Lactiplantibacillus plantarum subsp. plantarum and Fructooligosaccharides Combination Inhibits the Growth, Adhesion, Invasion, and Virulence of Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen responsible for many food outbreaks worldwide. This study aimed to investigate the single and combined effect of fructooligosaccharides (FOS) and Lactiplantibacillus plantarum subsp. plantarum CICC 6257 (L. plantarum) on the growth, adhesion, invasion, and virulence of gene expressions of Listeria monocytogenes 19112 serotype 4b (L. monocytogenes). Results showed that L. plantarum combined with 2% and 4% (w/v) FOS significantly (p < 0.05) inhibited the growth of L. monocytogenes (3–3.5 log10 CFU/mL reduction) at the incubation temperature of 10 °C and 25 °C. Under the same combination condition, the invasion rates of L. monocytogenes to Caco-2 and BeWo cells were reduced more than 90% compared to the result of the untreated group. After L. plantarum was combined with the 2% and 4% (w/v) FOS treatment, the gene expression of actin-based motility, sigma factor, internalin A, internalin B, positive regulatory factor A, and listeriolysin O significantly (p < 0.05) were reduced over 91%, 77%, 92%, 89%, 79%, and 79% compared to the result of the untreated group, respectively. The inhibition level of the L. plantarum and FOS combination against L. monocytogenes was higher than that of FOS or L. plantarum alone. Overall, these results indicated that the L. plantarum and FOS combination might be an effective formula against L. monocytogenes.

Effect of Enterocins A and B on the Viability and Virulence Gene Expression of Listeria monocytogenes in Sliced Dry-Cured Ham

Dry-cured ham can be contaminated with Listeria monocytogenes during its industrial processing. The use of bacteriocins could ensure the safety of such meat products, but their effect on pathogen physiology is unknown. Therefore, the impact of enterocins A and B on the L. monocytogenes population, and the expression patterns of five genes (inlA, inlB, clpC, fbpA and prfA) related to adhesion/invasion and virulence regulation have been monitored in sliced dry-cured ham during 30 d of storage in refrigeration (4 °C) and temperature-abuse conditions (20 °C). L. monocytogenes strains S2 (serotype 1/2a) and S7-2 (serotype 4b) counts were reduced by 0.5 and 0.6 log units immediately after the application of enterocins A and B, a decrease lower than previously reported. Differences in gene expression were found between the two strains. For strain S2, expression tended to increase for almost all genes up to day seven of storage, whereas this increase was observed immediately after application for strain S7-2; however, overall gene expression was repressed from day one onwards, mainly under temperature-abuse conditions. L. monocytogenes strains investigated in the present work exhibited a mild sensitivity to enterocins A and B in sliced dry-cured ham. Bacteriocins caused changes in the expression patterns of virulence genes associated with adhesion and invasion, although the potential virulence of surviving cells was not enhanced.

Occurrence of Multidrug-Resistant Listeria monocytogenes in Environmental Waters: A Menace of Environmental and Public Health Concern

The occurrence and antibiogram profiles of Listeria monocytogenes in environmental waters in the Eastern Cape Province, South Africa, were investigated. Seventy-eight samples from rivers, wastewater, and irrigation water were collected at different geographical locations within the province from February to September 2019. The procedure of the International Organization for Standardization EN ISO 11290:2017 part 1 and 2 was adopted for the isolation of Lm. The counts of presumptive Lm ranged from 2.0 × 103 CFU/100 ml to 3.6 × 105 CFU/100 ml. About 39.74% of the samples were positive for Lm, and the isolates were confirmed as serotype 1/2a (78.95%) and serotype 4b (21.05%). About 68.42% of the isolates demonstrated biofilm-forming potentials. Ten virulence determinants including the plcA, inlA, and inlB were detected in all the isolates; however, inlC (85.97%), inlJ (80.70%), actA (59.65%), prfA (92.98%), plcB (89.47%), hly (80.70%), and mpl (73.68%) were less prevalent. The antibiogram profiles of confirmed Lm isolates revealed high susceptibilities (&gt;50%) to all antibiotics ranging from 52.63% (cefotetan) to 100% (ampicillin) except for sulfamethoxazole (35.09%), erythromycin (26.32%), streptomycin (38.60%), oxytetracycline (45.61%), and amoxicillin (49.12%). Conversely, high resistance rates against sulfamethoxazole (63.16%), oxytetracycline (54.39%), and amoxicillin (50.88%) were observed. Isolates exhibited 52 resistance patterns against 22 antibiotics tested ranging from 1 to 15 antibiotics, and 82.46% showed multidrug-resistance phenotypes against the antibiotics. The antibiotic resistance index (ARI) of river water (0.18) was less than the permissible (0.2) Krumperman threshold, whereas that of irrigation and wastewater were higher than the Krumperman threshold. The multiple/antibiotic resistance index ((M)ARI) of each of the isolates ranged from 0.05–0.68. Resistance determinants (21) encoding resistance against sulphonamides, β-lactamase, phenicols, aminoglycosides, and tetracyclines were detected among the phenotypically resistant Lm isolates investigated which justifies the phenotypic resistance observed in this study. The abundance of resistance determinants in Lm recovered from environmental waters in this study suggests that the aquatic environments may serve as a channel for the dissemination of antimicrobial-resistant Lm to other niches including the food chain.

Transcriptomic Response of L. monocytogenes to Co-Culture with S. cerevisiae

The aim of the present study was to assess the transcriptomic response of L. monocytogenes during co-culture with three S. cerevisiae strains. For this purpose, BHI broth was inoculated with 7 log CFU·mL−1 L. monocytogenes serotype 4b strain LQC 15257, isolated from a strawberry sample and 4 log CFU·mL−1 S. cerevisiae strains Y32, Y34 and Y37, isolated from spontaneous olive fermentation. Sampling took place after 24 and 48 h incubation at 5 and 20 °C. RNA was extracted, stabilized and the transcription of virulence associated genes prfA, sigB, hly, plcA, plcB, inlA, inlB, inlC and inlJ, was assessed by RT-qPCR. Co-culture with the yeast strains mostly affected the transcription of sigB and inlJ, the upregulation of which during growth at 5 °C for 24 h, reached 10.13 and 9.76 log2(fold change), respectively. Similarly, the effect that incubation time had on the relative transcription of the genes under study was dependent on the co-cultivating yeast strain. On the other hand, the effect of the yeast strain was less pronounced when the relative transcription of the genes under study was assessed between 20 °C and 5 °C. In that case, incubation temperature seemed to have an important effect since, in the 79.2% of the samples analyzed, upregulation was evident, irrespective of yeast strain presence. These results highlight the complex trophic relationships that take place during co-existence between L. monocytogenes and S. cerevisiae.

Listeria monocytogenes Serogroup 1/2 Strains Have a Competitive Growth Advantage over Serotype 4b during Refrigerated Storage of an Artificially Contaminated Ready-To-Eat Pork Meat Product

Listeria monocytogenes is the bacterial causative agent of listeriosis, a life-threatening disease for humans, mainly transmitted through contaminated food. Human clinical isolates of the pathogen are frequently identified as serotype 4b strains; interestingly, however, serotype 4b (lineage I) is normally underrepresented among the food isolates in which serotype 1/2a (lineage II) is usually prevalent. The present study aimed to assess in situ dominance dynamics for the most commonly detected serotypes of L. monocytogenes implicated in foodborne listeriosis cases. A four-strain mixture comprised of L. monocytogenes serogroup 1/2 (i.e., serotypes 1/2a, 1/2b, and 1/2c) and serotype 4b food isolates was inoculated on a sliced ready-to-eat pork meat product, and dominance rates for the pathogenic strains were estimated based on serotype recoveries by utilizing multiplex polymerase chain reaction (mPCR), during storage of the product at 4 °C and 10 °C. The cumulative mPCR results showed that serotype 4b decreased at both storage temperatures, with the most abrupt decrease being noticed during storage at 10 °C. Irrespective of the storage temperature applied, L. monocytogenes strains of serogroup 1/2 predominated at the end of the meat product’s storage period. Conclusively, the preliminary findings of this research suggested a competitive growth advantage of L. monocytogenes serogroup 1/2 strains over serotype 4b during the refrigerated shelf-life of foods, thus advancing our knowledge on the pathogen’s behavior and contributing toward elucidating the manifested underrepresentation of serotype 4b in favor of serogroup 1/2 strains among the food isolates of the pathogen, particularly those recovered during detection and/or enumeration of L. monocytogenes in meat and products thereof.

Environmental dissemination of pathogenic Listeria monocytogenes in flowing surface waters in Switzerland

Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. The aquatic environment may represent a potential source for the transmission of L. monocytogenes to animals and the food chain. The present study assessed the occurrence of L. monocytogenes in 191 surface water samples from rivers, streams and inland canals throughout Switzerland. Twenty-five (13%) of the surface water samples contained L. monocytogenes. Whole genome sequence (WGS) data were used to characterize the 25 isolates. The isolates belonged to major lineages I and II, with the majority assigned to either serotype 1/2a (48%), or 4b (44%). The predominant CCs identified were the hypervirulent serotype 4b clones CC1 and CC4, and the serotype CC412; all three have been implicated in listeriosis outbreaks and sporadic cases of human and animal infection worldwide. Two (8%) of the isolates belonged to CC6 which is an emerging hypervirulent clone. All isolates contained intact genes associated with invasion and infection, including inlA/B and prfA. The four CC4 isolates all harbored Listeria pathogenicity island 4 (LIPI-4), which confers hypervirulence. The occurrence of L. monocytogenes in river ecosystems may contribute to the dissemination and introduction of clinically highly relevant strains to the food chain.

The complete genome sequence of Listeria monocytogenes strain S2542 and expression of selected genes under high-pressure processing

Objectives The study aims to generate the whole genome sequence of L. monocytogenes strain S2542 and to compare it to the genomes of strains RO15 and ScottA. In addition, we aimed to compare gene expression profiles of L. monocytogenes strains S2542, ScottA and RO15 after high-pressure processing (HPP) using ddPCR. Results The whole genome sequence of L. monocytogenes S2542 indicates that this strain belongs to serotype 4b, in contrast to the previously reported serotype 1/2a. Strain S2542 appears to be more susceptible to the treatment at 400 MPa compared to RO15 and ScottA strains. In contrast to RO15 and ScottA strains, viable cell counts of strain S2542 were below the limit of detection after HPP (400 MPa/8 min) when stored at 8 °C for 24 and 48 h. The transcriptional response of all three strains to HPP was not significantly different.

Characterization of a Novel Group of Listeria Phages That Target Serotype 4b Listeria monocytogenes

Listeria monocytogenes serotype 4b strains are the most prevalent clinical isolates and are widely found in food processing environments. Bacteriophages are natural viral predators of bacteria and are a promising biocontrol agent for L. monocytogenes. The aims of this study were to characterize phages that specifically infect serotype 4b strains and to assess their ability to inhibit the growth of serotype 4b strains. Out of 120 wild Listeria phages, nine phages were selected based on their strong lytic activity against the model serotype 4b strain F2365. These nine phages can be divided into two groups based on their morphological characteristics and host range. Comparison to previously characterized phage genomes revealed one of these groups qualifies to be defined as a novel species. Phages LP-020, LP-027, and LP-094 were selected as representatives of these two groups of phages for further characterization through one-step growth curve and inhibition of serotype 4b L. monocytogenes experiments. Listeria phages that target serotype 4b showed an inhibitory effect on the growth of F2365 and other serotype 4 strains and may be useful for biocontrol of L.monocytogenes in food processing environments.

Identification and Characterization of a Novel Genomic Island Harboring Cadmium and Arsenic Resistance Genes in Listeria welshimeri

Listeria monocytogenes, the bacterial foodborne pathogen responsible for the severe disease listeriosis, frequently exhibits heavy metal resistance. Concurrent resistance to cadmium and arsenic in L. monocytogenes is strongly associated with the 35-kb chromosomal island LGI2. LGI2 has been encountered repeatedly among L. monocytogenes serotype 4b hypervirulent clones but, surprisingly, not among non-pathogenic Listeria spp. Here we describe a novel LGI2 variant, LGI2-3, in two L. welshimeri strains from an urban aquatic environment. Whole genome sequence analysis revealed that the genomes were closely related except for one prophage region and confirmed a chromosomally integrated LGI2-3. It harbored a cystathionine beta-lyase gene previously only encountered in LGI2-1 of L. monocytogenes clonal complex 1 but was otherwise most closely related to LGI2. LGI2-3 harbored a novel cadAC cassette (cadA7C7) that, like LGI2′s cadA4C4, was associated with lower-level tolerance to cadmium (MIC 50 μg/mL) than other cadAC cassettes (MIC ≥ 140 μg/mL). CadA sequence analysis identified two amino acids that may be important for mediating different levels of cadmium tolerance. Our findings clearly demonstrated the potential for LGI2-like islands to be harbored by non-pathogenic Listeria spp. and generate intriguing hypotheses on the genetic diversity mediated by this island and its transfer among Listeria spp.

Prevalence and antibiotic resistance profile of Listeria spp. associated with seafoods from fish catchment areas in Kerala, India

Background and Aim: Listeria monocytogenes is a ubiquitous, intracellular pathogen which has been implicated as a cause of several foodborne outbreaks. This study aimed to generate information on the prevalence and antibiotic resistance profile of Listeria species isolated from seafood. Materials and Methods: A total of 400 samples of fresh fish, 100 samples of dry fish and 200 samples each of crustaceans and mollusks were collected from the fish catchment areas. All the samples were subjected to isolation and identification of Listeria spp. by two-step enrichment in UVM broth and plating on selective agar media (PALCAM) and then subjected to molecular characterization. L. monocytogenes isolates obtained during the study were subjected to serotyping by multiplex polymerase chain reaction. The isolates were also subjected to antibiotic sensitivity test. Results: The prevalence of L. monocytogenes in seafoods in the present study was 0.55%. The isolates of L. monocytogenes were found to possess all virulence genes, namely, iap, hlyA, actA, prfA, plcA, and inlA. All the isolates belonged to serotype 4b. The occurrence of Listeria innocua was found to be more and was detected in 16.77% of seafoods samples. Antibiotic sensitivity test revealed that all isolates were resistant to cefixime but were sensitive to almost all other commonly used antibiotics. Conclusion: The presence of Listeria spp. in raw seafood samples augments the need for implementation of good hygienic practices during the handling and processing of seafoods to safeguard the health of the consumers.