ao aço
condicionado com água. Resultados similares foram encontrados por Helkeet al.
13
e
Speers & Gilmour
28
. Cunliffe et al.
7
estudaram os efeitos físico-químicos das
superfícies condicionadas com diferentes substâncias e encontraram uma menor
adesão quando a superfície foi condicionada com grupamentos hidrofílicos. Flint et
al.
8
também encontraram uma menor adesão quando trataram uma superfície com
soluções alcalinas. Assim, a aderência microbiana poderá ser alterada através das
alterações das cargas de superfície pelo uso de diferentes soluções como os
sanitizantes.
Normalmente, moléculas do meio são responsáveis pelo condicionamento,
contudo certos microrganismos também são capazes de condicionar a superfície.
Sasahara & Zottola
26
observaram que Listeria monocytogenes mostrou aderência ao
vidro significativamente maior quando crescia juntamente com Pseudomonas fragi.
Os autores concluíram que P. fragi pode colonizar primeiro e a EPS produzida por
este microrganismo ser a responsável pelo aumento da adesão de L.
monocytogenes.
Adesão Reversível e Irreversível
O segundo passo na formação do Biofilme é a aderência do microrganismo à
superfície condicionada. Este processo pode ser ativo ou passivo e depende da
motilidade bacteriana ou do transporte das células planctônicas por gravidade,
difusão ou forças dinâmicas do fluído onde se encontram.
19
Os mecanismos
envolvidos na adesão inicial são dependentes não só das propriedades físico-
químicos da superfície bacteriana, mas também da composição da superfície de
adesão, como a natureza eletroquímica e hidrofobicidade relativa, da rugosidade e
do filme condicionador. Para acontecer, deve haver forças atraentes entre a célula e
a superfície. Essas forças de atração devem ser grandes o suficiente para superar
qualquer força repulsiva. Forças de longo alcance e interações de alcance limitado
podem ter um papel significante em adesão.
14, 15
Inicialmente, as interações entre bactéria e substrato levam a uma adesão
reversível. Durante este estágio várias forças de longo alcance estão envolvidas
como forças de van der Waals, forças da dupla camada elétrica e interações
hidrofóbicas. Nesse estágio a bactéria ainda mostra movimentos Brownianos que
podem removê-la por forças exercidas pelo fluído. Para ocorrer adesão irreversível o
microrganismo deve ultrapassar as forças repulsivas, utilizando apêndices especiais
como flagelo, fímbrias, pili e exopolissacarídeos. Na adesão irreversível várias forças
de curto alcance estão envolvidas como interações dipolo-dipolo, ligações de
hidrogênio, iônicas e covalentes e interações hidrofóbicas.
19
É bastante difícil
determinar quais são as forças ou interações mais importantes para a aderência.
Embora a motilidade ajude na colonização dos microrganismos Gram-
negativos, não é uma condição prévia para formação do biofilme, pois várias
bactérias que não apresentam motilidade como Streptococcus, Staphylococcus, e
Mycobacteria formam biofilmes rapidamente. Woodward et al.
32
estudaram o
envolvimento da fímbria SEF14 e SEF17 na adesão de Salmonella Enteritidis e
encontraram que em cepas mutantes, sem fimbrias, a adesão foi reduzida em 90%
quando comparada com cepas que possuíam fímbrias. Em estudo realizado com
adesão de Staphylococcus epidermidis em modelos animais, proteínas e adesinas
polissacarídicas foram relacionadas à aderência.
11
MORTON et al.
25
concluíram que
a adesão em ambientes naturais e indústrias é mediada por EPS, produzidas pelas
próprias células.
Produção de Substância Extracelular Polimérica
A EPS é constituída de polissacarídeos, ácidos nucléicos e proteínas que
servem de união entre as microcolônias que formam o biofilme. EPS é também
denominada de matriz extracelular, “substâncias polímeras“, “polissacarídeos
extracelulares” e “substância extracelular polimérica”.
O termo glicocálice, cápsula ou camada limosa em muitos casos também se
referem a EPS. A EPS produzida pelos microrganismos é importante na adesão
inicial do microrganismo, bem como em sua permanência, por protegerem as células
da desidratação e auxiliarem na captura de nutrientes. A EPS conserva e concentra
enzimas digestivas liberadas pelas bactérias, aumentando assim a eficiência
metabólica das células. Constitui uma barreira física e protege da ação de agentes
antibacterianos.
5, 16, 23, 25
A composição química da EPS é específica de cada cepa. O polímero
polissacarídeo alginato, produzido por P. aeruginosa é o componente melhor
estudado de biofilme e parece ter um papel importante na estrutura do biofilme,
determinando a complexidade estrutural em forma de cogumelo.
11
Em biofimes
orais, as bactérias são capazes de sintetizar dextranos e levanos utilizando a
sacarose da alimentação como substrato.
30
V. cholerae produz EPS rica em
galactose e glicose. A composição da EPS excretada também pode variar conforme
varia a composição do meio.
Ainda não ficou estabelecido se os ácidos nucléicos e as proteínas
encontradas na EPS têm um papel estrutural ou somente são resíduos da lise
celular. O DNA extracelular parece ter importância na estrutura, pois quando os
biofilmes jovens (<60 h) são expostos a DNAse ocorre o rompimento de
microcolônias. A utilização de enzimas poderia ser útil na limpeza de superfícies pela
possibilidade de remoção ou desestabilização dos biofilmes devido à alteração das
propriedades da EPS.
11
Embora os polissacarídeos sejam os componentes mais estudados e
caracterizados da EPS, os dados disponíveis sugerem que a EPS produzida por
diferentes espécies microbianas em condições de crescimento diversas, apresenta
grande diversidade em relação à sua constituição. A dificuldade encontrada na
caracterização química da EPS se deve a problemas encontrados durante a
separação e extração da EPS de células bacterianas e à complexidade da análise de
polissacarídeos altamente ramificados e com uma grande variedade de
acoplamentos.
11
Crescimento, Desenvolvimento e Desprendimento do biofilme
Após a adesão irreversível, as células crescem e se multiplicam utilizando os
nutrientes presentes no filme condicionador e no fluído que as envolve. Durante este
período as células também produzem EPS, a qual auxilia na ancoragem das células
à superfície. Estudos atuais sugerem que EPS de alto peso molecular não age
diretamente como adesina. Outros fatores, possivelmente polissacarídeos de baixo
peso molecular, medeiam o processo de colonização inicial seguida da ação de EPS
de peso molecular mais alto.
11, 15, 20
O desenvolvimento do biofilme acontece através de vários mecanismos,
podendo ocorrer pela redistribuição de células aderidas na superfície e pela
motilidade dos microrganismos na própria superfície. Fatores ambientais, como troca
do substrato do meio, também alteram o desenvolvimento do biofilme. Applegate &
Bryers
2
observaram que quando havia baixa disponibilidade de oxigênio e carbono
ocorria mudança no biofilme e a troca de substrato fazia com as células se
desprendessem da superfície. O crescimento do biofilme resulta da divisão dos
microrganismos, ss células filhas se espalham formando um agrupamento de células
de maneira semelhante à formação de colônia em placas de ágar. Esse tipo de
crescimento foi monitorado microscopicamente e foi possível medir a expansão
radial de microcolônias de Mycobacterium fortuitum em uma superfície de silicone.
11
Em uma superfície com grande quantidade de bactérias densamente
condensadas, o crescimento pode ser prejudicado, com posterior morte celular,
devido à incapacidade de obtenção de nutrientes e ou pelo acúmulo de metabólitos
tóxicos liberados. Entretanto, isso não acontece, pois os biofilmes são estruturas
altamente organizadas. As células bacterianas produzem moléculas sinalizadoras,
também denominadas quorum-sensing, que permitem a comunicação entre elas, e
dessa forma possibilitando, que as bactérias cresçam e sobrevivam em ambientes
hostis.
Os processos de rompimento do biofilme acontecem a qualquer hora durante
o desenvolvimento, resultando na liberação e ressuspensão dos microorganismos do
biofilme para a fase planctônica (livre) do sistema
20, 25
e posterior disseminação do
biofilme.
Resistência aos sanitizantes
Biofilmes podem ser uma fonte de contaminação permanente dos alimentos,
uma vez que não podem ser removidos durante o procedimento de limpeza de
plantas de processamento de alimentos.
17
Vários estudos mostraram que microrganismos estabilizados em um biofilme
apresentam maior resistência ao tratamento por sanitizantes do que as células
planctônicas em suspensão.
1, 17, 19, 22, 25, 27, 31
Esta resistência tem sido atribuída a
diversas propriedades que afetam a atividade do sanitizante, incluindo temperatura,
pH, presença de matéria orgânica
25
, fatores relacionados com o microrganismo
como quantidade de nutrientes, fase de crescimento e sensibilidade ao sanitizante.
Além disso, fatores relacionados com o biofilme, que levam à diminuição da difusão
do sanitizante na matriz de EPS ou a presença de enzimas que degradam
substâncias antimicrobianas podem também afetar a resistência.
19
Algumas revisões que descrevem os mecanismos de proteção da EPS à ação
dos sanitizantes sugerem que esta ação não está relacionada somente com a
constituição química do agente antimicrobiano em questão. É importante também
outros fatores, como absorção do agente antimicrobiano pelos microrganismos
presentes na superfície do biofilme.
31
A eficácia dos sanitizantes na redução de bactérias aderidas depende do tipo
de microrganismo, da natureza do sanitizante e da superfície de adesão. O uso de
misturas de sanitizantes pode auxiliar no controle das bactérias aderidas.
25, 27
Gândara & Oliveira
9
estudaram os efeitos da higienização e remoção do
Streptococcus thermophilus do aço e encontraram que para melhorar a eficiência da
limpeza foram necessárias diferentes etapas de limpeza e tipos de detergentes. O
uso de detergentes e enxágüe com água a baixa pressão pode remover ou
desestabilizar o material extracelular que envolve a população aderida, reduzindo a
área coberta por EPS, mas pode não afetar o número de células viáveis.
10
Salmonella e L. monocytogenes quando aderidas são mais resistentes aos
sanitizantes em relação às células planctônicas.
17, 21, 27
Os agentes sanificantes de
uso comum precisam ser usados em concentrações 5 a 10 vezes maiores que as
utilizadas para a desinfecção de bactérias livres.
15, 18, 24, 31
Isso foi constatado por
Luppens et al.
22
que observaram a necessidade do uso de concentração 50 e 600
vezes maior de cloreto de benzalcônio e hipoclorito de sódio, respectivamente, para
a destruição de S. aureus em biofilme quando comparado com os microrganismos
em suspensão.
A maior resistência aos sanitizantes observada muitas vezes nas bactérias
Gram negativas é atribuída à membrana externa, que atua como uma barreira à
entrada de sanitizantes.
25
Como alguns biofilmes são compostos por diferentes
bactérias Gram positivas e Gram negativas, o estudo da composição do biofilme
pode facilitar a escolha de um produto efetivo ou indicar o uso de combinações de
detergentes.
10
Embora os microrganismos aderidos se mostrem mais resistentes que células
livres aos sanitizantes, não significa que o trabalho para reduzir a presença de
células livres não seja essencial. Além disso, a eficácia dos sanificantes é reduzida
pela presença de matéria orgânica, portanto é necessário uma limpeza adequada
para remoção de sujidades e células aderidas antes da aplicação do sanitizante.
Outro fator importante é testar anteriormente a eficácia dos sanitizantes sobre
microrganismos aderidos. Em alguns casos é necessária alteração no protocolo de
limpeza e sanitizacão, visando o controle da contaminação por biofilmes.
Conclusão
Biofilmes podem ser uma fonte constante de contaminação de alimentos, ter
um impacto negativo na qualidade dos produtos finais e causar corrosão dos
equipamentos. Por esta razão é importante que em um protocolo de limpeza e
desinfecção na indústria de alimentos a presença de biofilme seja sempre
considerada e a sua remoção realizada o mais cedo possível. Alguns autores
sugerem diferentes etapas e tipos de detergentes e sanitizantes, além de um
aumento do tempo destinado à fase de limpeza para uma eficaz remoção de células
aderidas. Esses cuidados e o monitoramento do ambiente de processamento de
alimentos são essencias para evitar os vários problemas associados à formação de
biofilmes.
Abstract
Microbial Biofilm and resistance to sanitizers: a review
Biofilms are structures highly organized in which microorganisms grow and therefore
can survive to hostile environments. They are defined as complex community of
microorganisms usually encased in an extracellular matrix of polymeric substances
(EPS) attached to a surface. Biofilms can be formed and be constant sources of
product contaminations if poor cleaning and disinfection practices do not remove
microorganisms from food processing surfaces. The cells embedded in biofilm are
more resistant to sanitizers than free cells. However, the sanitizers’ effectiveness
depend on the type of microorganism, the type of sanitizer and the surface
attachment. The understanding of biofilms formation is important for food quality and
safety. The aim of this study was to review biofilms formation and its resistance to
sanitizers.
Keywords: adhesion,EPS, sanitizer
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Adhesion of Salmonella Enteritidis to materials used in kitchen surfaces
KELLY OLIVEIRA
3
, TEREZA OLIVEIRA
1
, PILAR TEIXEIRA
2
, JOANA
AZEREDO
2
AND ROSÁRIO OLIVEIRA
2
*
1Universidade Estadual de Londrina, Centro de Ciências Agrárias, Departamento de
Tecnologia de Alimentos e Medicamentos, C.P. 6001, CEP:87051-970, Londrina, PR, Brasil.
2Centro de Engenharia Biológica - CEB, Universidade do Minho, Campus de Gualtar, 4710-
057 Braga, Portugal
3Centro Universitário de Maringá – CESUMAR, Maringá-PR, Brasil
* Author for correspondence:
e-mail: [email protected]
tel: 351 253604409
fax: 351 253678986
Key words: Adhesion;
Salmonella Enteritidis; kitchen surfaces; cross contamination
ABSTRACT
Contamination of kitchen surfaces due to bacteria present in foodstuff is one of the main
causes of foodborne outbreaks. Pathogenic bacteria as
Salmonella species can be transferred
from contaminated food to kitchen surfaces and from there to non contaminated food. This
phenomenon is commonly assigned as cross contamination. Salmonella infections are an
important cause of foodborne disease and
Salmonella Enteritidis is the most commonly isolate
in the last years.
This study attempted to investigate the factors that are involved in the adhesion of four
Salmonella Enteritidis isolates to different materials (polyethylene, polypropylene and granite)
used as cover surfaces or as other utensils in kitchens. Surface hydrophobicity and roughness
were determined in order to assess the differences in the extent of adhesion. The main
conclusion to be drawn is that
Salmonella sp. adhesion is strongly strain dependent, despite
the similar degree of hydrophobicity displayed by all the strains assayed.
INTRODUCTION
Considerable research interest has been focused on the ability of bacteria to adhere to solid
surfaces, with subsequent formation of stable biofilms, and its relevance to environmental,
medical, dental and industrial applications has been increasingly studied (
7). The attachment
of microorganisms and subsequent development of biofilms could be a continuous source of
contamination to foods coming in contact with them when formed on contact surfaces. During
preparation of naturally contaminated food, potential pathogens are frequently spread to hands
and food processing surfaces. Those cells adhered to surfaces of domestic kitchen are not
usually removed by the normal cleaning procedure and therefore could be a source of
contamination of other foods coming in contact with such surfaces and objects.
Salmonella sp.
are important pathogenic bacteria which are of considerable significance to the food
processing industry (
3,17,12,18). Salmonella infections are an important cause of food borne
bacterial disease (
19). In many countries Salmonella Enteritidis has been the most commonly
isolate in food borne diseases in the last years (1, 20, 9).
Several studies have shown that many sites in the kitchen become contaminated when
food harboring bacteria is prepared and this may be an important source of
Salmonella
infections in the home (31, 5, 13). The occurrence of Salmonella sp. in chicken carcasses can
change between 0.024 to 85.0%, which demonstrates that chicken carcasses are a strong
potential source of bacterial contamination of utensils and kitchen surfaces.
Materials that retain fewer microorganisms after cleaning would be the hygienic choice
and present the minor risk of cross contamination. The wear of surfaces will affect their finish
and potentially their hygienic status (
27). Ceramics are commonly used as hygienic and
impervious floor coverings. Chopping boards are more prone to cross-contamination,
particularly from juices of raw meat and poultry remaining on the surface resulting in transfer
of microorganisms to other foods subsequently prepared on the same surface (10). In recent
years there has been a steady rise in the use of plastic materials in food industry (
15) and
many studies have been conducted to evaluate biofilm formation by Salmonella spp. on plastic
surfaces (6, 17, 12, 19).
Biofilms have the potential to act as a chronic source of microbial contamination, which
may compromise food quality and represent a significant health hazard. To control these
problems, it has been recognized that a greater understanding of the interaction between
microorganisms and food-processing surfaces is required (
2). The factors involved in bacterial
colonization of inert support materials in kitchen remain poorly understood. Knowledge of the
mechanisms governing adhesion and formation of the first layer on such supports is, however,
of particular relevance to the prevention of food borne disease. Under these considerations, the
aim of this work was to study the factors that are involved in the adhesion of
Salmonella
Enteritidis to different materials (polyethylene, polypropylene and granite) used as cover
surfaces or as other utensils in kitchens.
MATERIALS AND METHODS
Bacterial strains and growth
Four strains of
Salmonella Enteritidis were selected for this study: two Salmonella
Enteritidis (Salmonella Enteritidis EMB and Salmonella Enteritidis MUSC), were previously
isolated from poultry,
Salmonella EMB were isolated from water of packaged chicken and
Salmonella Enteritidis MUSC from chicken breast. The other two Salmonella (Salmonella
Enteritidis
PC and Salmonella Enteritidis AL) were isolated from patients with food borne
disease outbreaks.
All bacterial isolates were maintained in trypticase soy agar (TSA). Every strain was
subcultured twice in trypticase soy broth (TSB) at 37°C in an orbital shaker (130 rpm),
overnight. The cells were then harvested by centrifugation at 5000 g for 10 min and washed
three times with phosphate buffer saline (PBS 0,1M pH 7). The pellets were resuspended in
PBS to an inoculum level of 10
8
CFU/ml, determined by optical density.
Materials
The test surfaces were: polyethylene utilized in cutting boards, polypropylene from a basin
and granite (Pedras Salgadas, Portugal) commonly utilized as bench cover in domestic
kitchens. For the adhesion experiments, the materials were cut in coupons of 0.8 cm x 0.8 cm
in the case of polypropylene and 1.8 cm x 1.8 cm for polyethylene and granite. For contact
angle measurements, materials were cut in slides of 7.0 cm x 2.5 cm, washed in a solution of a
commercial detergent (Sonasol Pril, Henkel Ibérica S.A., Portugal) in ultrapure water for 30
min and then thoroughly rinsed in ultrapure water (to remove any remaining detergent),
followed by immersion in ethanol 90 % over 30 min to completely degrease the surface.
Hydrophobicity and free energy of adhesion
Hydrophobicity was evaluated through contact angle measurements and using the
approach of van Oss and co-workers (
22,26,25). In this approach, the degree of
hydrophobicity of a given material (1) is expressed as the free energy of interaction between
two entities of that material when immersed in water (w) -
1w1
G∆ . If the interaction between
the two entities is stronger than the interaction of each entity with water the material is
considered hydrophobic (
1w1
G∆< 0), conversely, for a hydrophilic material
1w1
G∆> 0.
1w1
G∆
can be calculated through the surface tension components of the interacting entities.
The surface tension is considered to have two components, one apolar due to Lifshitz-van der
Waals interactions (
LW
γ
) and one polar due to Lewis acid-base interactions (
AB
γ
). The latter
comprises two parameters:
+
γ
and
-
γ
the electron acceptor and electron donor, respectively.
The surface tension components of a solid material are obtained by measuring the contact
angles of three pure liquids (one apolar and two polar), with well known surface tension
components (24).
Contact angle measurement
Contact angle measurements (at least 25 determinations for each liquid and for each
material and microorganism) were performed automatically with the aid of an image analysis
system (G2/G40) installed in a standard contact angle apparatus (Kruss-GmbH). The images
were transmitted by a video camera to a 486 DX4 100 MHz personal computer for evaluation.
All the measurements were performed at room temperature. In the case of bacterial cells the
measurements were performed on a cell lawn using the sessile drop method described by
Busscher et al. (4). Briefly, bacteria were deposited on a cellulose acetate membrane filter
(pore diameter of 0.45 µm) by filtration of the suspension using negative pressure. To
standardize the moisture content, the filters were then transferred onto agar in Petri dishes
containing 1% (w/v) agar with 10% (v/v) glycerol. Measurements of water contact angles
were carried out at 25°C and three liquids with different polarities were used, water (W),
formamide (F) and α-bromonaphthalene (α-B)
Contact angle measurements on the materials was determined by the same technique, with
direct measurements of contact angles on polyethylene, polypropylene and granite surfaces,
after degreasing and cleaning.
Adhesion assays
The coupons of the materials were immersed in 2 ml of each bacterial suspension
containing 10
8
CFU/ml. After 1 h at 37
o
C with constant shaking at 100 rpm, the coupons were
rinsed twice with PBS to remove poorly adhered bacteria. An aliquot of 20µl/ml of a 4´,6-
diamidino-2-phenylindole (DAPI)
solution was added to each well containing the plates and
incubated for 30 min in the dark. After this time, the wells were rinsed with sterile distilled
water and the adherent microorganisms were quantified by automatic enumeration using
epifluorescence microscopy. Thirty fields per coupon were scanned and the fluorescent cells
were enumerated. Computerized image analysis software (Image-Pro Plus, Media
Cybernetics) was used for the quantitative estimation of the adherent cells. All experiments
were done in triplicate.
Roughness
The surface roughness of the materials studied was evaluated by a non-contact laser stylus
tracing (Perthometer S4P – Perthen GmbH, Gottingen, Germany), to determine the R
a
and R
z
,
values. The R
a
value provides the arithmetical average value of all departures from the mean
line throughout the sampling length, the R
z
value is the sum of the height of the highest peak
plus the lowest valley depth within a sampling length. The default evaluation length consists
of five sample lengths.
Statistical analysis
The resulting data were analyzed using SPSS software (Statistical Package for the Social
Sciences). One-way ANOVA with Bonferroni test was used to compare the number of
adhered cells. All tests were performed with a confidence level of 95%.
RESULTS AND DISCUSSION
Figure 1 presents the number of cells of Salmonella Enteritidis (EMB, MUSC, PC, AL)
adhered to the materials tested. Statically, the extent of adhesion of the different strains to the
materials assayed was different (p<0.05). Salmonella EMB adhered in greater extent to granite
while Salmonella MUSC adhered in greater extent to polypropylene and in a less extent to
granite. The extent of adhesion of Salmonella AL was almost the same to all the materials
tested and Salmonella PC adhered in a greater extent to granite, followed by polypropylene
and, at last, by polyethylene. The source of Salmonella spp. isolates does not seem to affect
the ability of adhesion. Stepanovic et al. (18) also refer that the source of Salmonella spp.
isolates (from humans, animals or foods) did not affect biofilm formation.
Several studies report different extents of adhesion of Salmonella spp. and, generally, it
was found that Salmonella species adhere in greater extent to the more hydrophobic materials
(17, 8). Joseph and co-authors (12) studied the ability of biofilm formation of two poultry
Salmonella isolates to plastic, cement and stainless steel and observed that biofilm formation
of both isolates were very similar with the highest density being on plastic followed by cement
and stainless steel. The ongoing epidemic of Enteritidis may be related, in part, to the success
of the bacterium in passing down the food chain with adherence to inanimate surfaces
contributing to persistence as well as communicability (30). Enteritidis strains may adhere to
surfaces such as eggs, food-processing equipment, animal carcasses and farm-yard
implements over a wider range of environmental conditions (30). Of the many serotypes of S.
enterica, Enteritidis is unique in possessing the ability to elaborate SEF14 and SEF17
fimbriae, both of which contribute to adherence although under different environmental
conditions. Stepanovic et al (18) demonstrated that both Salmonella spp. and Listeria
monocytogens have higher ability for biofilm formation on plastic surfaces and, generally,
Salmonella spp. produces more biofilm in nutrient-poor medium. This fact aggravates the
phenomenon of cross contamination in food manipulation.
0
2000
4000
6000
8000
10000
12000
14000
16000
Salmonella MUS C Salmonella EMB Salmo nella AL Salmonella P C
Cells/mm
2
Granite
Polyethylene
Polypropylene
Figure 1 – Number of adhered cells/mm
2
of Salmonella Enteritidis strains to the different
materials studied.
It is well known that bacterial surface hydrophobicity, surface charge, cell density and the
presence of exopolysaccharides are determinant factors in the adhesion process. For example,
Sinde and Carballo (17) referred that differences found in the degree of attachment of
Salmonella and L. monocytogenes indicate that there must be other factors on the surface of
the bacteria, rather than hydrophobicity, contributing to bacteria attachment to food contact
surfaces. In the other hand, Walker et al. (29) studied the effect of pH, temperature and
contact surface on the elaboration of fimbriae (SEF 21, SEF 14 e SEF 17) and flagella and
found differences among the four strains assayed. Hood and Zottola (11) observed that both
growth media and surface conditioning were significant factors affecting the level of
adherence. Surface hydrophobicity and roughness were determined in order to explain the
differences in the extent of adhesion.
The water contact angles on bacterial lawns as well as surface tension components and
hydrophobicity of the strains studied are presented in Table 1. The water contact angle value
gives preliminary information about the degree of surface hydrophobicity. The sample is
considered hydrophobic or hydrophilic if the angle is higher or lower than 65
o
, respectively
(28). According to this criterion, all Salmonella strains assayed are hydrophilic, with values of
water contact angles ranging from 9.7
o
to 14.0º and being somewhat lower than those reported
by Sinde and Carballo (17) (25.4 – 35.0) for other Salmonella strains. The different serovars
studied can explain this fact (17). Teixeira et al. (21) also observed a great variation of
hydrophobicity among strains of the same bacterial species. The changeable complexity of the
cellular surface results in hydrophobic or hydrophilic appendices and other macromolecular
components that can confer different behaviours according to the method of evaluation. In
practice, the non-uniformity of bacterial surface can result in an apparently hydrophilic
bacterium in an assay and hydrophobic in another one (7). Affinity techniques, like microbial
adhesion to hydrocarbons - MATH (16) - are more prone to variability and by such techniques
hydrophobicity is only assessed qualitatively (14). Using the approach of van Oss (23), it is
possible to determine the absolute degree of hydrophobicity of any substance (1) vis-à-vis
water (w), which can be precisely expressed in applicable S.I. units. Accordingly, all
Salmonella strains studied were similarly hydrophilic (Table 1), which is in consonance with
the classification obtained through the water contact angles.
Table 1 - Values of water contact angles (in degrees), values of the components of surface
tension (
γ
LW
, γ+, γ-, γ
AB
) and degree of hydrophobicity (∆G
1w1
) of bacterial cells (in mJ/m
2
).
Strain
W
ater contact angle (±SD) γ
LW
γ+ γ- γ
AB
∆G
1w1
Salmonella MUSC 13.5 (1.6) 26.00 4,60 51.30 30.99 31.70
Salmonella EMB
10.9 (2.23) 39.89 0.97 55.99 1.94 34.12
Salmonella
AL
9.7 (1.95) 39.50 1.05 55.84 15.32 33.79
Salmonella PC
14.0 (4.42) 38.06 1.22 54.48 16.31 32.28
The values of the contact angles (in degrees) as well as the values of the surface tension
components and the degree of hydrophobicity (
1w1
G
∆
) of the materials assayed are presented
in Table 2. Water contact angles of the materials were statistically different (P<0.05) among
them. According to the results, all the materials are hydrophobic (∆G
sws
<0). The polymers
present values of the free energy of self-interaction in water very similar (-52.12 mJ/m
2
and -
49.96 mJ/m
2
, respectively), while granite presents a lower value (-4.7 mJ/m
2
), displaying a
less hydrophobic character. Considering the surface tension parameters, granite is a surface
predominantly electron donor (higher values of
−
γ
), with a low electron acceptor parameter
(
+
γ
). Its
−
γ
is much higher than the
−
γ
of the other surfaces in study. Probably this fact is
due to the polar groups formed by O and N, which are electron donors, while polymer
surfaces are formed only by carbon and hydrogen atoms without polar groups as can be
observed by the γ
AB
parameter that corresponds to the polar component. However, in the
present situation it is not possible to hypothesize about a specific role of Lewis acid-base
interactions in the adhesion process. At least it is not possible to establish any correlation
between the electron donor and electron acceptor capabilities of the interacting surfaces.
Table 2 - Water contact angle (in degrees), values of the surface tension components (γ
LW
, γ+,
γ-, γ
AB
) and degree of hydrophobicity (∆G
1w1
) of the materials assayed (in mJ/m
2
).
Surface
Water contact angle (±SD) γ
LW
γ+ γ- γ
AB
∆G
1w1
Granite
53.4 (±3.6)
41.12 0.28 26.26 5.4 -4.72
Polyethylene
74.3 (±8.3)
39.89 0.50 5.56 3.35 -49.96
Polypropylene
87.8 (
±
3.4)
41.93 0.06 7.76 1.42 -52.12
The values of surface roughness (Ra and Rz) are shown in Table 3. Polyethylene was the
roughest material (with a higher value of Ra - longitudinal 36.02 µm and transversal 30.91 µm
- and of Rz -longitudinal 195.96 µm and transversal 145.3 µm), but it was the material
displaying less extent of bacterial colonization. Flint et al. (8) referred that the adhesion of
thermo-resistant Streptococci to stainless steel with surface roughness (Ra) values ranging
between 0.5 and 3.3
µ
m was largely independent of the substrate topography although
bacterial entrapment may occur at Ra values of 0.9 µm. Barnes et al. (
2) compared the
adhesion of
Staphylococcus aureus to polished stainless steel and to rougher stainless steel
and they observed a greater number of
S. aureus adhered to the rougher surface. According to
the same authors, scanning electron microscopy micrographs showed that organisms did not
orient themselves exclusively along polishing lines. As a matter of fact, it has been widely
suggested that surface roughness may play an important role in the adhesion of
microorganisms by protecting them from shear forces and increasing the available surface
area. However, for a microbial cell to be entrapped due to surface roughness it is necessary to
have enough space available between two consecutive peaks of surface topography for the cell
to sit there. It has to be noticed that the same value of Ra can correspond to different surface
topographies. Actually, Ra measures the average height and depth of peaks and valleys but not
the distance between them. As adhesion is dependent on the number of contact points between
the interacting surfaces, it might be the distance between peaks that also determines the “peak
density” (i.e. low
vvvvv
or high vvvvvvv), which is responsible for the extent of contact
between the microbial cell and the surface. This means that a higher number of peaks close
together will promote more contact points between the surface and the cell sitting on it. Under
this reasoning, the most common parameter used to express surface roughness is not the most
appropriate to assess the effect of roughness on microbial adhesion.
Table 3 - Roughness of the surfaces studied (µm).
Ra Rz
Surface
Longitudinal Transversal Longitudinal Transversal
Granite 32.38 24.88 155.16 114.36
Polyethylene
36.02 30.91 195.96 145.3
Polypropylene
6.16 0.19 39.6 4.84
As the adhesion process is “multi-factorial” involving several physico-chemical and
microbiological factors, for a better understanding, it would be necessary to investigate the
role of cell wall proteins as well as fimbriae and flagella. Furthermore, other structures such as
pili, polysaccharides, capsules or "
slime layers" have also been related to the adhesion
process.
Considering all the tentative explanations based on physico-chemical properties of
bacterial cells and surfaces, it is not possible to establish any direct correlation to elicit the
hypothesis of a reasonable model of adhesion. The main conclusion to be drawn is that
Salmonella spp. adhesion is strongly strain dependent, despite the similar degree of
hydrophobicity displayed by all the strains assayed.
ACKNOWLEDGEMENTS
Kelly Oliveira fully acknowledges CAPES/ Brazil for the grant BEX 0891/01-0.
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