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Listeria monocytogenes Isolated from Illegally Imported Food Products into the European Union Harbor Different Virulence Factor Variants.

ABSTRACT: Unregulated international flow of foods poses a danger to human health, as it may be contaminated with pathogens. Recent studies have investigated neglected routes of pathogen transmission and reported the occurrence of Listeria monocytogenes in food illegally imported into the European Union (EU), either confiscated at four international airports or sold illegally on the Romanian black market. In this study we investigated the genotype diversity and the amino acid sequence variability of three main virulence factors of 57 L. monocytogenes isolates. These isolates were derived from 1474 food samples illegally imported into the EU and originated from 17 different countries. Multilocus sequence typing revealed 16 different sequence types (STs) indicating moderate genotype diversity. The most prevalent STs were ST2, ST9, and ST121. The pulsed-field gel electrophoresis (PFGE) analysis resulted in 34 unique pulsotypes. PFGE types assigned to the most prevalent STs (ST2, ST9, and ST121) were highly related in their genetic fingerprint. Internalin A (InlA) was present in 20 variants, including six truncated InlA variants, all harbored by isolates of ST9 and ST121. We detected eight ST-specific listeriolysin O (LLO) variants, and among them, one truncated form. The actin-assembly-inducing protein ActA was present in 15 different ST-specific variants, including four ActA variants with an internal truncation. In conclusion, this study shows that L. monocytogenes, isolated from illegally imported food, have moderate genotype diversity, but diverse virulence factors variants, mainly of InlA.

SUBMITTER: Rychli K

PROVIDER: S-EPMC6162745 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AbstractA comprehensive understanding of foodborne pathogen diversity in preharvest environments is necessary to effectively track pathogens on farms and identify sources of produce contamination. As such, this study aimed to characterize Listeria diversity in wildlife feces and agricultural water collected from a New York state produce farm over a growing season. Water samples were collected from a pond (n = 80) and a stream (n = 52). Fecal samples (n = 77) were opportunistically collected from areas <5 m from the water sources; all samples were collected from a <0.5-km2 area. Overall, 86 (41%) and 50 (24%) of 209 samples were positive for Listeria monocytogenes and Listeria spp. (excluding L. monocytogenes), respectively. For each positive sample, one L. monocytogenes or Listeria spp. isolate was speciated by sequencing the sigB gene, thereby allowing for additional characterization based on the sigB allelic type. The 86 L. monocytogenes and 50 Listeria spp. isolates represented 8 and 23 different allelic types, respectively. A subset of L. monocytogenes isolates (n = 44) from pond water and pond-adjacent feces (representing an ∼5,000-m2 area) were further characterized by pulsed-field gel electrophoresis (PFGE); these 44 isolates represented 22 PFGE types, which is indicative of considerable diversity at a small spatial scale. Ten PFGE types were isolated more than once, suggesting persistence or reintroduction of PFGE types in this area. Given the small spatial scale, the prevalence of L. monocytogenes and Listeria spp., as well as the considerable diversity among isolates, suggests traceback investigations may be challenging. For example, traceback of finished product or processing facility contamination with specific subtypes to preharvest sources may require collection of large sample sets and characterization of a considerable number of isolates. Our data also support the adage "absence of evidence does not equal evidence of absence" as applies to L. monocytogenes traceback efforts at the preharvest level.Highlights

Project description:World Health Organization classified Listeria monocytogenes as a major notable foodborne pathogen associated with high mortality and hospitalization. The study reports the prevalence, antibiogram, virulence determination and genetic characterization of L. monocytogenes from different food products. A total of 250 food samples, fifty samples each from raw milk, ice cream, minced meat, fish fillet and sausage were collected from the Menoufiya governorate in Egypt. L. monocytogenes was detected in 17 (6.8%) of the tested food samples including minced meat (14%), fish fillet (8%), sausage (6%) and raw milk (6%). The antimicrobial susceptibility assay of 17 L. monocytogenes isolates against seventeen antibiotics belonging to eight antibiotics classes revealed a high susceptibility to norfloxacin (82.3%), amoxicillin-clavulanic acid (76.4%), cefotaxime (70.5%), erythromycin (64.6%), amoxicillin (64.6%), gentamicin (58.7%) and vancomycin (58.7%). While, high resistance was observed against oxytetracycline (76.4%), trimethoprim-sulfamethoxazole (76.4%), chloramphenicol (70.5%), doxycycline (64.6%), levofloxacin (41.2%) and azithromycin (41.2%). Of note, all L. monocytogenes isolates were multidrug-resistant. The multiplex PCR successfully amplified L. monocytogenes in all tested isolates. Screening of the five virulence-related genes revealed the hlyA and iap as the most prevalent genes followed by actA gene, however, the inlA and prfA genes were not detected in any of the studied isolates. The partial 16S rRNA gene sequencing of three L. monocytogenes isolates showed a high nucleotide similarity (99.1-99.8%) between the study isolates and various global clones, and phylogenetic analysis clustered these L. monocytogenes strains with other Listeria species including L. welshimeri, L. seeligeri and L. innocua. This study demonstrates the impact of L. monocytogenes as a major contaminant of various food products and suggests more attention to the awareness and hygienic measures in the food industry.

Project description:BackgroundListeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.Methodology/principal findingsThe objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp(LAP)) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp(LAP) for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp(LAP) pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp(LAP) also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp(LAP) cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.Conclusions/significanceWild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp(LAP) blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.

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Listeria monocytogenes Isolated from Illegally Imported Food Products into the European Union Harbor Different Virulence Factor Variants.

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