Listeria Pathogenicity Island Research Articles

EⅡB Mutation Reduces the Pathogenicity of Listeria monocytogenes by Negatively Regulating Biofilm Formation Ability, Infective Capacity, and Virulence Gene Expression

To explore the role of the membrane permease ⅡB (EⅡB) gene of Listeria pathogenicity island 4 (LIPI-4) in the virulence of Listeria monocytogenes, both an EⅡB deletion strain (∆EⅡB) and a complemented strain were constructed. In vitro experiments demonstrated that EⅡB deletion affected the biofilm formation ability of the wild-type strain (Lm928). Moreover, this deletion decreased the intracellular proliferation abilities of L. monocytogenes. Mice infected with ∆EⅡB survived longer and experienced less weight loss on days 1, 2, and 3 post-infection. The bacterial load in the liver tissue of ∆EⅡB-infected mice was significantly reduced, and a considerable decrease in the blood levels of inflammatory cytokines IL-β, IL-6, IL-10, and TNF-α were observed. Following EⅡB deletion, 65% (13/20) of genes were downregulated, 25% (5/20) were upregulated, and 10% (2/20) showed no change. These findings suggest that EⅡB deletion may reduce both the in vivo and in vitro virulence levels as well as the biofilm formation ability of Lm928 by downregulating the transcription levels of genes associated with virulence and biofilm formation. These findings provide a foundation for further examining the pathogenic mechanisms of LIPI-4 and EⅡB in L. monocytogenes.

Genomic Insights into Listeria monocytogenes: Organic Acid Interventions for Biofilm Prevention and Control.

Listeria monocytogenes is an important pathogen that has been implicated in foodborne illness. The aim of the present study was to investigate the diversity of virulence factors associated with the mechanisms of pathogenicity, persistence, and formation of biofilm L. monocytogenes by tandem analysis of whole-genome sequencing. The lineages that presented L. monocytogenes (LmAV-2, LmAV-3, and LmAV-6) from Hass avocados were lineages I and II. Listeria pathogenicity island 1 (LIPI-1) and LIPI-2 were found in the isolates, while LIPI-3 and Listeria genomic island (LGI-2) only was in IIb. Stress survival island (SSI-1) was identified in lineage I and II. In the in silico analysis, resistance genes belonging to several groups of antibiotics were detected, but the bcrABC and transposon Tn6188 related to resistance to quaternary ammonium salts (QACs) were not detected in L. monocytogenes. Subsequently, the anti-L. monocytogenes planktonic cell effect showed for QACs (MIC = 6.25 ppm/MBC = 100 ppm), lactic acid (MBC = 1 mg/mL), citric acid (MBC = 0.5 mg/mL) and gallic acid (MBC = 2 mg/mL). The anti-biofilm effect with organic acids (22 °C) caused a reduction of 4-5 log10 cfu/cm2 after 10 min against control biofilm L. monocytogenes formed on PP than SS. This study is an important contribution to understanding the genomic diversity and epidemiology of L. monocytogenes to establish a control measure to reduce the impact on the environment and the consumer.

The Slaughterhouse as Hotspot of CC1 and CC6 Listeria monocytogenes Strains with Hypervirulent Profiles in an Integrated Poultry Chain of Italy.

In Europe, very few studies are available regarding the diversity of Listeria monocytogenes (L. monocytogenes) clonal complexes (CCs) and sequence types (ST) in poultry and on the related typing of isolates using whole genome sequencing (WGS). In this study, we used a WGS approach to type 122 L. monocytogenes strains isolated from chicken neck skin samples collected in two different slaughterhouses of an integrated Italian poultry company. The studied strains were classified into five CCs: CC1-ST1 (21.3%), CC6-ST6 (22.9%), CC9-ST9 (44.2%), CC121-ST121 (10.6%) and CC193-ST193 (0.8%). CC1 and CC6 strains presented a virulence gene profile composed of 60 virulence genes and including the Listeria Pathogenicity Island 3, aut_IVb, gltA and gltB. According to cgMLST and SNPs analysis, long-term persistent clusters belonging to CC1 and CC6 were found in one of the two slaughterhouses. The reasons mediating the persistence of these CCs (up to 20 months) remain to be elucidated, and may involve the presence and the expression of stress response and environmental adaptation genes including heavy metals resistance genes (cadAC, arsBC, CsoR-copA-copZ), multidrug efflux pumps (mrpABCEF, EmrB, mepA, bmrA, bmr3, norm), cold-shock tolerance (cspD) and biofilm-formation determinants (lmo0673, lmo2504, luxS, recO). These findings indicated a serious risk of poultry finished products contamination with hypervirulent L. monocytogenes clones and raised concern for the consumer health. In addition to the AMR genes norB, mprF, lin and fosX, ubiquitous in L. monocytogenes strains, we also identified parC for quinolones, msrA for macrolides and tetA for tetracyclines. Although the phenotypical expression of these AMR genes was not tested, none of them is known to confer resistance to the primary antibiotics used to treat listeriosis The obtained results increase the data on the L. monocytogenes clones circulating in Italy and in particular in the poultry chain.

Whole-genome sequencing of Listeria monocytogenes isolated from the first listeriosis foodborne outbreak in South Korea.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans with severe symptoms. In South Korea, listeriosis had only been reported sporadically among hospitalized patients until the first foodborne outbreak occurred in 2018. In this study, a L. monocytogenes strain responsible for this outbreak (FSCNU0110) was characterized via whole genome sequencing and compared with publicly available L. monocytogenes genomes of the same clonal complex (CC). Strain FSCNU0110 belonged to multilocus sequence typing (MLST)-based sequence type 224 and CC224, and core genome MLST-based sublineage 6,178. The strain harbored tetracycline resistance gene tetM, four other antibiotic resistance genes, and 64 virulence genes, including Listeria pathogenicity island 1 (LIPI-1) and LIPI-3. Interestingly, llsX in LIPI-3 exhibited a characteristic SNP (deletion of A in position 4, resulting in a premature stop codon) that was missing among all CC224 strains isolated overseas but was conserved among those from South Korea. In addition, the tetM gene was also detected only in a subset of CC224 strains from South Korea. These findings will provide an essential basis for assessing the characteristics of CC224 strains in South Korea that have shown a potential to cause listeriosis outbreaks.

Whole-genome sequencing of Listeria innocua recovered from retail milk and dairy products in Egypt.

The similarity of the Listeria innocua genome with Listeria monocytogenes and their presence in the same niche may facilitate gene transfer between them. A better understanding of the mechanisms responsible for bacterial virulence requires an in-depth knowledge of the genetic characteristics of these bacteria. In this context, draft whole genome sequences were completed on five L. innocua isolated from milk and dairy products in Egypt. The assembled sequences were screened for antimicrobial resistance and virulence genes, plasmid replicons and multilocus sequence types (MLST); phylogenetic analysis of the sequenced isolates was also performed. The sequencing results revealed the presence of only one antimicrobial resistance gene, fosX, in the L. innocua isolates. However, the five isolates carried 13 virulence genes involved in adhesion, invasion, surface protein anchoring, peptidoglycan degradation, intracellular survival, and heat stress; all five lacked the Listeria Pathogenicity Island 1 (LIPI-1) genes. MLST assigned these five isolates into the same sequence type (ST), ST-1085; however, single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed 422-1,091 SNP differences between our isolates and global lineages of L. innocua. The five isolates possessed an ATP-dependent protease (clpL) gene, which mediates heat resistance, on a rep25 type plasmids. Blast analysis of clpL-carrying plasmid contigs showed approximately 99% sequence similarity to the corresponding parts of plasmids of L. monocytogenes strains 2015TE24968 and N1-011A previously isolated from Italy and the United States, respectively. Although this plasmid has been linked to L. monocytogenes that was responsible for a serious outbreak, this is the first report of L. innocua containing clpL-carrying plasmids. Various genetic mechanisms of virulence transfer among Listeria species and other genera could raise the possibility of the evolution of virulent strains of L. innocua. Such strains could challenge processing and preservation protocols and pose health risks from dairy products. Ongoing genomic research is necessary to identify these alarming genetic changes and develop preventive and control measures.

Whole-Genome Sequencing-Based Characterization of Clinical Listeria monocytogenes Isolates in China, 2013-2019.

Invasive listeriosis is a rare but serious foodborne disease that causes maternal-neonatal, central nervous system, and bloodstream infections. The aim of this study was to assess the whole-genome sequencing (WGS)-based genetic diversity of clinical Listeria monocytogenes isolates over a 7-year period and prove the effect of WGS application in food vehicle investigation. A total of 360 isolates were recovered during 2013 and 2019 through the national listeriosis special surveillance program. Two hundred twenty-six isolates (62.8%) were associated with pregnancy. All isolates belonged to lineage I (214 isolates) or lineage II (146 isolates), with 4 serogroups (46.9% IIb, 39.7% IIa, 12.5% IVb, and 0.8% IIc). All isolates were in 25 clonal complexes (CCs) and 3 singletons, with CC87, CC8, and CC5 being the most common causes of human listeriosis. All clinical isolates were positive for Listeria pathogenicity island 1 (LIPI-1), LIPI-3 was present in 21.4% of isolates and LIPI-4 was detected in 29.2% of isolates. LIPI-4-positive isolates, including CC87, sequence type (ST)619, ST382, CC4, and CC2, have been shown to confer hypervirulence. Fifteen isolates harbored at least one antimicrobial encoding gene, including tet (M), mef (A), msr (D), and dfr (G). The sublineage designations were consistent with CC designations, and 215 distinct cgMLST types (CTs) were classified, the most abundant being CT58 and CT750. In summary, there is a high level of genetic diversity among the clinical isolates. WGS has strengthened listeriosis surveillance and will be implemented for other foodborne bacteria in the National Molecular Tracing Network for Foodborne Disease.

Contrasting Genetic Diversity of Listeria Pathogenicity Islands 3 and 4 Harbored by Nonpathogenic Listeria spp.

Listeria monocytogenes causes the severe foodborne disease listeriosis. Several clonal groups of L. monocytogenes possess the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Here, we investigated the prevalence and genetic diversity of LIPI-3 and LIPI-4 among 63 strains of seven nonpathogenic Listeria spp. from the natural environment, i.e., wildlife (black bears [Ursus americanus]) and surface water. Analysis of the whole-genome sequence data suggested that both islands were horizontally acquired but differed considerably in their incidence and genetic diversity. LIPI-3 was identified among half of the L. innocua strains in the same genomic location as in L. monocytogenes (guaA hot spot) in a truncated form, with only three strains harboring full-length LIPI-3, and a highly divergent partial LIPI-3 was observed in three Listeria seeligeri strains, outside the guaA hot spot. Premature stop codons (PMSCs) and frameshifts were frequently noted in the LIPI-3 gene encoding listeriolysin S. On the other hand, full-length LIPI-4 without any PMSCs was found in all Listeria innocua strains, in the same genomic location as L. monocytogenes and with ~85% similarity to the L. monocytogenes counterpart. Our study provides intriguing examples of genetic changes that pathogenicity islands may undergo in nonpathogenic bacterial species, potentially in response to environmental pressures that promote either maintenance or degeneration of the islands. Investigations of the roles that LIPI-3 and LIPI-4 play in nonpathogenic Listeria spp. are warranted to further understand the differential evolution of genetic elements in pathogenic versus nonpathogenic hosts of the same genus. IMPORTANCE Listeria monocytogenes is a serious foodborne pathogen that can harbor the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Intriguingly, these have also been reported in nonpathogenic L. innocua from food and farm environments, though limited information is available for strains from the natural environment. Here, we analyzed whole-genome sequence data of nonpathogenic Listeria spp. from wildlife and surface water to further elucidate the genetic diversity and evolution of LIPI-3 and LIPI-4 in Listeria. While the full-length islands were found only in L. innocua, LIPI-3 was uncommon and exhibited frequent truncation and genetic diversification, while LIPI-4 was remarkable in being ubiquitous, albeit diversified from L. monocytogenes. These contrasting features demonstrate that pathogenicity islands in nonpathogenic hosts can evolve along different trajectories, leading to either degeneration or maintenance, and highlight the need to examine their physiological roles in nonpathogenic hosts.

Assessment of the influence of selected stress factors on the growth and survival of Listeria monocytogenes

BackgroundListeria monocytogenes are Gram-positive rods, which are the etiological factor of listeriosis. L. monocytogenes quickly adapts to changing environmental conditions. Since the main source of rods is food, its elimination from the production line is a priority. The study aimed to evaluate the influence of selected stress factors on the growth and survival of L. monocytogenes strains isolated from food products and clinical material.ResultsWe distinguished fifty genetically different strains of L. monocytogenes (PFGE method). Sixty-two percent of the tested strains represented 1/2a-3a serogroup. Sixty percent of the rods possessed ten examined virulence genes (fbpA, plcA, hlyA, plcB, inlB, actA, iap, inlA, mpl, prfA). Listeria Pathogenicity Island 1 (LIPI-1) was demonstrated among 38 (76.0%) strains. Majority (92.0%) of strains (46) were sensitive to all examined antibiotics. The most effective concentration of bacteriophage (inhibiting the growth of 22 strains; 44.0%) was 5 × 108 PFU. In turn, the concentration of 8% of NaCl was enough to inhibit the growth of 31 strains (62.0%). The clinical strain tolerated the broadest pH range (3 to 10). Five strains survived the 60-min exposure to 70˚C, whereas all were alive at each time stage of the cold stress experiment. During the stress of cyclic freezing-defrosting, an increase in the number of bacteria was shown after the first cycle, and a decrease was only observed after cycle 3. The least sensitive to low nutrients content were strains isolated from frozen food. The high BHI concentration promoted the growth of all groups.ConclusionsData on survival in stress conditions can form the basis for one of the hypotheses explaining the formation of persistent strains. Such studies are also helpful for planning appropriate hygiene strategies within the food industry.

Whole-genome sequencing reveals genomic characterization of Listeria monocytogenes from food in China.

Listeria monocytogenes is a foodborne bacterium that could persist in food and food processing environments for a long time. Understanding the population structure and genomic characterization of foodborne L. monocytogenes is essential for the prevention and control of listeriosis. A total of 322 foodborne L. monocytogenes isolates from 13 geographical locations and four food sources in China between 2000 and 2018 were selected for whole-genome sequencing. In silico subtyping divided the 322 isolates into five serogroups, 35 sequence types (STs), 26 clonal complexes (CCs) and four lineages. Serogroup IIa was the most prevalent serogroup and ST9 was the most prevalent ST of foodborne L. monocytogenes strains isolated in China. The in-depth phylogenetic analysis on CC9 revealed that ST122 clone might be original from ST9 clone. Furthermore, 23 potentially relevant clusters were identified by pair-wised whole-genome single nucleotide polymorphism analysis, indicating that persistent- and/or cross-contamination had occurred in markets in China. ST8 and ST121 were the second and third top STs of L. monocytogenes in China, which had heterogeneity with that of L. monocytogenes isolates from other countries. The antibiotic resistance genes aacA4, tetM, tetS, dfrG carried by different mobile elements were found in L. monocytogenes strains. One lineage II strain carrying Listeria Pathogenicity Island 3 was first reported. In addition, a novel type of premature stop codon in inlA gene was identified in this study. These findings revealed the genomic characteristics and evolutionary relationship of foodborne L. monocytogenes in China on a scale larger than previous studies, which further confirmed that whole-genome sequencing analysis would be a helpful tool for routine surveillance and source-tracing investigation.

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.

Whole-genome sequence of a putative pathogenic Bacillus sp. strain SD-4 isolated from cattle feed

ObjectivesThe present study describes the draft genome sequence of a novel Bacillus sp. strain SD-4 isolated from animal feed. The study aims to get a deeper insight into antimicrobial resistance and secondary metabolite biosynthetic gene clusters (BGCs) and the association between them. MethodsThe strain SD-4 was preliminarily evaluated for antibacterial activities, motility, biofilm formation, and enterotoxin production using in vitro assays. The genome of strain SD-4 was sequenced using the Illumina HiSeq 2500 platform with paired-end reads. The reads were assembled and annotated using SPAdes and PGAP, respectively. The genome was further analysed using several bioinformatics tools, including TYGS, AntiSMASH, RAST, PlasmidFinder, VFDB, VirulenceFinder, CARD, PathogenFinder, MobileElement finder, IslandViewer, and CRISPRFinder. ResultsIn vitro assays showed that the strain is motile, synthesises biofilm, and produces an enterotoxin and antibacterial metabolites. The genome analysis revealed that the strain SD-4 carries antimicrobial resistance genes (ARGs), virulence factors, and beneficial secondary metabolite BGCs. Further genome analysis showed interesting genome architectures containing several mobile genetic elements, including two plasmid replicons (repUS22 and rep20), five prophages, and at least four genomic islands (GIs), including one Listeria pathogenicity island LIPI-1. Moreover, the strain SD-4 is identified as a putative human pathogen. ConclusionThe genome of strain SD-4 harbours several BGCs coding for biologically active metabolites. It also contains antimicrobial resistance genes and is identified as a potential human pathogen. These results can be used to better comprehend antibiotic resistance in environmental bacteria that are not influenced by human intervention.

Characterization of Listeria monocytogenes isolated from wildlife in central New York.

Background Listeria monocytogenes (Lm) present in farming soil and food‐processing facilities threatens food safety, but little is known about the carriage of Lm by wildlife.ObjectivesWe estimated the prevalence of faecal Lm shedding among wildlife admitted to a veterinary medical teaching hospital in central New York and characterized a subset of the Lm isolates.MethodsWildlife samples were collected between May 2018 and December 2019. We characterized the Lm isolates by assessing the growth at three temperatures approximating the body temperatures of reptiles (25°C), mammals (37°C), and birds (42°C) and identifying genotypic characteristics related to transmission and virulence.ResultsThe apparent prevalence of faecal Lm shedding was 5.6% [18/324; 95% confidence interval (CI), 3.3%–8.6%]. Among 13 isolates that represented two lineages and 11 clonal complexes, three and five isolates were grouped into the same SNP clusters with human clinical isolates and environmental isolates, respectively. However, specific SNP difference data showed that Lm from wildlife was generally not closely related (>22 SNP differences) to Lm from human clinical sources and the food‐processing environment. While the stress response locus SSI‐2 was absent, SSI‐1 was found in four isolates. Virulence genes prfA, plcA, hly, mpl, actA, plcB, inlA, inlB, inlC, inlE, inlH, inlJ, and inlK were present, without any premature stop codons, in all isolates. Virulence loci Listeria pathogenicity island 3 (LIPI‐3) and LIPI‐4, which have been linked to hypervirulence, and inlG were found in four, three, and seven isolates, respectively.ConclusionsWildlife represents a potential reservoir for genetically diverse and putatively hypervirulent Lm strains. No statistically significant association between growth parameters and hosts was observed. However, compared to lineage I isolates, lineage II isolates showed significantly (p < 0.05) faster growth at 25°C and significantly slower growth at 42°C, suggesting that wildlife Lm isolates that belong to lineages I and II differ in their ability to grow at 25°C and 42°C.

Systematic review ofListeria monocytogenesfrom food and clinical samples in Chinese mainland from 2010 to 2019

AbstractListeria monocytogenes (L. monocytogenes), a foodborne pathogen, can cause human listeriosis. Listeriosis is a potentially fatal gastrointestinal illness, which is closely related to the spread of food to humans. We review the literature published during 2010–2019 to better understand the prevalence of L. monocytogenes in food products, incidence of human listeriosis, and their characteristics in Chinese mainland. We found the main sequence types (STs) strains from foods are similar globally, and the prevalence of L. monocytogenes from raw meat was the highest among all food products. The most common STs in food products and clinical cases were ST9 (serogroup I.2) strains and ST87 (serogroup II.2) strains, respectively. The ST87 strains being the most common STs of clinical cases might be related to the existence of Listeria pathogenicity island 4 gene and Chinese eating habits for ready-to-eat foods, among which the prevalence of ST87 strain was the highest in ready-to-eat foods. Therefore, more research should be conducted to explore the reasons for the L. monocytogenes isolates differences in food and clinical sources.

Application of Whole Genome Sequencing to Understand Diversity and Presence of Genes Associated with Sanitizer Tolerance in Listeria monocytogenes from Produce Handling Sources.

Recent listeriosis outbreaks linked to fresh produce suggest the need to better understand and mitigate L. monocytogenes contamination in packing and processing environments. Using whole genome sequencing (WGS) and phenotype screening assays for sanitizer tolerance, we characterized 48 L. monocytogenes isolates previously recovered from environmental samples in five produce handling facilities. Within the studied population there were 10 sequence types (STs) and 16 cgMLST types (CTs). Pairwise single nucleotide polymorphisms (SNPs) ranged from 0 to 3047 SNPs within a CT, revealing closely and distantly related isolates indicative of both sporadic and continuous contamination events within the facility. Within Facility 1, we identified a closely related cluster (0–2 SNPs) of isolates belonging to clonal complex 37 (CC37; CT9492), with isolates recovered during sampling events 1-year apart and in various locations inside and outside the facility. The accessory genome of these CC37 isolates varied from 94 to 210 genes. Notable genetic elements and mutations amongst the isolates included the bcrABC cassette (2/48), associated with QAC tolerance; mutations in the actA gene on the Listeria pathogenicity island (LIPI) 1 (20/48); presence of LIPI-3 (21/48) and LIPI-4 (23/48). This work highlights the potential use of WGS in tracing the pathogen within a facility and understanding properties of L. monocytogenes in produce settings.

Genomic characteristics of Listeria monocytogenes isolated during mushroom (Agaricus bisporus) production and processing

Listeria monocytogenes is a foodborne pathogen ubiquitously found in nature and which has been isolated from food and food processing environments. This study aimed to characterize L. monocytogenes strains isolated from the production and processing environments of frozen sliced mushrooms (Agaricus bisporus). An analysis was executed along the mushroom processing chain including one mushroom grower and two mushroom processing factories. A total of 153 L. monocytogenes strains were isolated, which could be grouped in three PCR serogroups, namely, serogroup 1/2a-3a (39.2%), serogroup 1/2b-3b-7 (34.0%) and serogroup 4b-4d-4e (26.8%). A selection of 44 L. monocytogenes strains isolated from the processing environment after cleaning and disinfection (C&D) and from frozen sliced mushrooms was genotyped by whole genome sequencing (WGS), because these strains pose a potential risk for product contamination after C&D and for human consumption. Multilocus sequence typing (MLST) revealed 11 clonal complexes (CCs), with strains belonging to CC1, CC4, CC37 and CC87 being detected in both processing factories. Comparative WGS analysis of the 44 strains showed the presence of Listeria pathogenicity island 1 (LIPI-1) with a disrupted version of actA in all CC1, CC4, CC5, CC59 strains, and all but one CC224 strains. Notably, both inlA and inlB were detected as full-length loci in every strain, except for inlA in a CC6 strain that harbored a three amino acid deletion. LIPI-3 was detected in all CC1, CC4, CC6 and CC224 strains, while LIPI-4 was detected in all CC4 and CC87 strains. In addition, antibiotic susceptibility tests showed susceptibility towards fourteen antibiotics tested. The bcrABC operon was found in one CC5 strain, that showed a higher tolerance towards benzalkonium chloride than any other strain tested with confluent growth till 12.5 μg/ml for the CC5 strain compared to 2.5 μg/ml for the other strains. This study highlights that the ecology of L. monocytogenes in the frozen sliced mushroom production chain is highly diverse, and shows the importance of hygienic measures to control L. monocytogenes along the frozen sliced mushroom production chain.

Conditions of In Vitro Biofilm Formation by Serogroups of Listeria monocytogenes Isolated from Hass Avocados Sold at Markets in Mexico.

Listeria monocytogenes is an important pathogen that has been implicated in foodborne illnesses and the recall of products such as fruit and vegetables. This study determines the prevalence of virulence-associated genes and serogroups and evaluates the effects of different growth media and environmental conditions on biofilm formation by L. monocytogenes. Eighteen L. monocytogenes isolates from Hass avocados sold at markets in Guadalajara, Mexico, were characterized by virulence-associated genes and serogroup detection with PCR. All isolates harbored 88.8% actA, 88.8% plcA, 83.3% mpl, 77.7% inlB, 77.7% hly, 66.6% prfA, 55.5% plcB, and 33.3% inlA. The results showed that 38.8% of isolates harbored virulence genes belonging to Listeria pathogenicity island 1 (LIPI-1). PCR revealed that the most prevalent serogroup was serogroup III (1/2b, 3b, and 7 (n = 18, 66.65%)), followed by serogroup IV (4b, 4d–4e (n = 5, 27.7%)) and serogroup I (1/2a–3a (n = 1, 5.5%)). The assessment of the ability to develop biofilms using a crystal violet staining method revealed that L. monocytogenes responded to supplement medium TSBA, 1/10 diluted TSBA, and TSB in comparison with 1/10 diluted TSB (p < 0.05) on polystyrene at 240 h (p < 0.05). In particular, the biofilm formation by L. monocytogenes (7.78 ± 0.03–8.82 ± 0.03 log10 CFU/cm2) was significantly different in terms of TSBA on polypropylene type B (PP) (p < 0.05). In addition, visualization by epifluorescence microscopy, scanning electron microscopy (SEM), and treatment (DNase I and proteinase K) revealed the metabolically active cells and extracellular polymeric substances of biofilms on PP. L. monocytogenes has the ability to develop biofilms that harbor virulence-associated genes, which represent a serious threat to human health and food safety.

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.

Prevalence and Genetic Diversity of Listeria monocytogenes Isolated From Retail Pork in Wuhan, China.

Listeria monocytogenes is a ubiquitous bacteria and causative agent of zoonotic listeriosis with high mortality. The consumption of contaminated animal-derived foods has been linked with both epidemic and sporadic listeriosis. In this work, a total of 64 L. monocytogenes isolates from 259 pork samples sold in 11 supermarket chains were identified and characterized by comparative whole-genome analysis. All isolates were delineated into eight clonal complexes (CCs), namely CC2, CC8, CC9, CC11, CC155, CC121, CC204, and CC619, spanning two lineages (I and II) and carrying 3–5 antibiotic-resistant genes (fosX, lnu, mprF, tetM, and dhfR). It is noted that Listeria pathogenicity island (LIPI)-1, LIPI-3, and LIPI-4 were distributed in all ST619 isolates from two supermarket chains that were closely related with clinical isolates (<40 SNP). Some of the isolates from different supermarket chains with 0 SNP difference indicated a common pork supply source. Notably, 57.81% of the strains carried types IB, IIA, or IIIB CRISPR-Cas system, CC121 isolates carried both types IB and IIA CRISPR-Cas systems, Cas proteins of CC155 isolates located between two CRISPR loci, each CC has unique organization of Cas proteins as well as CRISPR loci. CRISPR-Cas system-based subtyping improved discrimination of pork-derived L. monocytogenes isolates. Comparisons at the genome level contributed to understand the genetic diversities and variations among the isolates and provided insights into the genetic makeup and relatedness of these pathogens.

Nosocomial cross-infection of hypervirulent Listeria monocytogenes sequence type 87 in China.

BackgroundTo investigate the epidemiological and phenotypic characteristics and molecular relatedness of L. monocytogenes, which were cultured from the blood and cerebrospinal fluid (CSF) samples isolated from two neonates.MethodsIn the present case study, two infected neonates were interviewed and epidemiological investigation performed. The phenotypic characteristics and molecular relatedness of L. monocytogenes was characterized by serotyping, pulsed-field gel electrophoresis and whole-genome sequencing (WGS).ResultsThe field investigation found that the two neonates were born in the same hospital (Hospital B) and admitted to the neonatal department through different channels within half an hour by different nurses, where they were weighed and placed in different but adjacent incubators. Then they were cared for by the same group of nurses that evening. It is worth noting that there was no record of sanitation of the neonatal incubator of neonate-1. The serotype of the two isolated L. monocytogenes were 1/2b, with an indistinguishable pulsotypes and were sequence type (ST) 87. WGS showed that there were no core SNP differences identified. In order to explore the genomic traits associated with L. monocytogenes virulence genes, we identified the Listeria pathogenicity island 4 and found that the genome was devoid of any stress islands. There are no positive results from the environmental samples. Considering the genomic data together with epidemiological evidence and clinical symptoms, insufficient surface cleaning along with the nursing staff caring for these neonates was considered as cross-infection factors.ConclusionsTo our knowledge, this is the first report of a nosocomial cross-infection of L. monocytogenes ST87 between two neonates, which carries the recently identified gene cluster expressing the cellobiose-family phosphotransferase system (PTS-LIPI-4) between two neonates. The test results of environmental samples in the hospital indicate that strict sterilization and patient isolation measures cannot be emphasized enough in neonatal nursing.

Genetic diversity and profiles of genes associated with virulence and stress resistance among isolates from the 2010-2013 interagency Listeria monocytogenesmarket basket survey.

Whole genome sequencing (WGS) was performed on 201 Listeria monocytogenes isolates recovered from 102 of 27,389 refrigerated ready-to-eat (RTE) food samples purchased at retail in U.S. FoodNet sites as part of the 2010–2013 interagency L. monocytogenes Market Basket Survey (Lm MBS). Core genome multi-locus sequence typing (cgMLST) and in-silico analyses were conducted, and these data were analyzed with metadata for isolates from five food groups: produce, seafood, dairy, meat, and combination foods. Six of 201 isolates, from 3 samples, were subsequently confirmed as L. welshimeri. Three samples contained one isolate per sample; mmong the 96 samples that contained two isolates per sample, 3 samples each contained two different strains and 93 samples each contained duplicate isolates. After 93 duplicate isolates were removed, the remaining 102 isolates were delineated into 29 clonal complexes (CCs) or singletons based on their sequence type. The five most prevalent CCs were CC155, CC1, CC5, CC87, and CC321. The Shannon’s diversity index for clones per food group ranged from 1.49 for dairy to 2.32 for produce isolates, which were not significantly different in pairwise comparisons. The most common molecular serogroup as determined by in-silico analysis was IIa (45.6%), followed by IIb (27.2%), IVb (20.4%), and IIc (4.9%). The proportions of isolates within lineages I, II, and III were 48.0%, 50.0% and 2.0%, respectively. Full-length inlA was present in 89.3% of isolates. Listeria pathogenicity island 3 (LIPI-3) and LIPI-4 were found in 51% and 30.6% of lineage I isolates, respectively. Stress survival islet 1 (SSI-1) was present in 34.7% of lineage I isolates, 80.4% of lineage II isolates and the 2 lineage III isolates; SSI-2 was present only in the CC121 isolate. Plasmids were found in 48% of isolates, including 24.5% of lineage I isolates and 72.5% of lineage II isolates. Among the plasmid-carrying isolates, 100% contained at least one cadmium resistance cassette and 89.8% contained bcrABC, involved in quaternary ammonium compound tolerance. Multiple clusters of isolates from different food samples were identified by cgMLST which, along with available metadata, could aid in the investigation of possible cross-contamination and persistence events.