The magnitude of cell invasion and cell-to-cell spread of Listeria monocytogenes is correlated with serotype-specific traits.

Listeriosis is a foodborne disease caused by the Gram-positive bacterium Listeria monocytogenes, a pathogen that modulates its intracellular survival via vacuolar escape and cytosolic replication. In the present study, we examined the ability of 58 L. monocytogenes isolates recovered in Brazil (beef, clinical and environmental samples, from 1978 to 2013) to invade, replicate and spread in a human intestinal epithelial cell line (Caco-2). Premature stop codons were common in the inlA gene of serotype 1/2c strains from beef and environment samples, associated with decreased Caco-2 cell invasion when compared to other serotypes. The isolates varied widely in their intracellular doubling times, and there was no clear relationship between serotypes and samples origin. Serotype 1/2a isolates were generally impaired in their ability to spread between Caco-2 cells, with an average 30 % smaller focus area than the 10403S reference strain. However, most isolates of serotype 1/2b exhibited enhanced cell-to-cell spread, with an average 35 % increase in focus area. Our findings are consistent with serotype being a better predictors of cell invasion potential and cell spread compared with sample origin of isolates, although the most invasive isolates were primarily isolated from beef. Additionally, we have identified isolates that could provide novel insight into the pathogenicity of L. monocytogenes that may not be revealed by studying common laboratory reference strains.

Genetic Profiles and Invasion Ability of Listeria monocytogenes Isolated from Bovine Carcasses in Southern Brazil.

ABSTRACT: The goals of this study were to evaluate the persistence and the virulence potential of Listeria monocytogenes isolated from beef carcasses obtained in processing facilities in the southern region of Rio Grande do Sul, Brazil, based on pulsed-field gel electrophoresis (PFGE), invasion ability in human colorectal carcinoma cells (HCT-116), internalin A (InlA) expression by Western blot, and identification of mutation points in inlA. PFGE profiles demonstrated that L. monocytogenes isolates were grouped based on their previously identified lineages and serogroups (lineage I: serogroup IIb, n = 2, and serogroup IVb, n = 5; lineage II: serogroup IIc, n = 5). Isolates with indistinguishable genetic profiles through this method were obtained from different slaughterhouses and sampling steps, with as much as a 3-year interval. Seven isolates showed high invasion ability (2.4 to 7.4%; lineage I, n = 6, and lineage II, n = 1) in HCT and expressed InlA. Five isolates showed low cell invasion ability (0.6 to 1.4%; lineage I, n = 1, and lineage II, n = 4) and did not express InlA, and two of them (lineage II, serogroup IIc) presented mutations in inlA that led to premature stop codon type 19 at position 326 (GAA → TAA). The results demonstrated that most L. monocytogenes isolates from lineage I expressed InlA and were the most invasive in HCT, indicating their high virulence potential, whereas most isolates from lineage II showed attenuated invasion because of nonexpression of InlA or the presence of premature stop codon type 19 in inlA. The obtained results demonstrated that L. monocytogenes with indistinguishable PFGE profiles can persist or be reintroduced in beef processing facilities in the studied region and that differences in their virulence potential are based on their lineages and serogroups.

Listeria monocytogenes From Farm to Fork in a Brazilian Pork Production Chain.

ABSTRACT: Listeria monocytogenes contamination was assessed in different steps of a pork production chain. Ten lots of pigs were sampled at termination barns, at slaughter (after bleeding, after buckling, after evisceration, and after final washing), at processing (knives, deboning tables, and employees' hands), and of end products (ribs, shoulder, ham, and sausage). All samples (n = 670) were subjected to L. monocytogenes detection, and the obtained isolates (n = 18, identified as Listeria spp.) were characterized by their biochemical characteristics, serogroups, virulence genes, pulsed-field gel electrophoresis profiles, antibiotic resistances (ampicillin, penicillin, gentamicin, and sulfamethoxazole-trimethoprim), and adhesion abilities. The results revealed the low occurrence of Listeria spp. in the evaluated pork production chain. However, four tested sausage samples (40%) were positive for Listeria spp., with L. monocytogenes identified in two (20%) of these samples. Ten isolates were identified as L. monocytogenes (eight from serogroup 1/2a or 3a and two from serogroup 4b, 4d, or 4e): all isolates were also positive for the virulence-related genes hlyA, iap, plcA, actA, inlA, inlB, inlC, and inlJ and susceptible to the tested antibiotics. One sausage sample was contaminated by both serogroups 1/2a or 3a and 4b, 4d, or 4e. Isolates from serogroup 1/2a or 3a obtained during visits 5 and 6 presented distinct genetic profiles by pulsed-field gel electrophoresis, indicating that contamination may come from different sources. The adhesion potential exhibited by Listeria spp. isolates (n = 18) ranged from weak (serogroup 4b, 4d, or 4e) to moderate (L. innocua and L. monocytogenes serogroup 1/2a or 3a). Despite the low occurrence of L. monocytogenes, pathogenic serogroups were detected in sausages, demanding control measures by the industry.

Short communication: Potential use of passion fruit (Passiflora cincinnata) as a biopreservative in the production of coalho cheese, a traditional Brazilian cheese.

Passion fruit (Passiflora cincinnata Mast.) is a native fruit from the Caatinga, a typical ecoregion in northeastern Brazil, and it has potential for use by the food and pharmaceutical industries. In this study, we characterized the antimicrobial activity of P. cincinnata and its application in the production of coalho cheese, a traditional Brazilian product. Aqueous extract of P. cincinnata exhibited high inhibitory activity against Listeria spp. (n = 4, reference strains), Staphylococcus aureus (n = 3, reference strains), and multidrug-resistant Staph. aureus (n = 8), and low inhibitory activity against lactic acid bacteria (LAB, n = 3, reference strains). Based on these results, we produced coalho cheese using goat milk with and without (control) passion fruit. Cheeses were stored at 10°C for 14 d and populations of mesophilic aerobes, Staph. aureus, and presumptive LAB were monitored at d 1, 7 and 14. The passion fruit cheese had lower counts of mesophilic aerobes, Staph. aureus (after 7 and 14 d), and presumptive LAB (after 14 d) than the control cheese. Adding ground passion fruit contributed to a reduction of Staph. aureus counts in goat cheese, although these differences were not significant. These results indicated the inhibitory potential of passion fruit and its potential use for controlling microbial populations in a cheese model; further studies are needed to characterize the active molecules that are responsible for such activity.

Whole-genome sequencing reveals Listeria monocytogenes diversity and allows identification of long-term persistent strains in Brazil.

Advances in whole-genome sequencing (WGS) technologies have documented genetic diversity and epidemiology of the major foodborne pathogen Listeria monocytogenes (Lm) in Europe and North America, but data concerning South America are scarce. Here, we examined the population structure and genetic diversity of this major foodborne pathogen collected in Brazil. Based on core genome multilocus sequence typing (cgMLST), isolates from lineages I (n = 22; 63%) and II (n = 13; 37%) were distributed into 10 different sublineages (SLs) and represented 31 new cgMLST types (CTs). The most prevalent SLs were SL9 (n = 9; 26%), SL3 (n = 6; 17%) and SL2 and SL218 (n = 5; 14%). Isolates belonging to CTs L2-SL9-ST9-CT4420 and L1-SL315-ST520-CT4429 were collected 3 and 9 years apart, respectively, revealing long-term persistence of Lm in Brazil. Genetic elements associated with stress survival were present in 60% of isolates (57% SSI-1 and 3% SSI-2). Pathogenic islands were present in 100% (LIPI-1), 43% (LIPI-3) and 6% (LIPI-4) of the isolates. Mutations leading to premature stop codons were detected in the prfA and inlA virulence genes. This study is an important contribution to understanding the genomic diversity and epidemiology of Lm in South America. In addition, the results highlight the importance of using WGS to reveal Lm long-term persistence.

Lactic Acid Bacteria (LAB) and Their Bacteriocins as Alternative Biotechnological Tools to Control Listeria monocytogenes Biofilms in Food Processing Facilities.

Bacteriocins are antimicrobial peptides produced by bacteria Gram-negative and Gram-positive, including lactic acid bacteria (LAB), organisms that are traditionally used in food preservation practices. Bacteriocins have been shown to have an aptitude as biofilm controlling agents in Listeria monocytogenes biofilms, a major risk for consumers and the food industry. Biofilms protect pathogens from sanitization procedures, allowing them to survive and persist in processing facilities, resulting in the cross-contamination of the end products. Studies have been undertaken on bacteriocinogenic LAB, their bacteriocins, and bioengineered bacteriocin derivatives for controlling L. monocytogenes biofilms on different surfaces through inhibition, competition, exclusion, and displacement. These alternative strategies can be considered promising in preventing the development of resistance to conventional sanitizers and disinfectants. Bacteriocins are "friendly" antimicrobial agents, and with high prevalence in nature, they do not have any known associated public health risk. Most trials have been carried out in vitro, on food contact materials such as polystyrene and stainless steel, while there have been few studies performed in situ to consolidate the results observed in vitro. There are strategies that can be employed for prevention and eradication of L. monocytogenes biofilms (such as the establishment of standard cleaning procedures using the available agents at proper concentrations). However, commercial cocktails using alternatives compounds recognized as safe and environmental friendly can be an alternative approach to be applied by the industries in the future.

Combined effect of bacteriocin produced by Lactobacillus plantarum ST8SH and vancomycin, propolis or EDTA for controlling biofilm development by Listeria monocytogenes / Efecto combinado de la bacteriocina producida por Lactobacillus plantarum ST8SH y la vancomicina, el própolis o el EDTA para controlar el desarrollo del biofilm producido por Listeria monocytogenes

The Listeria monocytogenes strains selected in the present study exhibited similar behavior in biofilm formation, independently of the tested conditions (bacteriocin from L. plantarum ST8SH, vancomycin, propolis (a natural antimicrobial product) and EDTA (chelating agent)), individual or in associations. The individual application of vancomycin had better inhibitory activity than that of propolis and EDTA; however, the association of the previously mentioned antimicrobial agents with bacteriocins resulted in better performance. However, when we compared the effects of vancomycin, propolis and EDTA, we could clearly observe that the combined application of bacteriocin and vancomycin was more effective than the combination of bacteriocin and propolis, and bacteriocin and EDTA. Considering the current need to reduce the use of antimicrobials and chemical substances in food processing, propolis can represent an alternative to improve the inhibitory effect of bacteriocins against L. monocytogenes biofilm formation, based on the obtained results. In general, high concentrations of bacteriocin produced by L. plantarum ST8SH were more effective in biofilm inhibition, and similar results were observed for vancomycin and propolis; however, all tested EDTA concentrations had similar effect on biofilm formation.

Las cepas de Listeria monocytogenes seleccionadas en el presente estudio presentaron comportamientos similares en la formación de biofilms, independientemente de los tratamientos a las que fueron sometidas (bacteriocina de Lactobacillus plantarum ST8SH, vancomicina, própolis (produto natural antimicrobiano) y EDTA (agentes quelante)), individual o en combinaciones. La aplicación individual de vancomicina presentó una mejor actividad inhibitoria frente a las aplicaciones individuales de própolis y de EDTA; sin embargo, la combinación de estos agentes antimicrobianos con las bacteriocinas resultó en un mejor desempeño. Se observó claramente que la aplicación combinada de bacteriocina y vancomicina fue más efectiva para controlar el desarrollo de biofilm en comparación con la combinación de la bacteriocina y el própolis o de la combinación de la bacteriocina y el EDTA. Considerando la necesidad actual de reducir el uso de sustancias antimicrobianas y químicas en el procesamiento de alimentos y sobre la base de los resultados obtenidos, se puede afirmar que el própolis representa una alternativa para mejorar el efecto inhibitorio de bacteriocinas contra la formación de biofilm de L. monocytogenes. En general, altas concentraciones de la bacteriocina producida por L. plantarum ST8SH fueron más eficaces en la inhibición del biofilm, y se observaron resultados similares para la vancomicina y el própolis; sin embargo, todas las concentraciones de EDTA evaluadas tuvieron un efecto similar en la formación de biofilm.

Combined effect of bacteriocin produced by Lactobacillus plantarum ST8SH and vancomycin, propolis or EDTA for controlling biofilm development by Listeria monocytogenes.

The Listeria monocytogenes strains selected in the present study exhibited similar behavior in biofilm formation, independently of the tested conditions (bacteriocin from L. plantarum ST8SH, vancomycin, propolis (a natural antimicrobial product) and EDTA (chelating agent)), individual or in associations. The individual application of vancomycin had better inhibitory activity than that of propolis and EDTA; however, the association of the previously mentioned antimicrobial agents with bacteriocins resulted in better performance. However, when we compared the effects of vancomycin, propolis and EDTA, we could clearly observe that the combined application of bacteriocin and vancomycin was more effective than the combination of bacteriocin and propolis, and bacteriocin and EDTA. Considering the current need to reduce the use of antimicrobials and chemical substances in food processing, propolis can represent an alternative to improve the inhibitory effect of bacteriocins against L. monocytogenes biofilm formation, based on the obtained results. In general, high concentrations of bacteriocin produced by L. plantarum ST8SH were more effective in biofilm inhibition, and similar results were observed for vancomycin and propolis; however, all tested EDTA concentrations had similar effect on biofilm formation.

Genotypic and antimicrobial characterization of pathogenic bacteria at different stages of cattle slaughtering in southern Brazil.

Meat can be contaminated in different stages of the slaughtering process and the identification of these stages is the starting point to implement adequate control measures. The objectives of this study were to assess the presence of pathogenic microorganisms in cattle carcasses, to identify the most important contamination points of the slaughtering process, and to evaluate the possible risk factors related to them in a cattle slaughterhouse. To this aim, 108 cattle carcasses were sampled at three stages of the slaughtering process: Point 1 (hides after bleeding); Point 2 (carcasses after hide removal); and Point 3 (carcasses immediately after division). Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Livingstone were isolated from the carcasses. Phenotypic and genotypic characterization indicated that there was cross-contamination among animals, since bacteria with identical genotypic and phenotypic profiles were isolated from different animals at the same sampling day. Furthermore, this is the first report about the isolation of E. coli O157:H7 in a bovine slaughterhouse from southern Brazil.