Biofilm growth by Listeria monocytogenes on stainless steel and expression of biofilm-related genes under stressing conditions.

This research was carried out to investigate the differences in adhesion and growth during biofilm formation of L. monocytogenes from different sources and clonal complexes. Biofilm by L. monocytogenes (isolates CLIST 441 and 7: both lineage I, serotype 1/2b, CC3; isolates 19 and 508: both lineage II, serotype 1/2c, CC9) was grown on stainless steel coupons under different stressing conditions (NaCl, curing salts and quaternary ammonium compounds-QAC), to determine the expression of different genes involved in biofilm formation and stress response. CLIST 441, which carries a premature stop codon (PMSC) in agrC, formed high-density biofilms in the presence of QAC (7.5% w/v) or curing salts (10% w/v). Reverse Transcriptase-qPCR results revealed that L. monocytogenes isolates presented differences in transcriptional profile of genes related to biofilm formation and adaptation to environmental conditions. Our results demonstrated how L. monocytogenes can survive, multiply and form biofilm under adverse conditions related to food processing environments. Differences in transcriptional expression were observed, highlighting the role of regulatory gene networks for particular serotypes under different stress responses.

Interference of the acid stress on the expression of llsX by Listeria monocytogenes pathogenic island 3 (LIPI-3) variants.

Listeria monocytogenes harbor different virulence factors, with a highly heterogeneous distribution between distinct lineages and serotypes. The Listeria Pathogenicity Island 3 (LIPI-3), mainly described in lineage I, encodes for Listeriolysin S (LLS), a virulence factor expressed by L. monocytogenes in the gastrointestinal tract during in vivo infections. The aim of this study was to carry out a comparative genotypic analysis of LIPI-3 identified in L. monocytogenes isolates obtained in Brazil and subjected to whole genomic sequencing (WGS). In addition, transcription of llsX expression under different acid stress conditions was evaluated by RT-PCR. Homologues of the eight LIPI-3 genes (llsAGHXBYDP) were identified in 15 isolates (all from lineage I) representative of different sequence types: ST1 (n = 3), ST3 (n = 6), ST218 (n = 5) and ST288 (n = 1). Single nucleotide polymorphism (SNP) analysis revealed that genetic variation resulted in modification of the final peptide LLS for ST218 (serogroup IVb-v1) and ST288 (serogroup IIb). Selected strains from ST3 and ST288 were subjected to acid stress conditions and the expression of llsX, a LIPI-3 gene, was observed: only F2365 (4b/ST1) presented llsX expression after six hours of acid stress, indicating relevant differences when compared to isolates IIb (ST3 and 288). The results highlight the presence of genomic variations on LIPI-3 and llsX expression under acid stress conditions, demanding further studies to evaluate if these mutations have an impact on L. monocytogenes virulence in vivo.

Distribution, adhesion, virulence and antibiotic resistance of persistent Listeria monocytogenes in a pig slaughterhouse in Brazil.

Listeria monocytogenes is a relevant pathogen usually associated with meat and ready-to-eat products. This study aimed to assess the distribution, adhesion, virulence and antibiotic resistance of L. monocytogenes in a pork production chain. Environment, carcass and food samples (n = 894) were obtained from different steps of a pork production chain over a 6-month period (10 samplings), including from farms and the slaughterhouse (reception, slaughtering, processing, storage and end products). L. monocytogenes was detected in samples from the reception (lairage floor, 1/10), slaughtering (drains, 2/20) and cutting room stages (conveyor belts in the final packing stage - 11/20, knife - 1/40, and cutting boards - 1/20). Positive results for conveyor belts were recorded in seven consecutive samplings. L. monocytogenes isolates (n = 87) were characterized as belonging to serogroup IVb and presented positive PCR results for inlA, inlB, inlC, inlJ, hlyA, plcA, actA and iap. Isolates were selected according to the original samples (n = 31) and subjected to Pulsed Field Gel Electrophoresis (PFGE), demonstrating their high clonal identity (98.4-100%). According to PFGE results and their original samples, isolates were selected (n = 16) and subjected to phenotypic assay to assess their adhesion potential and tested for resistance against 15 antibiotics; all tested isolates presented weak adhesion potential and were resistant to ampicillin. The present study demonstrated the persistence of L. monocytogenes in the pork processing facility, indicating the potential risk for cross-contamination with a potential virulent and resistant clone.

Occurrence and characterization of Listeria monocytogenes from beef jerky processing line.

Beef jerky is a ready-to-eat product that does not require refrigeration at the point of sale. Here, we evaluated the occurrence of Listeria monocytogenes in the production process of beef jerky, the presence of virulence genes and the genomic relatedness of the isolates, to assess the safety of the final product. The raw material, surfaces with and without contact with the product and the final product were evaluated along the beef jerky processing line. The samples were evaluated by VIDAS immunoassay system, and the L. monocytogenes isolates were confirmed and evaluated for the presence of several virulence genes by PCR. Listeria monocytogenes was identified in six of the 84 samples (7.14%), and no genetic relationship was observed among isolates. Samples of raw material (2/7), food contact surface (1/56), and work surfaces without contact with food (3/14) presented contamination by L. monocytogenes. The final product was not contaminated, demonstrating that barriers to multiplication of pathogens used during the production process were effective for its control.

Yersinia enterocolitica in a Brazilian pork production chain: Tracking of contamination routes, virulence and antimicrobial resistance.

This study aimed to track Yersinia enterocolitica contamination in a pork production chain in Minas Gerais, Brazil, and to characterize the virulence and antibiotic resistance of isolates. Samples were collected from four different steps of the pork production chain (pig farm, carcass, processing environment and end product; n = 870), and tested for the presence of Y. enterocolitica. The pathogen was detected in 8 samples (palatine tonsils = 5; mesenteric lymph nodes = 2; carcass after bleeding = 1), from which 16 isolates were obtained and identified as Y. enterocolitica bioserotype 4/O:3. XbaI macrorestriction allowed the clustering of isolates in 5 pulsetypes, and the identification of identical profiles of Y. enterocolitca isolated from different samples. All isolates were positive for the virulence related genes ail, virF, myfA, ystA, tccC, ymoA, hreP and sat, and negative for ystB, ystC, fepA, fepD and fes. Considering 17 antibiotics from 11 classes, only ciprofloxacin and kanamycin were effective against all isolates, and three multidrug resistance profiles were identified among them, with simultaneous resistance to 9 of 11 classes. All isolates presented positive results for emrD, yfhD and marC, related to multidrug resistance. The results of this study demonstrated the contamination routes of Y. enterocolitica within the assessed pork production chain, and highlighted the pathogenic potential and antibiotic resistance of this foodborne pathogen.

Hygiene and Safety in the Meat Processing Environment from Butcher Shops: Microbiological Contamination and Listeria monocytogenes.

The quality and safety of meat products can be estimated by assessing their contamination by hygiene indicator microorganisms and some foodborne pathogens, with Listeria monocytogenes as a major concern. To identify the main sources of microbiological contamination in the processing environment of three butcher shops, surface samples were obtained from the hands of employees, tables, knives, inside butcher displays, grinders, and meat tenderizers (24 samples per point). All samples were subjected to enumeration of hygiene indicator microorganisms and detection of L. monocytogenes, and the obtained isolates were characterized by their serogroups and virulence genes. The results demonstrated the absence of relevant differences in the levels of microbiological contamination among butcher shops; samples with counts higher than reference values indicated inefficiency in adopted hygiene procedures. A total of 87 samples were positive for Listeria spp. (60.4%): 22 from tables, 20 from grinders, 16 from knives, 13 from hands, 9 from meat tenderizers, and 7 from butcher shop displays. Thirty-one samples (21.5%) were positive for L. monocytogenes, indicating the presence of the pathogen in meat processing environments. Seventy-four L. monocytogenes isolates were identified, with 52 from serogroups 1/2c or 3c and 22 from serogroups 4b, 4d, 4a, or 4c. All 74 isolates were positive for hlyA, iap, plcA, actA, and internalins (inlA, inlB, inlC, and inlJ). The establishment of appropriate procedures to reduce microbial counts and control the spread of L. monocytogenes in the final steps of the meat production chain is of utmost importance, with obvious effects on the quality and safety of meat products for human consumption.

Antibiotic Resistance of Listeria monocytogenes Isolated from Meat-Processing Environments, Beef Products, and Clinical Cases in Brazil.

The present study aimed to assess the antimicrobial resistance and the presence of virulence markers in 137 Listeria monocytogenes isolates obtained from meat-processing environments, beef products, and clinical cases. All isolates were subject to molecular serogrouping and their antibiotic resistance profiles were assessed against 12 antimicrobials. In addition, isolates were subjected to detection of virulence marker genes (inlA, inlC, inlJ). The isolates were classified into serogroups 4b, 4d, 4a, or 4c (46%), 1/2c or 3c (27%), 1/2a or 3a (13.9%), and 1/2b or 3b (13.1%). All tested isolates presented sensitivity to the majority of the tested antimicrobials, but most of them presented resistance or intermediate resistance to clindamycin (88.3%) and oxacillin (73.7%). Virulence markers were detected in all isolates, demanding further analysis to better characterize their pathogenic potential.