Formación de biopelículas por Listeria monocytogenes aisladas de queso fresco de mercados del Cercado de Lima

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Abstract

Introducción. Listeria monocytogenes, bacteria de importancia en salud pública por causar la listeriosis y sus graves consecuencias, se asocia al consumo de alimentos que durante su producción se pueden contaminar con bacterias que se liberan de biopelículas formadas en tuberías, superficies y equipos. Objetivo. Analizar muestras de queso fresco con el fin de aislar e identificar L. monocytogenes y evaluar su capacidad formadora de biopelículas en dos medios de cultivo. Diseño. Estudio experimental, prospectivo. Institución. Instituto de Investigación en Química Biológica, Microbiología y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad Nacional Mayor de San Marcos, Lima, Perú. Material. 75 muestras de queso fresco provenientes de diez mercados del Cercado de Lima. Métodos. En el análisis microbiológico se empleó metodologías del Manual de Bacteriología Analítica de la Food and Drug Administration (FDA). Para determinar la capacidad formadora de biopelículas se aplicó el método de microplaca descrito por Djordjevic modificado por Borucki. Se usó la prueba de Kruskal-Wallis. Principales medidas de resultados. Identificación de L. monocytogenes. Resultados. Se identificó L. monocytogenes en 14 muestras (18,7%), valor que representó un riesgo potencial para la salud de los consumidores. Se halló nueve cepas con capacidad de formar biopelículas que, según la densidad óptica a 595 nm, fueron clasificadas en formadoras débiles y moderadas, con diferencias significativas dependiendo del medio de cultivo utilizado, siendo el caldo infusión cerebro corazón (BHI) el más efectivo para esta bacteria. Conclusiones. Se identificó L. monocytogenes en 18,7% (14/75) de las muestras de quesos frescos adquiridas en 10 mercados del Cercado de Lima. De estas cepas, 64,3% (9/14) tuvieron capacidad formadora de biopelículas.

Translated abstract

Introduction: L. monocytogenes is a bacterium of importance in public health because it causes listeriosis, which has serious consequences. It is associated with the consumption of foods that can be contaminated during its production with bacteria released from pipes, surfaces and equipment biofilms. Objectives: To analyze cheese samples in order to isolate and identify L. monocytogenes and evaluate its ability for biofilm formation in two culture media. Design. Experimental, prospective study. Setting: Instituto de Investigación en Química Biológica, Microbiología y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad Nacional Mayor de San Marcos, Lima, Peru. Material: 75 samples of fresh cheese from ten markets of Cercado de Lima. Methods: The microbiological analysis used methods described in the Bacterial Analytical Manual of the Food and Drug Administration. The microplate method described by Djordjevic modified by Borucki was used to determine the ability of biofilm formation. The Kruskal-Wallis test was applied. Main outcome measures: Identification of L. monocytogenes. Results: L. monocytogenes was identified in 14 samples (18.7%), a value representing potential risk to the consumers' health. Nine biofilm-forming strains were found and classified according to their optical density at 595 nm in weak and moderate formers, with significant differences depending on the culture medium used. The brain heart infusion (BHI) was the most effective one. Conclusions: L. monocytogenes was identified in 18.7% (14/75) of samples of fresh cheese obtained in ten of Cercado de Lima's markets. Of these strains, 64.3% (9/14) were biofilm formers.

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Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation.

D Djordjevic, M Wiedmann, L A McLandsborough (2002)

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32 degrees C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.

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Variation in biofilm formation among strains of Listeria monocytogenes.

Frank Loge, D Call, Jason Peppin … (2003)

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.

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What is the incubation period for listeriosis?

Véronique Goulet, Lisa A King, Véronique Vaillant … (2013)

Background Listeriosis is a foodborne infection with a low incidence but a high case fatality rate. Unlike common foodborne diseases, the incubation period can be long. The first incubation periods were documented during a large listeriosis outbreak published in 1987 by Linnan and al. in the New England Journal of Medicine (range: 3 days to 70 days). Data on the incubation period of listeriosis are scarce. Our study aim was to estimate precisely the incubation period of listeriosis using available data since 1987. Methods We estimated the incubation period of listeriosis using available published data and data from outbreak investigations carried out by the French National Institute for Public Health Surveillance. We selected cases with an incubation period calculated when a patient had a single exposure to a confirmed food source contaminated by Listeria monocytogenes. Results We identified 37 cases of invasive listeriosis (10 cases with central nervous system involvement (CNS cases), 15 bacteraemia cases and 12 pregnancy-associated cases) and 9 outbreaks with gastroenteritis. The overall median incubation period of invasive listeriosis was 8 days (range: 1–67 days) and differed significantly by clinical form of the disease (p<0.0001). A longer incubation period was observed for pregnancy-associated cases (median: 27.5 days; range: 17–67 days) than for CNS cases (median: 9 days; range: 1–14 days) and for bacteraemia cases (median: 2 days; range: 1–12 days). For gastroenteritis cases, the median incubation period was 24 hours with variation from 6 to 240 hours. Conclusions This information has implications for the investigation of food borne listeriosis outbreaks as the incubation period is used to determine the time period for which a food history is collected. We believe that, for listeriosis outbreaks, adapting the exposure window for documenting patients’ food histories in accordance with the clinical form of infection will facilitate the identification of food products as the source of contamination. We therefore propose to take an exposure window of 14 days before the diagnosis for CNS and bacteraemia cases, and of 6 weeks before the diagnosis, for pregnancy-associated cases.