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Braz. J. Microbiol. vol.38 no.4 São Paulo Oct./Dec. 2007
Listeria spp. associated to different levels of autochthonous microbiota in meat, meat products and processing plants
Listeria spp. associado a diferentes níveis da microbiota autóctone de carne, produtos cárneos e plantas de processamento
Márcia de Aguiar Ferreira BarrosI,II* ; Luís Augusto NeroIII; Aline Villas-Bôas ManoelII; Loredana d'OvídioII; Lívia Cavaletti da SilvaII; Bernadette Dora Gombossy de Melo FrancoIV; Vanerli BelotiII
IFaculdade de Agronomia e Medicina Veterinária, Universidade de Brasília, Brasília, DF, Brasil
IIDepartamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, Londrina, PR, Brasil
IIIDepartamento de Veterinária, Universidade Federal de Viçosa, Viçosa, MG, Brasil
IVDepartamento de Alimentos e Nutrição Experimental, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil
High levels of microbial contamination, commonly found in animal origin foods and food processing environments, are able to hinder the growth of pathogens in these products and interfere in the results of laboratory analyses for detection of these pathogens. With the aim of verifying the possible interference of the autochthonous microbiota encountered in meat and meat products and processing plants over the presence of Listeria spp., 443 samples, collected from 11 meat retail establishments, were submitted to microbiological analysis to determine the levels of mesophilic aerobes, total coliforms and Escherichia coli and the presence of Listeria spp., according to the methodology proposed by the USDA. The results did not show evident interference of the autochthonous microbiota over Listeria spp., once the genus was detected even in the meat, meat products and environmental samples with high levels of contamination by mesophilic aerobes and coliforms.
Key-words: meat; Listeria spp., autochthonous microbiota
Altos níveis de contaminação microbiana, usualmente encontrados em alimentos de origem animal e nos ambientes de processamento, podem inibir a multiplicação de microrganismos patogênicos nesses produtos e interferir nos resultados das análises laboratoriais para o isolamento desses patógenos. Com o objetivo de verificar as possíveis interferências da microbiota autóctone encontrada na carne, produtos cárneos e plantas de processamento sobre a presença de Listeria spp., 443 amostras, coletadas em 11 estabelecimentos processadores, foram submetidas a análises microbiológicas para determinação dos níveis de contaminação por aeróbios mesófilos, coliformes totais e Escherichia coli e para verificação da presença de Listeria spp., de acordo com a metodologia proposta pelo USDA. Os resultados obtidos não mostraram uma interferência evidente da microbiota autóctone sobre Listeria spp., uma vez que esse gênero foi detectado mesmo nas amostras de carne e produtos cárneos e amostras ambientais e de superfície de equipamentos que apresentaram altos níveis de contaminação por aeróbios mesófilos e coliformes.
Palavras-chave: carne, Listeria spp.; microbiota autóctone
Listeria monocytogenes emergence as a foodborne pathogen dates from 1980, with the occurrence of many outbreaks and sporadic cases of listeriosis associated with the consumption of contaminated foods (3,7,20,28). The invasive form of this pathogen is considered too risky especially to newborns, elderly and immune-compromised individuals, who may present septicemia, encephalitis, meningitis and abortion, and high levels of mortality (11,12,29). Among Listeria species, only L. monocytogenes is recognized as a human pathogen (7). Most foods are tested for the presence of non-pathogenic Listeria spp only, as an indication of the possible presence of L. monocytogenes (5,6,26,30).
The occurrence of Listeria spp. and L. monocytogenes in foods is highly variable worldwide (7,9,24). In many regions the reported occurrence is low, especially in foods with high levels of microbial contaminants (15,16,24). According to Jay (15,16), levels of contaminants higher than 106 CFU/g create unsatisfactory conditions for the growth of pathogens in foods. The dynamic of the interference depends on the presence of specific antagonistic microorganisms and also on the ability of producing inhibitory substances.
In meat and meat products, the authochtonous microbiota, which includes the natural microbiota of beef and the contaminants determined by the hygienic procedures during slaughter and processing (1,10,13,16,23,33), may include strains capable of producing a variety of antimicrobial compounds that may impair the survival or inhibit the growth of L. monocytogenes (2,4,18,22). Moreover, the contaminants can also interfere on the effectiveness of the laboratory isolation and detection methods, leading to erroneous results about the presence of L. monocytogenes in the product (31).
Several methodologies are available for isolation of Listeria spp. in foods. All methods employ enrichment phases at different temperatures using different supplements, selective agents and incubation conditions (6,19,27,31). Selective enrichment steps are necessary to inhibit the growth of the competitive microbiota and optimize the multiplication of Listeria spp., leading to their amplification to required level for proper detection. As an example, the University of Vermont Listeria Enrichment broth (UVM), recommended by the United Stated Department of Agriculture (32), is an enrichment medium which contains nalidixic acid and acriflavine as supplements to inhibit Gram negative and Gram positive microorganisms, respectively. This culture medium is considered the most suitable for testing foods where high levels of competitive microbiota are expected (25,30).
In beef, despite the occurrence of antagonistic microorganisms in the autochthonous microbiota (2,4,16), there is no evidence of the direct interference of these microbiota on Listeria spp. isolation procedures (6,21). The aim of this study was to verify the influence of the autochthonous microbiota in meat and meat products and in the meat processing environment on the detection of Listeria spp., using the detection protocol recommended by USDA (32).
MATERIAL AND METHODS
Samples and dilutions
Eleven meat retail establishments (one slaughterhouse and ten butcher shops) located in Londrina region, north of Paraná State, Brazil, inspected by Brazilian Agriculture (the slaughterhouse) or Health (butcher shops) Authorities, were randomly selected. In these establishments, a total of 443 samples were taken (Table 1), comprising 148 surface samples collected from the equipments (knives, mincers, boxes, etc), 65 samples collected from the environment (drains, floors, etc) and 230 meat and meat product samples. Surface samples were obtained with sterile swabs and sterile templates to limit the specific area shown in Table 1. Swabs were transferred to flasks containing 45 mL of 0.1% buffered peptone water (Oxoid, Basingstoke, UK). Meat samples were collected in sterile bags. The samples were kept under refrigeration during the transport to the laboratory. For testing, 10 g of the collected samples were weighted and transferred to 90 mL of 0.1% buffered peptone water (Oxoid, Basingstoke, UK), and homogenized with a stomacher. The homogenates were submitted to serial decimal dilutions using sterile NaCl 0.85%.
The samples were tested for the presence of Listeria spp. according to the protocol proposed by USDA (32). In the pre-enrichment step, 5.0 mL of the peptone water containing the swab or 5.0 g of product were transferred to 45.0 mL of University of Vermont Listeria Enrichment broth (UVM) (Oxoid, Basingstoke, UK), and incubated at 30ºC for 24h. One mL of the UVM broth was transferred to tubes containing 9 mL of Fraser broth (Oxoid, Basingstoke, UK), and incubated at 35ºC for 24h. Samples that presented darkening were streaked on Modified Oxford agar (BD Diagnostic Systems, Franklin Lakes, NJ, USA) and incubated at 35ºC for 24h. When Listeria typical colonies were present (small, grey to black, surrounded by blackening halo), they were submitted to identification by API-Listeria (bioMérieux S.A., Marcy l'Etoile, France). The final result was expressed as presence or absence of Listeria spp. in the sampled food or area (Table 1).
The autochthonous microbiota was determined by enumerating the following groups of microorganisms: mesophilic aerobes (MA), total coliforms (TC) and Escherichia coli (EC). From each sample, two decimal dilutions were selected and plated in Petrifilm plates (Petrifilm AC for MA, and Petrifilm EC for TC and EC) and incubated at 35ºC for 24-48h. After incubation, the colonies formed in the plated area were enumerated and expressed as Colony Forming Units per gram or cm2 (CFU/g or cm2).
For data analysis, the counts of MA, TC and EC were converted in log10. The numbers of Listeria spp positive samples according to the source (equipments, environments or products) and mean counts of MA, TC or EC were compared by Tukey Honest Significant Difference for unequal n, at a level of significance of 0.05. The frequencies of positive and negative results for Listeria spp. were also calculated considering three levels of contamination by MA, TC and EC. All analyses were processed using the software Statistica 6.0 (StatSoft, Inc., Tulsa, OK, USA).
RESULTS AND DISCUSSION
One hundred and sixty seven samples were positive for Listeria spp: 76 samples from equipments, 23 from environments and 68 from products. The prevalence of species was as follows: L. innocua (131), L. monocytogenes (21), L. welshimeri (12), L. seeligeri (02) and L. grayi (01). Fig. 1 shows the positive and negative results for Listeria spp in the samples, the mean counts of MA, TC and EC, and the significance of the correlations between presence of Listeria spp and the counts of MA, TC and EC. The frequencies of positive or negative samples for Listeria spp. according to the levels of contamination by MA, TC and EC are shown in Figs. 2, 3 and 4, respectively.
In general, samples with positive results for Listeria spp. presented higher mean counts of indicators microorganisms than the Listeria spp. negative samples. In equipments, 44.7% of the Listeria spp. positive samples presented MA counts over 5 log CFU/cm2, while in the environment Listeria spp. positive samples, this percentage was 73.9% and in products, 41.2%. In the average, 47.3% of the Listeria spp. positive samples presented MA counts higher than 5 log CFU/cm2, while for Listeria spp. negative samples this percentage was 24.3% (Fig. 2). The same situation was observed for total coliform counts (Fig. 3) and for E. coli (Fig. 4). All these differences were significant (P < 0.05). However, it must be noted that for E.coli counts, the majority of results were lower than the limit of the detection method (2 log CFU per gram or cm2), so the significance of the differences between Listeria spp. positive and negative results could not be calculated properly.
These results suggest that the autochthonous microbiota in meat products and meat processing environment did not affect the survival or growth of Listeria spp. in them. The possible interferences of the autochthonous food microbiota over Listeria spp. and L. monocytogenes are widely variable due to several factors, including microbiological composition, preserving temperatures and inhibitory substances production. A common microorganism present in meat and meat processing plants, Pseudomonas fluorescens, was already described as improving (22), inhibiting (4) and causing no effect (8) on L. monocytogenes growth. Those distinct behaviors of food accompanying microbiota were also observed by Buchanan and Bagi (2).
According to Jay (15,16), fresh meats with MA counts between 104 and 106 CFU/g are probably not vehicles of foodborne pathogens, due the autochthonous presence of inhibitory microorganisms. However, the results of the present study indicate that even at these levels the isolation of Listeria spp., including the pathogenic specie L. monocytogenes, is possible.
The utilization of UVM broth in the enrichment step may explain the absence of interference of the autochthonous microbiota of the meat products on the isolation of Listeria spp. This culture media is known as the most suitable to isolate Listeria spp. in foods with high levels of microbiological contamination, due to its effective inhibitory activity by the added supplements (19,30). Some authors even suggest that UVM promotes a better recovery of L. innocua than L. monocytogenes (5,26), especially due to a higher growth velocity of the first (21). However, Duffy et al. (6) demonstrated that this culture media did not promote significant differences in the recovery of both Listeria species.
In food processing surfaces and utensils, a contamination level of 104 CFU/cm2 is considered enough to begin biofilm formation (13). In this study, several samples presented counts higher than 104 CFU/cm2, suggesting the possibility of biofilm formation on equipment surfaces, and even in products. The importance of these findings is relevant because these contamination levels were found in several Listeria spp. positive samples. The literature reports studies indicating that L. monocytogenes is able to grow and some autochthonous strains, such as Flavobacterium spp., can even improve its development (17). Listeria spp. are able to produce glycocalyx in stainless steel surfaces within 48 h, promoting self protection against environmental microorganisms that can produce inhibitory substances (14,33).
In conclusion, the results of the present study suggest that the autochthonous microbiota of meat and meat processing plants have very little, if any, influence on the isolation of Listeria spp., even when the counts of hygiene indicator microorganisms surpass 5 log CFU/cm2 or g. The official methodology for the isolation of Listeria spp from meat and equipment surfaces was shown to be appropriate, with no interference of the autochthonous microbiota.
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Submitted: September 15, 2006; Returned to authors for corrections: July 04, 2007; Approved: September 20, 2007.
* Corresponding Author. Mailing address: Faculdade de Agronomia e Medicina Veterinária, Universidade de Brasília, Campus Darcy Ribeiro, Caixa Postal 4508, 70910 970, Brasília, DF, Brasil. Tel.: +55 61 3307 2803, ramal 20. Fax: +55 61 3273 6593. E-mail: firstname.lastname@example.org