Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Salmonella Isolated from Retail Meats in South Korea.

Earlier studies have validated the isolation of extended-spectrum beta-lactamase-producing Salmonella (ESBL-Sal) strains from food. While poultry is recognized as a reservoir for Salmonella contamination, pertinent data regarding ESBL-Sal remains limited. Consequently, the Ministry of Food and Drug Safety has isolated Salmonella spp. from retail meat and evaluated their antibiotic susceptibility and genetic characteristics via whole-genome sequencing. To further elucidate these aspects, this study investigates the prevalence, antibiotic resistance profiles, genomic characteristics, and homology of ESBL-Sal spp. obtained from livestock-derived products in South Korean retail outlets. A total of 653 Salmonella spp. were isolated from 1,876 meat samples, including 509 beef, 503 pork, 555 chicken, and 309 duck samples. The prevalence rates of Salmonella were 0.0%, 1.4%, 17.5%, and 28.2% in the beef, pork, chicken, and duck samples, respectively. ESBL-Sal was exclusively identified in poultry meat, with a prevalence of 1.4% in the chicken samples (8/555) and 0.3% in the duck samples (1/309). All ESBL-Sal strains carried the blaCTX-M-1 gene and exhibited resistance to ampicillin, ceftiofur, ceftazidime, nalidixic acid, and tetracycline. Eight ESBL-Sal isolates were identified as S. Enteritidis with sequence type (ST) 11. The major plasmid replicons of the Enteritidis-ST11 strains were IncFIB(S) and IncFII(S), carrying antimicrobial resistance genes (ß-lactam, tetracycline, and aminoglycoside) and 166 virulence factor genes. The results of this study provide valuable insights for the surveillance and monitoring of ESBL-Sal in South Korean food chain.

Efficacy of chemical sanitizers against Bacillus cereus on food contact surfaces with scratch and biofilm.

This study was performed to investigate the efficacy of chemical sanitizers (viz., chlorine, chlorine dioxide, alcohol, and quaternary ammonium compound) against Bacillus cereus on five food contact materials under different conditions (smooth vs. scratched and with vs. without biofilms). After incubating materials in B. cereus suspension, cell adhesion on a smooth surface (10 cm2) was in the following ascending order: stainless steel (7.36 ± 0.08 log CFU), glass (7.51 ± 0.26 log CFU), polyethylene (7.66 ± 0.30 log CFU), polypropylene (7.76 ± 0.30 log CFU), and wood (8.02 ± 0.33 log CFU). The efficacy of sanitizers was dramatically reduced in the presence of a biofilm on all materials. Among four different chemical sanitizers, chlorine showed the best bactericidal activity against B. cereus on the surface with scratch and biofilm. Selection of adequate materials, maintenance of a smooth surface, and inhibition of biofilm formation are good practices for food safety.

Pathogen enrichment device (PED) enables one-step growth, enrichment and separation of pathogen from food matrices for detection using bioanalytical platforms.

The bottleneck for accurate detection of foodborne pathogens is separation of target analytes from complex food matrices. Currently used sample preparation methods are cumbersome, arduous and lengthy; thus, a user-friendly system is desirable. A hand-held sample preparation system designated pathogen enrichment device (PED) was built that contains a growth chamber, filters, and an ion exchange cartridge to deliver bacteria directly onto the detection platforms. Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes were used as model pathogens. Spinach, ground beef, hotdogs, and eggs were used as model foods to evaluate PED performance, and results were compared with traditional bag enrichment method. Bacterial cells were inoculated at 1, 10, and 100 CFU/g of the sample and enriched in PED using appropriate pathogen-specific selective enrichment broths. The bacterial cell counts in both PED and stomacher-bag were comparable and the pH in PED-recovered cell suspension was close to neutral whereas the pH of cell suspension in the stomacher-bag was slightly acidic. The bacterial recovery from the PED was 79-100% and was directly detected by lateral-flow immunoassay (LFIA), quantitative PCR (qPCR) and light scattering sensor with sample-to-result time of 8-24h with a detection limit of 1CFU/g. In qPCR, the amplified PCR products appeared in 4-5 cycles earlier with PED-enriched cultures compared to the cultures enriched in stomacher-bag. The hand-held PED proved to be a one-step procedure for enrichment and recovery of homogenous particle-free bacterial cells for detection using immunological, molecular or biosensor-based platforms.

Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier.

BACKGROUND: Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown. METHODS: Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells. RESULTS: Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates. CONCLUSION: This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate.

Expression of LAP, a SecA2-dependent secretory protein, is induced under anaerobic environment.

Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase homolog (lmo1634) in Listeria monocytogenes, promotes bacterial adhesion to intestinal epithelial cells in vitro. Investigation of the effect of anaerobiosis, an intrinsic gastrointestinal condition, on LAP expression and LAP-mediated infection should elucidate its significance during intestinal infection. The influence of anaerobiosis on LAP expression was determined by growing L. monocytogenes wild type (WT), and lap-deficient (KB208) and -complemented (CKB208) strains anaerobically and monitoring LAP in secreted, cell wall, and whole-cell protein fractions. The effect of anaerobiosis on LAP-mediated infection was evaluated in cell culture adhesion assays and mouse infection models. Additionally, the role of secretory system SecA2 in LAP secretion was investigated. Anaerobic growth induced significant increases in level of lap transcript and protein secretion, and secretion was SecA2-dependent. Anaerobiosis facilitated greater LAP-mediated adhesion of L. monocytogenes to cultured intestinal cells. Oral administration of WT, KB208 and CKB208 to mice confirmed that LAP is essential for full virulence, and anaerobically-grown WT exhibited greater translocation to liver and spleen relative to aerobically-grown organisms. LAP, a SecA2-dependent secreted virulence factor, plays an important role during intestinal infection, particularly when L. monocytogenes is subjected to an anaerobic environment.

SEL, a selective enrichment broth for simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes.

Multipathogen detection on a single-assay platform not only reduces the cost for testing but also provides data on the presence of pathogens in a single experiment. To achieve this detection, a multipathogen selective enrichment medium is essential to allow the concurrent growth of pathogens. SEL broth was formulated to allow the simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. The results were compared to those obtained with the respective individual selective enrichment broths, Rappaport-Vassiliadis (RV) for S. enterica, modified E. coli broth with 20 mg of novobiocin/liter for E. coli O157:H7, and Fraser broth for L. monocytogenes, and a currently used universal preenrichment broth (UPB). The growth of each pathogen in SEL inoculated at 10(1) or 10(3) CFU/ml was superior to that in the respective individual enrichment broth, except in the case of RV, in which Salmonella cells inoculated at both concentrations grew equally well. In mixed-culture experiments with cells of the three species present in equal concentrations or at a 1:10:1,000 ratio, the overall growth was proportional to the initial inoculation levels; however, the growth of L. monocytogenes was markedly suppressed when cells of this species were present at lower concentrations than those of the other two species. Further, SEL was able to resuscitate acid- and cold-stressed cells, and recovery was comparable to that in nonselective tryptic soy broth containing 6% yeast extract but superior to that in the respective individual selective broths. SEL promoted the growth of all three pathogens in a mixture in ready-to-eat salami and in turkey meat samples. Moreover, each pathogen was readily detected by a pathogen-specific immunochromatographic lateral-flow or multiplex PCR assay. Even though the growth of each pathogen in SEL was comparable to that in UPB, SEL inhibited greater numbers of nontarget organisms than did UPB. In summary, SEL was demonstrated to be a promising new multiplex selective enrichment broth for the detection of the three most prominent food-borne pathogens by antibody- or nucleic acid-based methods.