Internalin Genes Research Articles

Virulence Potential and Antimicrobial Resistance of Listeria monocytogenes Isolates Obtained from Beef and Beef-Based Products Deciphered Using Whole-Genome Sequencing

Listeria monocytogenes is a ubiquitous bacterial pathogen that threatens the food chain and human health. In this study, whole-genome sequencing (WGS) was used for the genomic characterization of L. monocytogenes (n = 24) from beef and beef-based products. Multilocus Sequence Type (MLST) analysis revealed that ST204 of CC204 was the most common sequence type (ST). Other sequence types detected included ST1 and ST876 of CC1, ST5 of CC5, ST9 of CC9, ST88 of CC88, ST2 and ST1430 of CC2, and ST321 of CC321. Genes encoding for virulence factors included complete LIPI-1 (pfrA-hly-plcA-plcB-mpl-actA) from 54% (13/24) of the isolates of ST204, ST321, ST1430, and ST9 and internalin genes inlABC that were present in all the STs. All the L. monocytogenes STs carried four intrinsic/natural resistance genes, fosX, lin, norB, and mprF, conferring resistance to fosfomycin, lincosamide, quinolones, and cationic peptides, respectively. Plasmids pLGUG1 and J1776 were the most detected (54% each), followed by pLI100 (13%) and pLM5578 (7%). The prophage profile, vB_LmoS_188, was overrepresented amongst the isolates, followed by LP_101, LmoS_293_028989, LP_030_2_021539, A006, and LP_HM00113468. Listeria genomic island 2 (LGI-2) was found to be present in all the isolates, while Listeria genomic island 3 (LGI-3) was present in a subset of isolates (25%). The type VII secretion system was found in 42% of the isolates, and sortase A was present in all L. monocytogenes genomes. Mobile genetic elements and genomic islands did not harbor any virulence, resistance, or environmental adaptation genes that may benefit L. monocytogenes. All the STs did not carry genes that confer resistance to first-line antibiotics used for the treatment of listeriosis. The characterization of L. monocytogenes in our study highlighted the environmental resistance and virulence potential of L. monocytogenes and the risk posed to the public, as this bacterium is frequently found in food and food processing environments.

Genomic characterization of Listeria monocytogenes recovered from dairy facilities in British Columbia, Canada from 2007 to 2017.

Listeria monocytogenes is a foodborne pathogen of concern in dairy processing facilities, with the potential to cause human illness and trigger regulatory actions if found in the product. Monitoring for Listeria spp. through environmental sampling is recommended to prevent establishment of these microorganisms in dairy processing environments, thereby reducing the risk of product contamination. To inform on L. monocytogenes diversity and transmission, we analyzed genome sequences of L. monocytogenes strains (n = 88) obtained through the British Columbia Dairy Inspection Program. Strains were recovered from five different dairy processing facilities over a 10 year period (2007-2017). Analysis of whole genome sequences (WGS) grouped the isolates into nine sequence types and 11 cgMLST types (CT). The majority of isolates (93%) belonged to lineage II. Within each CT, single nucleotide polymorphism (SNP) differences ranged from 0 to 237 between isolates. A highly similar (0-16 SNPs) cluster of over 60 isolates, collected over 9 years within one facility (#71), was identified suggesting a possible persistent population. Analyses of genome content revealed a low frequency of genes associated with stress tolerance, with the exception of widely disseminated cadmium resistance genes cadA1 and cadA2. The distribution of virulence genes and mutations within internalin genes varied across the isolates and facilities. Further studies are needed to elucidate their phenotypic effect on pathogenicity and stress response. These findings demonstrate the diversity of L. monocytogenes isolates across dairy facilities in the same region. Findings also showed the utility of using WGS to discern potential persistence events within a single facility over time.

Outcomes of the multicenter monitoring of the causative agent of invasive listeriosis in the metropolis

Introduction. Invasive listeriosis is a rare disease posing a threat to high-risk groups and often leading to a fatal outcome. Its causative agent is Listeria monocytogenes, a ubiquitous saprophyte that has turned into an important foodborne pathogen with the growing industry of semi-cooked and ready-to-eat products.
 The aim of the study is the characterization of L. monocytogenes isolates in the Moscow region and identification of possible causes of susceptibility to infection
 Materials and methods. The multicenter monitoring of L. monocytogenes was conducted in the Moscow metropolitan area, using bacteriological and genomic methods for description of the pathogen, medical history collection and detailed analysis of patient case summaries.
 Results. In the cohorts of patients with perinatal listeriosis (PL) and meningitis-septicemia (MS), invasive listeriosis had a year-round occurrence with slight upswings in MarchApril and JulyNovember. During the COVID-19 pandemic, in the MS group, the minimum age of patients decreased to 31 years and the proportion of deaths increased 1.57-fold compared to 20182019. During the pandemic, an increase in the diversity of L. monocytogenes genotypes was observed, along with changes in the spectrum of pathogen genotypes throughout the pandemic stages.
 During the monitoring, a total of 73 L. monocytogenes clinical isolates belonging to 24 genotypes were described. Seven genotypes belonged to the first phylogenetic lineage (PLI); 14 genotypes belonged to PLII. The PL cohort had the highest proportion of PLI genotypes (52%). In the MS cohort, the group of men had the widest diversity of genotypes, 6 of which were identical to genotypes of food isolates. In the analysed set of isolates, 12 new profiles of internalin genes were identified and described. The whole genome sequencing detected the presence of plasmids in 9 of 58 genomes of clinical isolates. The comparison of core genomes revealed an epidemic relationship between isolates of the same genotype for ST4, ST21, and ST425.
 Conclusion. The performed study presents a detailed description of the diversity and virulence of L. monocytogenes circulating in the Moscow metropolitan area, thus providing information for timely diagnosis and treatment of invasive listeriosis.

Comparative Genomics of Listeria Species Recovered from Meat and Food Processing Facilities.

ABSTRACTListeria species (spp.) are contaminants that can survive in food, on equipment, and on food processing premises if appropriate hygiene measures are not used. Homologous stress tolerance genes, virulence gene clusters such as the prfA cluster, and clusters of internalin genes that contribute to the pathogenic potential of the strains can be carried by both pathogenic and nonpathogenic Listeria spp. To enhance understanding of the genome evolution of virulence and virulence-associated properties, a comparative genome approach was used to analyze 41 genome sequences belonging to L. innocua and L. welshimeri isolated from food and food processing facilities. Genetic determinants responsible for disinfectant and stress tolerance were identified, including the efflux cassette bcrABC and Tn6188_qac_1 disinfectant resistance determinant, and stress survival islets. These disinfectant-resistant genes were more frequently found in L. innocua (12%) than in L. welshimeri (2%). Several isolates representing the presumed nonpathogenic L. innocua still carried virulence-associated genes, including LGI2, LGI3, LIPI-3, and LIPI-4 which were absent in all L. welshimeri isolates. The mobile genetic elements identified were plasmids (pLGUG1 and J1776) and prophages (PHAGE_Lister_vB_LmoS_188, PHAGE_Lister_LP_030_3, PHAGE_Lister_A118, PHAGE_Lister_B054, and PHAGE_Lister_vB_LmoS_293). The results suggest that the presumed nonpathogenic isolates especially L. innocua can carry genes relevant to the strain’s virulence and stress tolerance in the food and food processing facilities.IMPORTANCE This study provides genomic insights into the recently expanded genus in order to gain valuable information about the evolution of the virulence and stress tolerance properties of the genus Listeria and the distribution of these genetic elements pertinent to the pathogenic potential across Listeria spp. and clonal lineages in South Africa (SA).

Retrospective Investigation of the Whole Genome of the Hypovirulent Listeria monocytogenes Strain of ST201, CC69, Lineage III, Isolated from a Piglet with Fatal Neurolisteriosis.

Listeria monocytogenes (Lm), the causative agent for both human and animal listeriosis, is considered to be a rare but potentially fatal foodborne pathogen. While Lm strains associated with current cases of human listeriosis are now being intensely investigated, our knowledge of this microorganism which has caused listerial infection in the past is still extremely limited. The objective of this study was a retrospective whole-genome sequence analysis of the Lm collection strain, 4/52-1953, isolated in the middle of the 20th century from a piglet with listerial neuroinfection. The multi-locus sequence typing (MLST) analysis based on seven housekeeping genes (abcZ, bglA, cat, dapE, dat, ldh, and lhkA) showed that the Lm strain 4/52-1953 was assigned to the sequence type 201 (ST201), clonal complex 69 (CC69), and phylogenetic lineage III. The strain 4/52-1953, similarly to other ST201 strains, probably originated from the ST9, CC69 via ST157. At least eight different STs, ST69, ST72, ST130, ST136, ST148, ST469, ST769, and ST202, were identified as the descendants of the first generation and a single one, ST2290, was proved to be the descendant of the second generation. Among them there were strains either associated with some sporadic cases of human and animal listerial infection in the course of more than 60 years worldwide or isolated from food samples, fish and dairy products, or migratory birds. Phylogenetic analysis based on whole genomes of all the Lm strains available in the NCBI GenBank (n = 256) demonstrated that the strain 4/52-1953 belonged to minor Cluster I, represented by lineage III only, while two other major Clusters, II and III, were formed by lineages I and II. In the genome of the strain 4/52-1953, 41 virulence-associated genes, including the Listeria pathogenicity island 1 (LIPI-1), and LIPI-2 represented by two internalin genes, the inlA and inlB genes, and five genes related to antibiotic resistance, were found. These findings can help to make the emergence of both hyper- and hypovirulent variants, including those bearing antibiotic resistance genes, more visible and aid the aims of molecular epidemiology as well.

Soil Collected in the Great Smoky Mountains National Park Yielded a Novel Listeria sensu stricto Species, L. swaminathanii.

ABSTRACTSoil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI = 93.9%, isDDH = 55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) nonhemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity islands 1, 2, 3, and 4 as well as the internalin genes inlA and inlB. While the type strain contains an apparently intact catalase gene (kat), this strain is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species). Additional analyses identified a nonsynonymous mutation in a conserved codon of kat that is likely linked to the catalase-negative phenotype. Rapid species identification systems, including two biochemical and one matrix-assisted laser desorption/ionization, misidentified this novel species as either L. monocytogenes, L. innocua, or L. marthii. We propose the name L. swaminathanii, and the type strain is FSL L7-0020T (=ATCC TSD-239T).IMPORTANCE L. swaminathanii is a novel sensu stricto species that originated from a US National Park and it will be the first Listeria identified to date without official standing in the nomenclature. Validation was impeded by the National Park’s requirements for strain access, ultimately deemed too restrictive by the International Committee on Systematics of Prokaryotes. However, lack of valid status should not detract from the significance of adding a novel species to the Listeria sensu stricto clade. Notably, detection of non-monocytogenes sensu stricto species in a food processing environment indicate conditions that could facilitate the presence of the pathogen L. monocytogenes. If isolated, our data show a potential for L. swaminathanii to be misidentified as another sensu stricto, notably L. monocytogenes. Therefore, developers of Listeria spp. detection and identification methods, who historically only include validly published species in their validation studies, should include L. swaminathanii to ensure accurate results.

Occurrence, antimicrobial resistance, and genetic diversity of <i>Listeria monocytogenes</i> at fish-processing plants in Vietnam

In this study, the occurrence, serogroups, virulence genes, antibiotic susceptibility profiles, and diversity were estimated for Listeria monocytogenes isolated from Pangasius fish in two processing facilities in Vietnam. L. monocytogenes was most frequently detected in wash water samples at 20.8% (15 out of 72), and in Pangasius fish it was 16.6% (15 out of 90 fish samples). Serotypes 1/2b were dominant, and internalin genes (inlA, inlC, and inlJ) were found in all the L. monocytogenes isolates. Most of the isolates were resistant to cefoxitin, oxacillin, and fosfomycin. Genotyping of L. monocytogenes by random amplified polymorphic DNA analysis revealed three clusters of the isolates specific to the facilities to some extent. This study suggests a high risk of L. monocytogenes contamination from wash water to fish. Hence, maintaining the quality of water and designing sanitation procedures to reduce L. monocytogenes contamination are required to ensure food safety at Pangasius fish processing plants.

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Non-coding RNA regulates phage sensitivity in Listeria monocytogenes.

Non-coding RNAs (ncRNAs) have gained increasing attention as their diverse roles in virulence and environmental stress in Listeria monocytogenes have become clearer. The ncRNA rliB is an atypical member of the CRISPR family, conserved at the same genomic locus in all analyzed L. monocytogenes genomes and also in other Listeria species. In this study, rliB defective mutants (Lm3-22-ΔrliB) were constructed by homologous recombination. The growth cycle of Lm3-22-ΔrliB mutants was slower than that of wild-type Lm3-22. The sensitivity of Lm3-22-ΔrliB to the Listeria phage vB-LmoM-SH3-3 was significantly increased, and the efficiency of plaque formation was enhanced by 128 fold. Compared with wild type, the adhesion and invasion of Lm3-22-ΔrliB decreased significantly (9.3% and 1.33%, respectively). After 4 hours of infection, the proliferation of Lm3-22-ΔrliB in RAW264.7 cells also decreased significantly. Transcription level of invasion-related surface proteins showed that the internalin genes lmo0610 and lm0514, and the peptidoglycan binding protein gene lmo1799 in Lm3-22-ΔrliB were significantly increased. In addition, after interaction with phage, the transcription levels of inlA, lmo0610, lmo1799, lmo2085, and lmo0514 in Lm3-22-ΔrliB cells were significantly upregulated, while inlB was downregulated, compared with Lm3-22 control group with phage treatment. Therefore, rliB deletion effectively regulated the interaction between Listeria and phage, weaken its invasion ability, and provided a new theoretical basis for biocontrol of phage.

Whole Genome-Based Characterization of Listeria monocytogenes Isolates Recovered From the Food Chain in South Africa.

Listeria monocytogenes is an important foodborne pathogen which has the ability to adapt and survive in food and food processing facilities where it can persist for years. In this study, a total of 143 L. monocytogenes isolates in South Africa (SA) were characterized for their strain’s genetic relatedness, virulence profiles, stress tolerance and resistance genes associated with L. monocytogenes. The Core Genome Multilocus Sequence Typing (cgMLST) analysis revealed that the most frequent serogroups were IVb and IIa; Sequence Types (ST) were ST204, ST2, and ST1; and Clonal Complexes (CC) were CC204, CC1, and CC2. Examination of genes involved in adaptation and survival of L. monocytogenes in SA showed that ST1, ST2, ST121, ST204, and ST321 are well adapted in food processing environments due to the significant over-representation of Benzalkonium chloride (BC) resistance genes (bcrABC cassette, ermC, mdrL and Ide), stress tolerance genes (SSI-1 and SSI-2), Prophage (φ) profiles (LP_101, vB LmoS 188, vB_LmoS_293, and B054 phage), plasmids profiles (N1-011A, J1776, and pLM5578) and biofilm formation associated genes. Furthermore, the L. monocytogenes strains that showed hyper-virulent potential were ST1, ST2 and ST204, and hypo-virulent were ST121 and ST321 because of the presence and absence of major virulence factors such as LIPI-1, LIPI-3, LIPI-4 and the internalin gene family members including inlABCEFJ. The information provided in this study revealed that hyper-virulent strains ST1, ST2, and ST204 could present a major public health risk due to their association with meat products and food processing environments in SA.

Novel Sequence Types of Listeria monocytogenes of Different Origin Obtained in the Republic of Serbia.

Listeria monocytogenes, the causative agent of listeriosis, is amongst the major food-borne pathogens in the world that affect mammal species, including humans. This microorganism has been associated with both sporadic episodes and large outbreaks of human listeriosis worldwide, with high mortality rates. In this study, the main sequence types (STs) and clonal complexes (CCs) were investigated in all of the 13 L. monocytogenes strains originating from different sources in the Republic of Serbia in 2004–2019 and that were available in the BIGSdb-Lm database. We found at least 13 STs belonging to the phylogenetic lineages I and II. These strains were represented by ST1/ST3/ST9 of CC1/CC3/CC9, which were common in the majority of the European countries and worldwide, as well as by eight novel STs (ST1232/ST1233/ST1234/ST1235/ST1238/ST1236/ST1237/ST1242) of CC19/CC155/CC5/CC21/CC3/CC315/CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1), reported in the Republic of Serbia, the EU, for the first time. Our study confirmed the association of CC1 with cases of neuroinfection and abortions among small ruminants, and of CC3 and CC9 with food products of animal origin. The strains isolated in 2019 carried alleles of the internalin genes (inlA/inlB/inlC/inlE) characteristic of the most virulent strains from the hypervirulent CC1. These findings demonstrated the genetic relatedness between L. monocytogenes strains isolated in the Republic of Serbia and worldwide. Our study adds further information about the diversity of the L. monocytogenes genotypes of small ruminants and food products, as the strain distribution in these sources in Serbia had not previously been evaluated.

Listeria cossartiae sp. nov., Listeria farberi sp. nov., Listeria immobilis sp. nov., Listeria portnoyi sp. nov. and Listeria rustica sp. nov., isolated from agricultural water and natural environments.

A total of 27 Listeria isolates that could not be classified to the species level were obtained from soil samples from different locations in the contiguous United States and an agricultural water sample from New York. Whole-genome sequence-based average nucleotide identity blast (ANIb) showed that the 27 isolates form five distinct clusters; for each cluster, all draft genomes showed ANI values of <95 % similarity to each other and any currently described Listeria species, indicating that each cluster represents a novel species. Of the five novel species, three cluster with the Listeria sensu stricto clade and two cluster with sensu lato. One of the novel sensu stricto species, designated L. cossartiae sp. nov., contains two subclusters with an average ANI similarity of 94.9%, which were designated as subspecies. The proposed three novel sensu stricto species (including two subspecies) are Listeria farberi sp. nov. (type strain FSL L7-0091T=CCUG 74668T=LMG 31917T; maximum ANI 91.9 % to L. innocua ), Listeria immobilis sp. nov. (type strain FSL L7-1519T=CCUG 74666T=LMG 31920T; maximum ANI 87.4 % to L. ivanovii subsp. londoniensis ) and Listeria cossartiae sp. nov. [subsp. cossartiae (type strain FSL L7-1447T=CCUG 74667T=LMG 31919T; maximum ANI 93.4 % to L. marthii ) and subsp. cayugensis (type strain FSL L7-0993T=CCUG 74670T=LMG 31918T; maximum ANI 94.7 % to L. marthii ). The two proposed novel sensu lato species are Listeria portnoyi sp. nov. (type strain FSL L7-1582T=CCUG 74671T=LMG 31921T; maximum ANI value of 88.9 % to L. cornellensis and 89.2 % to L. newyorkensis ) and Listeria rustica sp. nov. (type strain FSL W9-0585T=CCUG 74665T=LMG 31922T; maximum ANI value of 88.7 % to L. cornellensis and 88.9 % to L . newyorkensis ). L. immobilis is the first sensu stricto species isolated to date that is non-motile. All five of the novel species are non-haemolytic and negative for phosphatidylinositol-specific phospholipase C activity; the draft genomes lack the virulence genes found in Listeria pathogenicity island 1 (LIPI-1), and the internalin genes inlA and inlB, indicating that they are non-pathogenic.

Analysis of sporadic cases of invasive listeriosis in a metropolis

Introduction. Listeriosis is a foodborne infection, especially dangerous for people in at-risk groups. Susceptibility to listeria infection is determined by a complex of reasons: environmental factors, host immune status, and pathogen virulence. The susceptibility to listeriosis can also be aggravated by previous infections, especially viral infections, which demonstrate a steadily increasing number of identified pathogens.The aim of our study was to present molecular and genetic characterization of pathogens causing sporadic invasive listeriosis in a megalopolis, primarily during the peak of influenza and ARVI incidence.Materials and methods. Listeria monocytogenes isolates were collected from 18 hospitalized patients at hospitals in Moscow, from November 2018 to October 2019. The first comparison group was represented by isolates from food products and fish preserves. The second comparison group included previously examined environmental isolates. The clinical isolates were examined by using multilocus sequence typing techniques, including the standard MLST scheme extended by loci of internalin genes. Isolates of the autochthonous genotype (ST7) were compared through whole-genome sequencing and subsequent analysis of the core genome (cgMLST).Results. In cases of invasive listeriosis, 44% of isolates were isolated from patients with listeriosis; 27% of isolates were obtained from patients with meningitis. L. monocytogenes of phylogenetic lineage II prevailed in these groups of cases that occurred when the epidemic threshold for influenza was crossed during the 2018/2019 season. Listeria pneumonia identified in the senior age group occurred during the season of autumn ARVI and was primarily caused by L. monocytogenes of phylogenetic lineage I. The examination of genomes of ST7 isolates demonstrated identity between the core genomes of bacteria isolated from the mother-infant pair. Out of ST7 food isolates most closely related to the clinical ones was the isolate from meat (23 locus differences, the common deletion in the MFS transporter locus). Analyzing invasive listeriosis, the comparison between the list of the identified genotypes and the data from European countries showed that each country had its own specific range of genotypes, though ST7 was detected in all the examined samples.Conclusions. Along with the monitoring of food manufacturing and storage, timely vaccination against seasonal respiratory infections and use of personal protective equipment in public spaces can reduce the risk of listeriosis incidence in at-risk groups.

Occurrence, Diversity of Listeria spp. Isolates from Food and Food-Contact Surfaces and the Presence of Virulence Genes.

This study evaluates the hazards posed by foodborne bacteria of the Listeria genus by analyzing the occurrence, diversity and virulence of Listeria spp.in food and food-manufacturing plants. Seventy-five isolates obtained from the routine analysis of 653 samples taken by three diagnostic laboratories in Northern Italy were genotypically differentiated by Repetitive Extragenic Palindrome (rep) PCR, with the GTG5 primer identified by sequencing the 16S rRNA gene and examined by specific PCR tests for the presence of L. monocytogenes virulence determinants occasionally found to occur in other species of the genus. Within this sample, 76% (n = 57) isolates were identified as L. innocua, 16% (n = 12) as L. monocytogenes, 6.6% (n = 5) as L. welshimeri and 1.3% (n = 1) as L. seeligeri. All L. monocytogenes isolates belonged to the serotype 1/2a and were predicted to be virulent for the presence of the inlJ internalin gene. Potentially virulent strains of L. innocua, L. seeligeri and L. welshimeri, carrying the L. monocytogenesinlA gene and/or hly gene, were identified, and most isolates were found to possess the toxin–antitoxin system mazEF for efficient adaptation to heat shock. Results indicated the need to reinforce food-contamination-prevention measures against all Listeria species by defining efficiently their environmental distribution.

Prevalence of Listeria Species in Dairy Cows and Pregnant Women with Reference to Virulotyping of Listeria monocytogenes in Egypt

This study was conducted to determine the prevalence of Listeria spp . in milk and feces of dairy cows as well as stool of pregnant women in Sharkia and Dakahlia Governorates, Egypt. Three hundred and fifty samples including; normal raw milk (n=200), mastitis milk (n=50), feces of dairy cattle (n=50) and stool of pregnant women (n=50) were randomly collected. Listeria spp. were isolated on Oxford agar and then subjected to biochemical and molecular identification. The overall isolation rate of Listeria spp. was 16%. The prevalence rates of Listeria monocytogenes were 8%, 4%, 2% and 4% in feces of dairy cows, normal milk, mastitis milk and stool of pregnant women, respectively. The prevalence of L. ivanovii and L. welshimeri in milk of dairy cows were 6% and 4%, respectively. L. monocytogenes isolates were molecularly confirmed to the species level by amplification of 16S rRNA gene. The distribution of internalin A (inlA) and internalin B (inlB) was determined in 15 identified L. monocytogenes isolates. The overall prevalence of inlA gene and inlB gene in L. monocytogenes isolates from different sources were 80% and 40%, respectively. Two L. monocytogenes isolates from normal raw milk and feces of dairy cows didn’t harbor both inlA and inlB genes. Listeria spp. isolated from normal milk, feces, mastitis milk and stool of pregnant women, potentially can cause human illness and abortion in pregnant women. Internalin (A&B) genes are considered the best indicator for virulence determination of L. monocytogenes isolated from different sources.

Characterization of internalin genes in Listeria monocytogenes from food and humans, and their association with the invasion of Caco-2 cells

BackgroundInternalins are surface proteins that are utilized by Listeria monocytogenes to facilitate its invasion into human intestinal epithelial cells. The expression of a full-length InlA is one of essential virulence factors for L. monocytogenes to cross the intestinal barrier in order to invade epithelial cells.ResultsIn this study, the gene sequences of inlA in 120 L. monocytogenes isolates from food (n = 107) and humans (n = 13) were analyzed. Premature stop codon (PMSC) mutations in inlA were identified in 51 isolates (50 from food and 1 from human). Six mutation types of PMSCs were identified. Among the 51 isolates with PMSCs in inlA, there were 44 serogroup 1/2c, 3c isolates from food, of which seven belonged to serogroups 1/2a, 3a. A total of 153,382 SNPs in 2247 core genes from 42 genomes were identified and used to construct a phylogenetic tree. Serotype 1/2c isolates with inlA PMSC mutations were grouped together. Cell culture studies on 21 isolates showed that the invasion to Caco-2 cells was significantly reduced among isolates with inlA PMSC mutations compared to those without PMSC mutations (P < 0.01). The PMSC mutations in inlA correlated with the inability of the L. monocytogenes isolates to invade Caco-2 cells (Pearson’s coefficient 0.927, P < 0.01).ConclusionOverall, the study has revealed the reduced ability of L. monocytogenes to invade human intestinal epithelial cells in vitro was linked to the presence of PMSC mutations in inlA. Isolates with PMSC mutations shared the same genomic characteristics indicating the genetic basis on the potential virulence of L. monocytogenes invasion.

Occurrence, serotypes, and characteristics of Listeria monocytogenes in meat and meat products in South Africa between 2014 and 2016

AbstractListeria monocytogenes is an intracellular pathogen that causes life‐threatening disease called listeriosis. However, in South Africa (SA), there is a dearth of information concerning the potential role of meat and meat products as potential sources for human listeriosis. The aim of the study was to determine the occurrence and level of L. monocytogenes found in meat and meat products in SA and to characterize L. monocytogenes strains according to serogroups, antimicrobial resistance profiles and virulence genes. A total of 2,017 samples from imported and locally produced raw, processed and ready to eat meat were collected from 2014 to 2016 across nine provinces of SA. The samples for L. monocytogenes were isolated using microbiological techniques and real‐time polymerase chain reaction (PCR). Antimicrobial resistance profiles of the isolates were determined by testing 19 antimicrobial impregnated discs using Kirby–Bauer disc diffusion method. Characterization in terms of serogroup typing and virulence profiling was done using conventional PCR. The overall occurrence of L. monocytogenes was 14.7% (296/2,017), which varied between meat collected on the domestic market (15.0%; 264/1,758) and directly at the three ports of entries (12.4%; 32/259). The contamination level of the positive samples ranged from 1 to 3.7 log CFU/g and 1 to 4.1 log CFU/g for samples collected from the domestic and imported meat, respectively. All positive isolates were serotyped by multiplex PCR, of which majority of the isolates belonged to molecular serogroup ½a‐3a (45.5%), followed by 4b‐4d‐4e (24.2%), and ½c‐3c (15.2%). Most of the isolates harbored the inlJ (98.7%) and ipa (95.6%) genes. However, at least one of the other internalin genes (inlB, inlC, and inlA) was present in most of the isolates. All the tested isolates showed resistance to at least 3 of the 19 antibiotics, with 5 (1.7%) of them displaying resistance to 13 of the 19 antibiotics. Resistance to streptomycin (99.0%), clindamycin (97.3%), fusidic acids (95.6%), nitrofurantoin (79.7%), and gentamycin (74.4%) was observed while high rates of sensitivity were observed for ampicillin (85.6%), kanamycin (84.6%), amikacin (77.6%), vancomycin (74.2%), and tetracycline (62.5%). The presence of L. monocytogenes in various meat products in SA pose a risk to human health. Therefore, the present research provides valuable baseline information that will help in the development of policies and regulations for monitoring of L. monocytogenes in meat and meat products in SA.Practical applicationsThis study is extensive and involved analysis of meat and meat products from all the nine provinces of South Africa. Furthermore, the study involved analysis of imported meat (from the three major ports of entry) that was destined for the South African market. The findings that were obtained are significant from academic, policy, and practical points of view. The findings provide empirical evidence of the contamination of a proportion of raw intact, raw processed, and ready‐to‐eat meat and products that are available on the South African market. Furthermore, our findings illustrate the levels of contamination, which is important for providing guidelines with respect to L. monocytogenes contamination of meats. Information of the predominant L. monocytogenes serotypes from this study provide important epidemiological data of the strains that are circulating in meat and meat products. The antimicrobial resistance results are important for contributing to guidelines on antimicrobial use.

Listeriosis: genotyping as a key for identification a possible source of infection

Objective. To perform a comparative analysis of clinical and food isolates of Listeria monocytogenes collected in the European part of Russia in 2018–2019. Materials and Methods. We used multilocus sequencing (MLST), supplemented by virulence loci, including fragments of internalin genes (MvLST, Multi-virulent-locus sequence typing), followed by phylogenetic analysis. Results. The main diagnoses for clinical isolates were prenatal and neonatal listeriosis and meningitis. Clinical isolates predominantly belonged to phylogenetic line II with the predominance of ST7, which was also the most abundant in food isolates. The second most common occurrence in food isolates was ST121, widely distributed in Europe. Isolates of phylogenetic line I in the group of clinical cultures in three cases were represented by ST6, detected during outbreaks of listeriosis in Europe 2015–2018 and South Africa in 2017–2018. Only in one isolate from food belonged to the phylogenetic lineage I. In general, the diversity of food isolate genotypes was significantly higher than clinical isolates. The analysis of virulence loci revealed a new internalin A allele and a new internalin genes profile (IP) in isolate ST7 from food. Conclusions. L. monocytogenes of the most common ST7 is autochthonous in Russia; cases of listeriosis caused by the ST6 bacterium are most likely imported. Based on the analysis of the diversity of ST and IP of L. monocytogenes identified in Russia, a rapid diagnosis scheme for epidemiological investigation is proposed.

CodY-Mediated c-di-GMP-Dependent Inhibition of Mammalian Cell Invasion in Listeria monocytogenes.

Elevated levels of the second messenger c-di-GMP suppress virulence in diverse pathogenic bacteria, yet mechanisms are poorly characterized. In the foodborne pathogen Listeria monocytogenes, high c-di-GMP levels inhibit mammalian cell invasion. Here, we show that invasion is impaired because of the decreased expression levels of internalin genes whose products are involved in invasion. We further show that at high c-di-GMP levels, the expression of the entire virulence regulon is suppressed, and so is the expression of the prfA gene encoding the master activator of the virulence regulon. Analysis of mechanisms controlling prfA expression pointed to the transcription factor CodY as a c-di-GMP-sensitive component. In high-c-di-GMP strains, codY gene expression is decreased, apparently due to the lower activity of CodY, which functions as an activator of codY transcription. We found that listerial CodY does not bind c-di-GMP in vitro and therefore investigated whether c-di-GMP levels affect two known cofactors of listerial CodY, branched-chain amino acids and GTP. Our manipulation of branched-chain amino acid levels did not perturb the c-di-GMP effect; however, our replacement of listerial CodY with the streptococcal CodY homolog, whose activity is GTP independent, abolished the c-di-GMP effect. The results of this study suggest that elevated c-di-GMP levels decrease the activity of the coordinator of metabolism and virulence, CodY, possibly via lower GTP levels, and that decreased CodY activity suppresses L. monocytogenes virulence by the decreased expression of the PrfA virulence regulon.IMPORTANCEListeria monocytogenes is a pathogen causing listeriosis, a disease responsible for the highest mortality rate among foodborne diseases. Understanding how the virulence of this pathogen is regulated is important for developing treatments to decrease the frequency of listerial infections in susceptible populations. In this study, we describe the mechanism through which elevated levels of the second messenger c-di-GMP inhibit listerial invasion in mammalian cells. Inhibition is caused by the decreased activity of the transcription factor CodY that coordinates metabolism and virulence.

Prevalence of Antibiotics Resistant Listeria monocytogenes Strains in Nigerian Ready-to-eat Foods.

The Listerial flora and Listeria monocytogenes bio-load associated with 411 ready-to-eat (RTE) foods sold at several locations in southern Nigeria was evaluated using phenotypic procedures which included serial dilution and pour plate techniques. Selected L. monocytogenes strains phenotypically identified from the RTE foods were further identified using virulence gene markers and 16srRNA amplification procedures. All the 90 L. monocytogenes strains cultured from the RTE foods were subjected to antibiogram analysis using disc diffusion. Some of the antibiotics employed included; Ceftazidine, cefuroxime, gentamicin, ofloxacin, augumentin, tetracycline and erythromycin. L. monocytogenes L. ivanovii, L. grayi, L.welshimeri, L. seeligeri and L. innocua were detected in the RTE foods. Haemolysin (hlyA) gene, Internalin gene (inlA) and invasive gene (iap) were detected in all L. monocytogenes isolates. L. monocytogenes LMEW70 with accession number KY053295 was 93% similar to L. monocytogenes L1846. All the L. monocytogenes isolates were resistant to amoxicillin, cloxacillin, augumentin and ceftazidime.