Listeria Monocytogenes Isolates Research Articles

Prevalence and Antibiotic Resistance of Listeria Monocytogenes in Chicken Meat Retailers in Yazd, Iran

Introduction: Listeria monocytogenes is a pathogen bacteria transmitted by food stuffs. Due to the lack of information about contamination of retail chicken meat to Listeria monocytogenes in Iran, this study aimed to determine the prevalence and antibiotic resistance of this bacterium in retail chicken meat in Yazd, Iran. Materials and Methods: This cross-sectional descriptive study was conducted from January 2018 to June 2018 on 811 randomly selected samples from four districts located in Yazd city, Iran. Isolation of Listeria monocytogenes was performed using enrichment and selective culture media as well as biochemical tests. The positive samples were confirmed by PCR assay. Results: In total, 247 samples (30.5%) were infected with one of the Listeria strains. Among the 247 samples, the isolates were 68 (27%) L. monocytogenes, 155 (63%) L. innocua, 5 (2%) L. seeligeri, 19 (8%) L. ivanovii. In the current study, the antibiotic resistance of positive samples was also evaluated that Listeria spp., especially Listeria monocytogenes were resistant to tetracycline and penicillin. Conclusions: The presence of this pathogenic microorganism in chicken meat can be a health risk, especially for pregnant women, the elderly, and those with immune deficiency. Considering the amount of contamination with L.monocytogenes in chicken and the high mortality rate caused by it, observance of the health principles and standards during the production, transportation, maintenance, and supply phases as well as training of employees in this industry are necessary.

An outbreak of multiple genotypes of Listeria monocytogenes in New Zealand linked to contaminated ready-to-eat meats-a retrospective analysis using whole-genome sequencing.

Four cases of listeriosis in a hospital (A) in New Zealand were identified in 2012. Pulsed-field gel electrophoresis (PFGE) used at the time identified four pulsotypes amongst the clinical isolates. Two of the pulsotypes matched to Listeria monocytogenes isolates obtained from ready-to-eat (RTE) meat samples from a RTE producer tested during a nationwide microbiological survey the month prior. The outbreak investigation confirmed that the RTE producer had supplied product to the hospital and additional testing confirmed the presence of L.monocytogenes in RTE meats from the hospital kitchen. Two further listeriosis cases presented in another hospital (B) with one clinical isolate identified as the same pulsotype as identified for one case in hospital A, but the epidemiology information concluded that the clinical cases from hospital B were not linked to the outbreak. Retrospective whole-genome sequencing confirmed that epidemiologically linked isolates belonging to three different genotypes for clinical cases from hospital A and RTE meats samples from the hospital kitchen differed by 0-1 core-genome locus or single nucleotide polymorphisms (SNP). The use of core-genome multilocus sequence typing and SNP analysis provided a greater degree of discrimination between isolates compared to PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a listeriosis outbreak associated with a hospital in New Zealand and attributed to contaminated ready-to-eat (RTE) meat supplied to the hospital by a single producer. Retrospective whole-genome sequence analysis of outbreak isolates was found to provide a greater degree of discrimination between isolates compared to pulsed-field gel electrophoresis and supported the conclusions made at the time of the outbreak. The multiple genotypes identified from clinical cases and the RTE meats obtained during the outbreak highlight the importance of epidemiological concordance alongside genotyping.

Listeria monocytogenes infections: Analysis of 41 patients

ObjectiveWe describe and analyze Listeria-related demographics and clinical features to determine the predisposing conditions for severe infections. MethodsWe performed a retrospective study using positive isolation of Listeria monocytogenes from blood, cerebrospinal fluid, and other organic fluids. Electronic health records were used to determine the epidemiological and clinical features of infections caused by L. monocytogenes. Mortality and sepsis were considered dependent variables in the statistical analyses. ResultsWe included 41 patients in an observation period of 15 years (2003–2018), with an annual incidence rate of 1.3 cases per 100,000 population. Three main population profiles were identified: newborns, pregnant women, and other adults (17.1%, 12.2%, and 82.9%, respectively). Neuroinvasive infection was present in 17 patients (41.5%). In both univariate and multivariate analyses, neurological infections, whether meningoencephalitis, rhombencephalitis, or brain abscesses, were the main risk factors for severe forms of Listeria-related infections (odds ratio 1.8, 95% CI 1.52–2.14, p=0.01). Malignancies, whether solid tumors or hematological neoplasms, immunosuppression, and chronic diseases were not related to either mortality or severe clinical syndromes. ConclusionInfections caused by L. monocytogenes were uncommon but could cause severe sepsis and mortality, especially in susceptible populations. Our study focused on neurological involvement and severe invasive forms of listeriosis. Neuroinvasive forms were the most important risk factors for severe illness but not for mortality.

Antimicrobial Susceptibility and Multilocus Sequence Typing of Listeria monocytogenes Isolated Over 11 Years from Food, Humans, and the Environment in Italy.

Due to the increasing number of studies reporting the detection of antimicrobial-resistant isolates of Listeria monocytogenes, we sought to determine the antimicrobial susceptibility of L. monocytogenes isolates collected in Italy and find potential correlations to their serotypes and multilocus sequence types (MLST). The antimicrobial susceptibility of 317 L. monocytogenes isolates collected from food, humans, and the environment from 1998 to 2009 was assessed by minimum inhibitory concentration (MIC). Serotyping and MLST was also performed on all isolates. Potential correlations among antimicrobial resistance profiles, serotyping, and MLST were statistically evaluated. Twenty-four percent of L. monocytogenes isolates were resistant to oxacillin, 28.7% intermediate to clindamycin, and 24.3% to ciprofloxacin. The majority of isolates with elevated MIC to oxacillin was of environmental origin and belonged to serotype 4b/4e and ST2. Isolates with intermediate MIC values to clindamycin and ciprofloxacin were mostly of food and human origin and belonged to serotype 4b/4e and ST9. Regarding the time frame of isolate collection, comparing the last 3 years (2007-2009) to previous years (1998-2006), an increase was observed in the percentage of resistant and intermediate isolates per year. This trend strongly suggests the need for increasing attention on the prevalence of antimicrobial resistance in L. monocytogenes in Italy. To predict future resistance trends, the monitoring of clinical intermediate resistance might represent a useful tool especially for antibiotics associated to multiple-step mechanisms of acquired resistance. A specific focus should be addressed to antimicrobial-resistant isolates of serotype 4b, repeatedly associated with food-borne outbreaks.

Characterization of Listeria Monocytogenes Originating from the Spanish Meat-Processing Chain.

Using agglutination techniques, 118 Listeria monocytogenes isolates from red meat and poultry were serotyped. Strains were ascribed to the serotypes 4b/4e (44.1% of the strains), 1/2 (a, b or c; 28.0%), 4c (6.8%), 4d/4e (5.9%) and 3 (a, b or c; 2.5%). Among these are the serotypes most frequently involved in cases of human listeriosis. The susceptibility of 72 strains to 26 antibiotics of clinical importance was determined by disc diffusion (Clinical and Laboratory Standards Institute; CLSI). High levels of resistance were observed to cefoxitin (77.8% of the strains showed resistance), cefotaxime (62.5%), cefepime (73.6%), nalidixic acid (97.2%), nitrofurantoin (51.4%) and oxacillin (93.1%). Less than 3% of the strains showed resistance to the antibiotic classes used in human listeriosis therapy (i.e., ampicillin, gentamicin, rifampicin, chloramphenicol, enrofloxacin, vancomycin, trimethoprim-sulfamethoxazole, erythromycin, and tetracycline). The influence of species and serotype on the growth kinetics (modified Gompertz equation) and on the adhesion ability (crystal violet staining) of nine isolates of L. monocytogenes (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, and 4d), and one strain of Listeria ivanovii were investigated. The maximum growth rate (ΔOD420-580/h) varied between 0.073 ± 0.018 (L. monocytogenes 1/2a) and 0.396 ± 0.026 (L. monocytogenes 4b). The isolates of L. monocytogenes belonging to serotypes 3a and 4a, as well as L. ivanovii, showed a greater (p < 0.05) biofilm-forming ability than did the remaining strains, including those that belong to the serotypes commonly implied in human listeriosis (1/2a, 1/2b, 1/2c and 4b). The need for training in good hygiene practices during the handling of meat and poultry is highlighted to reduce the risk of human listeriosis.

Influence of two different culture media on biofilm formation by Listeria monocytogenes isolated from a small-scale meat processing facility

In this study, 20 Listeria monocytogenes isolates detected in a food processing environment and food products were tested for biofilm-forming ability in two different culture media: Tryptone Soya Broth and Luria Bertani Broth. Statistical analysis of the data obtained was performed with the MINITAB software package, version 16.0. The two-sample t-test and confidence interval were used for data analysis. Significant differences between the isolates were observed in the ability to form biofilms.

Selective Isolation and Concentration of Foodborne Bacterial Pathogens in a Microfluidic Device

Listeria monocytogenes is a bacterium that is present in contaminated food products including dairy, vegetables and meat. It is a deadly foodborne pathogen that has contributed to multiple outbreaks, and the disease caused by this pathogen has high mortality rates. The conventional methods used for Listeria detection include standard microbiology or biochemical procedures, which require time-consuming sample pretreatment techniques that can take up to 72 hours. There is a growing need for methodologies that reduce the test time for Listeria detection from days to an hour or less, while also allowing for scalability and high-volume processing. The challenge lies in processing and pretreating large volumes of food samples to generate sufficient quantities of analyte for detection tests within a short time period, without compromising the selectivity of the test. We have previously worked on the development of separation and detection methods for bacterial cells [1, 2]. This work describes a multi-chamber microfluidic cell sorting device that uses magnetophoresis to detect, separate, and concentrate tagged Listeria monocytogenes from food samples. Bacteriophage-functionalized magnetic particles will be used to isolate the bacteria in a food sample for pre-concentration, which will allow for more rapid and selective cell sorting followed by impedimetric detection of Listeria in an electrochemical cell. Ghuman, A., et al., Magnetic Separation of Food-Borne Pathogens in a Microfluidic Device, in IBE 2018 Annual Conference. 2018.Zhou, Y., et al., Isolation and Separation of Listeria monocytogenes Using Bacteriophage P100-Modified Magnetic Particles. Colloids and Surfaces B: Biointerfaces, 2018. Under Review.

Outbreak of Listeria monocytogenes in South Africa, 2017-2018: Laboratory Activities and Experiences Associated with Whole-Genome Sequencing Analysis of Isolates.

In South Africa, a progressive increase in listeriosis cases was noted from mid-June 2017, heralding what was to become the world's largest listeriosis outbreak. A total of 1060 cases were reported for the period January 1, 2017 to July 17, 2018. We describe laboratory activities, experiences, and results of whole-genome sequencing (WGS) analysis of Listeria monocytogenes isolates associated with this outbreak. Bacteria were identified using the VITEK-2 COMPACT 15 microbial identification system. WGS was performed using Illumina MiSeq technology. WGS data were analyzed using CLC Genomics Workbench Software and free-to-use on-line analysis tools/pipelines. Multilocus sequence typing (MLST) showed that 91% of clinical isolates were sequence type 6 (ST6), determining that the outbreak was largely associated with L. monocytogenes ST6. Epidemiological and laboratory findings led to investigation of a large ready-to-eat processed meat production facility in South Africa, named Enterprise Foods. L. monocytogenes ST6 was found in environmental sampling swabs of the production facility and in ready-to-eat processed meat products (including polony, a product similar to bologna sausage) manufactured at the facility. ST6 isolates, sourced at the Enterprise Foods production facility and from Enterprise food products, were shown by single nucleotide polymorphism (SNP) analysis to be highly related to clinical isolates; these nonclinical ST6 isolates showed <10 SNP differences when compared to clinical ST6 isolates. Core-genome MLST showed that clinical ST6 isolates and Enterprise-related ST6 isolates had no more than 4 allele differences between each other, suggestive of a high probability of epidemiological relatedness. WGS data interpreted together with epidemiological data concluded that the source of the listeriosis outbreak was ready-to-eat processed meat products manufactured by Enterprise Foods. Listeriosis has now been added to the South African list of mandatory notifiable medical conditions. Surveillance systems have been strengthened to facilitate prevention and early detection of listeriosis outbreaks.

Comparative Analysis of Tools and Approaches for Source Tracking Listeria monocytogenes in a Food Facility Using Whole-Genome Sequence Data.

As WGS is increasingly used by food industry to characterize pathogen isolates, users are challenged by the variety of analysis approaches available, ranging from methods that require extensive bioinformatics expertise to commercial software packages. This study aimed to assess the impact of analysis pipelines (i.e., different hqSNP pipelines, a cg/wgMLST pipeline) and the reference genome selection on analysis results (i.e., hqSNP and allelic differences as well as tree topologies) and conclusion drawn. For these comparisons, whole genome sequences were obtained for 40 Listeria monocytogenes isolates collected over 18 years from a cold-smoked salmon facility and 2 other isolates obtained from different facilities as part of academic research activities; WGS data were analyzed with three hqSNP pipelines and two MLST pipelines. After initial clustering using a k-mer based approach, hqSNP pipelines were run using two types of reference genomes: (i) closely related closed genomes (“closed references”) and (ii) high-quality de novo assemblies of the dataset isolates (“draft references”). All hqSNP pipelines identified similar hqSNP difference ranges among isolates in a given cluster; use of different reference genomes showed minimal impacts on hqSNP differences identified between isolate pairs. Allelic differences obtained by wgMLST showed similar ranges as hqSNP differences among isolates in a given cluster; cgMLST consistently showed fewer differences than wgMLST. However, phylogenetic trees and dendrograms, obtained based on hqSNP and cg/wgMLST data, did show some incongruences, typically linked to clades supported by low bootstrap values in the trees. When a hqSNP cutoff was used to classify isolates as “related” or “unrelated,” use of different pipelines yielded a considerable number of discordances; this finding supports that cut-off values are valuable to provide a starting point for an investigation, but supporting and epidemiological evidence should be used to interpret WGS data. Overall, our data suggest that cgMLST-based data analyses provide for appropriate subtype differentiation and can be used without the need for preliminary data analyses (e.g., k-mer based clustering) or external closed reference genomes, simplifying data analyses needs. hqSNP or wgMLST analyses can be performed on the isolate clusters identified by cgMLST to increase the precision on determining the genomic similarity between isolates.

Isolation and Characterization of Clinical Listeria monocytogenes in Beijing, China, 2014-2016.

Listeria monocytogenes is an important foodborne pathogen with a significant impact on public health worldwide. A great number of outbreaks caused by L. monocytogenes has been reported, especially in the United States, and European countries. However, listeriosis has not yet been included in notifiable disease in China, and thus information on this infection has been scarce among the Chinese population. In this study, we described a 3-year surveillance of listeriosis in Beijing, China. Fifty-six L. monocytogenes strains isolated from 49 clinical infectious cases (27 pregnancy-associated infections and 22 non-pregnancy-associated infections) were analyzed by serotyping, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and antimicrobial susceptibility testing between 2014 and 2016 in Beijing. The predominant serogroups were 1/2a,3a and 1/2b,3b,7 which accounted for 92% of the overall isolates. Four strains were serogroup 4b,4d,4e, isolated from patients with pregnancy-associated infections. Based on PFGE, these isolates were divided into 32 pulsotypes (PTs) and 3 clusters associated with serogroups. Ten PTs were represented by more than one isolate with PT09 containing the most number of isolates. MLST differentiated the isolates into 18 STs, without new ST designated. The three most common STs were ST8 (18.4%), ST5 (16.3%), and ST87 (12.2%), accounting for 46.9% of the isolates. STs prevalent in other parts of the world were also present in China such as ST1, ST2, ST5, ST8, and ST9 which caused maternal fetal infections or outbreaks. However, the STs and serogroup distribution of clinical L. monocytogenes in Beijing, China was different from those in other countries. Strains of ST1 and ST2 were isolated from patients with pregnancy-associated infection, whereas none of ST155 isolates caused pregnancy-associated cases. Surveillance of molecular characterization will provide important information for prevention of listeriosis. This study also enhances our understanding of genetic diversity of clinical L. monocytogenes in China.

Isolation, Potential Virulence, and Population Diversity of Listeria monocytogenes From Meat and Meat Products in China.

Listeria monocytogenes is a globally notorious foodborne pathogen. This study aimed to qualitatively and quantitatively detect L. monocytogenes from meat and meat products in China and to establish their virulence profiles and population diversity. From 1212 meat and meat product samples, 362 (29.9%) were positive for L. monocytogenes. Of these positive samples, 90.6% (328/362) had less than 10 MPN/g, 5.5% (20/364) samples had 10–110 MPN/g, and 3.9% (14/362) of the positive samples had over 110 MPN/g. Serogroup analysis showed that the most prevalent serogroup of L. monocytogenes was I.1 (1/2a-3a), which accounted for 45.0% (123/458) of the total, followed by serogroup I.2 (1/2c-3c) that comprised 26.9%, serogroup II.1 (4b-4d-4e) that comprised 4.8%, and serogroup II.2 (1/2b-3b-7) that comprised 23.3%. A total of 458 isolates were grouped into 35 sequence types (STs) that belonged to 25 clonal complexes (CCs) and one singleton (ST619) by multi-locus sequence typing. The most prevalent ST was ST9 (26.9%), followed by ST8 (17.9%), ST87 (15.3%), ST155 (9.4%), and ST121 (7.6%). Thirty-seven isolates harbored the llsX gene (representing LIPI-3), and they belonged to ST1/CC1, ST3/CC3, ST288/CC288, ST323/CC288, ST330/CC288, ST515/CC1, and ST619, among which ST323/CC288, ST330/CC288, and ST515/CC1 were newly reported to carry LIPI-3. Seventy-five isolates carried ptsA, and they belonged to ST87/CC87, ST88/CC88, and ST619, indicating that consumers may be exposed to potential hypervirulent L. monocytogenes. Antibiotics susceptibility tests revealed that over 90% of the isolates were susceptible to 11 antibiotics; however, 40.0% of the isolates exhibited resistance against ampicillin and 11.8% against tetracycline; further, 45.0 and 4.6% were intermediate resistant and resistant to ciprofloxacin, respectively. The rise of antibiotic resistance in L. monocytogenes suggests that stricter regulations should be formulated to restrict the use of antibiotic agents in human listeriosis treatment and livestock breeding.

Isolation and characterization of Listeria monocytogenes from environmental and clinical sources by culture and PCR-RFLP methods

Due to the widespread distribution of Listeria monocytogenes in environmental and animal sources and serious clinical complications in human, this study was aimed to isolate L. monocytogenes from water and clinical specimens by culture and PCR methods and to investigate the presence of hlyA and inlA virulence genes. Water and clinical samples of vaginal and fecal were screened for the presence of L. monocytogenes by phenotypic and standard biochemical tests. PCR amplification was performed on extracted DNA using primers based on the hlyA and inlA genes. A 733-bp fragment of inlA gene was used for investigation of polymorphism using RFLP analysis. In total, 45 phenotypically and molecularly confirmed L. monocytogenes strains were isolated from different sources including 30 (16.7%) from water, 9 (11.3%) from vaginal swabs and 6 (7.5%) from fecal samples. RFLP analysis of PCR products using AluI and Tsp509I restriction enzymes, generated two profiles with 8 to 10 bands ranging in size from 15 to 210 bp. The majority of water and clinical isolates were classified in profile 2. We demonstrated 45 L. monocytogenes isolates from tested water and clinical samples by phenotypic and molecular tests. The majority of the isolates were classified in the same RFLP profile, showing the water as a potential source of clinical complications in patients in the region of study.

Genetic similarity, antibiotic resistance and disinfectant susceptibility of Listeria monocytogenes isolated from chicken meat and chicken-meat processing environment in Mato Grosso, Brazil

A trial was conducted to determine the genotypic and phenotypic profiles of Listeria monocytogenes (LM) isolated from chicken meat and chicken-meat processing environment in an export-authorized industrial facility in Brazil. A total of 37 LM isolates were separated according to their genetic similarity by pulsed-field gel electrophoresis (PFGE), classified by serotype and lineage using multiplex PCR, tested for antibiotic resistance using the disk-diffusion technique, and analyzed for disinfectant susceptibility using the minimal inhibitory concentration (MIC) approach. The majority of the isolates belonged to lineage II, serotypes 1/2a or 3a. Genotypically, isolates from packaged frozen carcasses and knives & boards of the cutting room were similar to those of carcasses after chiller. Isolates from packaged frozen boneless thighs, however, were very peculiar. Regardless the origin, high level of resistance to peracetic acid, ampicillin and sulfonamides were detected. Overall, our findings suggest that the prevalence of LM in the cutting room is not sustained by the continuous flow of new bacteria, but mainly by an already established microbiota that persists in the environment. Therefore, more effective strategies of cleaning and disinfection must be adopted by the chicken meat industry in order to reduce the prevalence of LM in the processing environment.

Listeria monocytogenes isolates from ready to eat plant produce are diverse and have virulence potential

Listeria monocytogenes is sporadically detected on a range of ready to eat fresh produce lines, such as spinach and rocket, and is a threat to public health. However, little is known about the diversity of L. monocytogenes present on fresh produce and their potential pathogenicity. In this work, fifteen Listeria monocytogenes isolates from the UK fresh produce supply chain were characterised using whole genome sequencing (WGS). Additionally, isolates were characterised based on their ability to form biofilm. Whole genome sequencing data was used to determine the sequence type of isolates based on multi-locus sequence typing (MLST), construct a core single nucleotide polymorphism (SNP) phylogeny and determine the presence of virulence and resistance associated genes. MLST revealed 9 distinct sequence types (STs) spanning 2 lineages (I & II) with one isolate belonging to the ST6 subtype, strains from which have been recently implicated in two large, food-associated L. monocytogenes outbreaks in South Africa and across Europe. Although most of the 15 isolates were different, comparison of core genome SNPs showed 4 pairs of ‘indistinguishable’ strains (<5 SNPs difference). Virulence profiling revealed that some isolates completely lacked the Listeria pathogenicity island-3 (LIPI-3) amongst other virulence factors. Investigation of the inlA gene showed that no strains in this study contained a premature stop codon (PMSC), an indicator of attenuated virulence. Assessment of biofilm production showed that isolates found in the fresh produce supply chain differ in their ability to form biofilm. This trait is considered important for L. monocytogenes to persist in environments associated with food production and processing. Overall the work indicates that a genetically diverse range of L. monocytogenes strains is present in the UK fresh produce supply chain and the virulence profiles found suggests that at least some of the strains are capable of causing human illness. Interestingly, the presence of some genetically indistinguishable isolates within the 15 isolates examined suggests that cross-contamination in the fresh produce environment does occur. These findings have useful implications in terms of food safety and for informing microbial surveillance programmes in the UK fresh produce supply chain.

Isolation and identification of Listeria monocytogenes utilizing DC insulator-based dielectrophoresis

Foodborne pathogens pose one of the greatest challenges facing public health in the modern day. One important pathogen, Listeria monocytogenes, is known to be challenging to detect and identify. Three serovars cause most of the Listeria related food-borne illnesses, which the Centers for Disease Control currently utilizes a combination of pulsed-field gel electrophoresis and whole genome sequencing for identification and the determination of clusters and outbreaks. There is a potential method for rapid collection of epidemiological information by exploiting the electrokinetic and dielectrophoretic properties of the L. monocytogenes serovars. Using dielectrophoresis, the three most commonly identified serovars of L. monocytogenes can be distinguished from each other. The electrokinetic and dielectrophoretic mobilities of each serovar was determined through a combination of electrokinetic velocity and dielectrophoretic trapping assessments, in conjunction with finite element multi-physics modeling. A mathematical model of the data, which defines the various factors of dielectrophoretic trapping, is utilized and verified based on the behavior of L. monocytogenes in the microchannel. The trapping condition for the serovars were evaluated as 2.8±0.2×109, 2.2±0.2×109, and 2.2±0.3×109Vm−2 and the electrokinetic mobility was assessed to be 19±0.7, 17±0.7, and 9.2±0.3×10−9m2V−1s−1 for the L. monocytogenes serovars 1/2a, 1/2b, and 4b, respectively.

LiSEQ - whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe.

We present the LiSEQ (Listeria SEQuencing) project, funded by the European Food Safety Agency (EFSA) to compare Listeria monocytogenes isolates collected in the European Union from ready-to-eat foods, compartments along the food chain (e.g. food-producing animals, food-processing environments) and humans. In this article, we report the molecular characterization of a selection of this data set employing whole-genome sequencing analysis. We present an overview of the strain diversity observed in different sampled sources, and characterize the isolates based on their virulence and resistance profile. We integrate into our analysis the global L. monocytogenes genome collection described by Moura and colleagues in 2016 to assess the representativeness of the LiSEQ collection in the context of known L. monocytogenes strain diversity.

Preliminary results on prevalence and persistence of Listeria monocytogenes in different dairy and meat processing plants in Central Italy

Purpose: Listeria monocytogenes is a relevant food - borne pathogen able to cause severe and fatal infections in humans. Understanding correlations among different Listeria monocytogenes strains and evaluating Listeria monocytogenes isolates capacity to persist in processing plant environments is an essential tool for food surveillance. Most outbreaks of human listeriosis seem to be linked to strains able to persist in the environment and to replicate in foods. The aim of this study was to investigate prevalence and persistence of Listeria monocytogenes strains subtypes in different dairy and meat processing plants in the Abruzzo and Molise Region. Methods & Materials: Totally 14 food processing plants have been selected for the study (7 for dairy products – DP - and 7 for meat products - MP). Sampling was performed in 4 session from March to October 2017, from 30 to 35 environmental sampling have been sampled in all the division of the plants, both from food contact surfaces (FCS) and non food contact surfaces (NFCS). Sampling sites were selected as niches for Listeria monocytogenes presence. Samples were tested with FSIS MLG8-10:2017 method. Listeria monocytogenes strains were serotyped using the method described in the U.S. FDA Bacteriological Analytical Manual (Bennett and Weaver, 2001). PFGE subtyping was performed according to the PulseNet protocol (Pulsenet USA, 2013) and macrorestriction patterns were analyzed using the BioNumerics 7.6 (Applied Maths, Kortrijk, Belgium). Results: Results are based on 1772 environmental samples tested, 135 (7.6%) samples were found positive for Listeria monocytogenes (94 NFCS and 41 FCS) in 4 DP and 6 MP. PFGE showed indistinguishable persistent strains in 2 DP and 3 MP in all sampling sessions and in one MP in 3 sampling sessions. Conclusion: The persistence of Listeria monocytogenes underlines the ability to persist in food producing plants, in favourable niches inside the processing environment. To reduce Listeria monocytogenes prevalence, Good Manufacturing Practices and Good Hygiene Practices are needed. Analysis are still ongoing to model the contamination path, NGS on isolated strains will be carried out to confirm the persistence of the strains and to find the presence of gene devoted to resistance and persistence.

Listeria monocytogenes risk associated with chicken at slaughter and biocontrol with three new bacteriophages

AbstractThe aim of this study was to isolate Listeria monocytogenes from chicken neck skins and lytic bacteriophages from poultry slaughterhouse wastewaters, and following the characterization of the isolates, biocontrol of L. monocytogenes was investigated on chicken drumsticks with the isolated phages. L. monocytogenes prevalence was detected 12.3% in the chicken samples and the dominant serotype was determined as 1/2a (92.5%). Expression levels of major virulence genes were revealed by real‐time RT‐PCR. Ten different DNA profiles were detected by ERIC‐PCR fingerprinting. According to the MIC results, LM‐P75 was defined as MDR by showing resistance to antibiotics in six different groups. Besides, five lytic listeriophages were isolated from wastewaters and treated with Cla1 and Sac1. Taking EoP, TEM, in vitro, and in vivo analyses results into consideration, three bacteriophages were used for the biocontrol assay. The application of the bacteriophages on drumsticks achieved a reduction up to 3.3 log CFU/ml in L. monocytogenes count in 3 hr of incubation at 4°C.Practical applicationsOur results showed that in spite of the developments in hygiene practices during slaughtering, chicken meat is still a potential source for L. monocytogenes. On the other hand, the phage cocktail that used in this study can be an effective tool to reduce L. monocytogenes in chicken carcasses at final wash or at cooling step in poultry slaughtering process, as well as in decontamination of chicken meat parts.

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.

Shared genome analyses of notable listeriosis outbreaks, highlighting the critical importance of epidemiological evidence, input datasets and interpretation criteria.

The persuasiveness of genomic evidence has pressured scientific agencies to supplement or replace well-established methodologies to inform public health and food safety decision-making. This study of 52 epidemiologically defined Listeria monocytogenes isolates, collected between 1981 and 2011, including nine outbreaks, was undertaken (1) to characterize their phylogenetic relationship at finished genome-level resolution, (2) to elucidate the underlying genetic diversity within an endemic subtype, CC8, and (3) to re-evaluate the genetic relationship and epidemiology of a CC8-delimited outbreak in Canada in 2008. Genomes representing Canadian Listeria outbreaks between 1981 and 2010 were closed and manually annotated. Single nucleotide variants (SNVs) and horizontally acquired traits were used to generate phylogenomic models. Phylogenomic relationships were congruent with classical subtyping and epidemiology, except for CC8 outbreaks, wherein the distribution of SNV and prophages revealed multiple co-evolving lineages. Chronophyletic reconstruction of CC8 evolution indicates that prophage-related genetic changes among CC8 strains manifest as PFGE subtype reversions, obscuring the relationship between CC8 isolates, and complicating the public health interpretation of subtyping data, even at maximum genome resolution. The size of the shared genome interrogated did not change the genetic relationship measured between highly related isolates near the tips of the phylogenetic tree, illustrating the robustness of these approaches for routine public health applications where the focus is recent ancestry. The possibility exists for temporally and epidemiologically distinct events to appear related even at maximum genome resolution, highlighting the continued importance of epidemiological evidence.