Monocytogenes Clinical Isolates Research Articles

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.

Isolation and identification of listeria in clinical material

Results from research on isolation, identification, and study of biological properties of L. monocytogenes clinical isolates and Listeria spp test strains are presented. Peculiarities of modern research methods for indicating and identifying pathogenic listeria to improve the quality of laboratory studies of clinical material are studied. The culture method provides reliable results of microbiological analyses upon detecting Listeria spp. The presented list and algorithm of the laboratory diagnostic methods can be used as a basis for elaborating regulatory documents for carrying out microbiological research on any biological material for the presence of bacteria of the genus Listeria spp. and L. monocytogenes species in it.

A Machine Learning Model for Food Source Attribution of Listeria monocytogenes.

Despite its low morbidity, listeriosis has a high mortality rate due to the severity of its clinical manifestations. The source of human listeriosis is often unclear. In this study, we investigate the ability of machine learning to predict the food source from which clinical Listeria monocytogenes isolates originated. Four machine learning classification algorithms were trained on core genome multilocus sequence typing data of 1212 L. monocytogenes isolates from various food sources. The average accuracies of random forest, support vector machine radial kernel, stochastic gradient boosting, and logit boost were found to be 0.72, 0.61, 0.7, and 0.73, respectively. Logit boost showed the best performance and was used in model testing on 154 L. monocytogenes clinical isolates. The model attributed 17.5 % of human clinical cases to dairy, 32.5% to fruits, 14.3% to leafy greens, 9.7% to meat, 4.6% to poultry, and 18.8% to vegetables. The final model also provided us with genetic features that were predictive of specific sources. Thus, this combination of genomic data and machine learning-based models can greatly enhance our ability to track L. monocytogenes from different food sources.

In vitro activity of iclaprim and comparator agents against Listeria monocytogenes clinical isolates from 2012 to 2018

ObjectivesThis study examined the in vitro activity of iclaprim and comparators against 40 Listeria monocytogenes clinical isolates mostly (95%) from patients with bloodstream infection (BSI) from the USA, Australia/New Zealand, Latin America and Europe collected between 2012–2018. MethodsAntimicrobial susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) interpretations were based on CLSI criteria. ResultsThe iclaprim MIC90 value for all L. monocytogenes was 0.015 μg/mL. The MIC50/90 values for iclaprim were 4-fold lower than trimethoprim, the only FDA-approved dihydrofolate reductase inhibitor, against all L. monocytogenes. ConclusionIclaprim demonstrated lower MIC values than trimethoprim against a collection (2012–2018) of L. monocytogenes clinical isolates mostly from patients with BSI from the USA, Australia/New Zealand, Latin America and Europe.

Identification of a major Listeria monocytogenes outbreak clone linked to soft cheese in Northern Italy \u2013 2009-2011

BackgroundMolecular subtyping and enhanced surveillance in Lombardy region identified a cluster of possibly related listeriosis cases from 2006 to 2010. This cluster grouped 31 isolates that belonged to serotype 1/2a and Sequence Type 38 (ST38) as defined by Multilocus Sequence Typing (MLST).MethodsOur study expanded the previous investigation to include cases from 2011 to 2014 and used Multi-Virulence-Locus Sequence Typing (MVLST) on all ST38 isolates to better understand their epidemiology and possibly identify a common source outbreak.ResultsOut of 306 L. monocytogenes clinical isolates collected, 43 (14.1%) belonged to ST38 with cases occurring in nine out of twelve Lombardy provinces. The ST38 isolates were split by MVLST into two Virulence Types (VTs): VT80 (n = 12) and VT104 (n = 31). VT104 cases were concentrated between 2009 and 2011 in two provinces, Bergamo and Milan. An epidemiologic investigation was performed and in one case, a matching VT104 isolate was retrieved from a soft cheese sample from a patient’s refrigerator.ConclusionsOur findings revealed a major listeriosis outbreak in Northern Italy linked to soft cheese in 2009–2011, which went undetected by local health authorities. Our study shows that integrating subtyping methods with conventional epidemiology can help identify the source of L. monocytogenes outbreak clones.

Characterisation of InlA truncation in Listeria monocytogenes isolates from farm animals and human cases in the province of Quebec

The introduction of Listeria monocytogenes into the food production chain is a concern, with numerous grouped cases of listeriosis associated with milk-derived or pork-derived products have been documented. Management of...

Overexpression of the novel MATE fluoroquinolone efflux pump FepA in Listeria monocytogenes is driven by inactivation of its local repressor FepR.

Whereas fluoroquinolone resistance mainly results from target modifications in gram-positive bacteria, it is primarily due to active efflux in Listeria monocytogenes. The aim of this study was to dissect a novel molecular mechanism of fluoroquinolone resistance in this important human pathogen. Isogenic L. monocytogenes clinical isolates BM4715 and BM4716, respectively susceptible and resistant to fluoroquinolones, were studied. MICs of norfloxacin and ciprofloxacin were determined in the presence or in the absence of reserpine (10 mg/L). Strain BM4715 was susceptible to norfloxacin (MIC, 4 mg/L) and ciprofloxacin (MIC, 0.5 mg/L) whereas BM4716 was highly resistant to both drugs (MICs 128 and 32 mg/L, respectively). Reserpine was responsible for a 16-fold decrease in both norfloxacin and ciprofloxacin MICs against BM4716 suggesting efflux associated resistance. Whole-genome sequencing of the strains followed by comparative genomic analysis revealed a single point mutation in the gene for a transcriptional regulator, designated fepR (for fluoroquinolone efflux protein regulator) belonging to the TetR family. The frame-shift mutation was responsible for the introduction of a premature stop codon resulting in an inactive truncated protein. Just downstream from fepR, the structural gene for an efflux pump of the MATE family (named FepA) was identified. Gene expression was quantified by qRT-PCR and demonstrated that fepA expression was more than 64-fold higher in BM4716 than in BM4715. The clean deletion of the fepR gene from BM4715 was responsible for an overexpression of fepA with resistance to norfloxacin and ciprofloxacin, confirming the role of FepR as a local repressor of fepA. In conclusion, we demonstrated that overexpression of the new MATE efflux pump FepA is responsible for fluoroquinolone resistance in L. monocytogenes and secondary to inactivation of the FepR repressor.

In vitro and intracellular activities of fosfomycin against clinical strains of Listeria monocytogenes

This study was designed to evaluate the potential role of fosfomycin as a therapeutic agent in human listeriosis. The in vitro activity of fosfomycin against 154 Listeria monocytogenes clinical isolates under conditions that mimic the induction of prfA expression was determined and was correlated with fosfomycin intracellular antimicrobial activity. In vitro, partial induction of prfA expression is achieved through bacterial growth in brain–heart infusion agar supplemented with activated charcoal (BHIC). A fosfomycin pharmacokinetic/pharmacodynamic breakpoint of ≤64mg/L was estimated using a Monte Carlo simulation to assess the success of an intravenous fosfomycin dose of 300mg/kg/day over 5000 individuals. Eighty strains (51.9%) were susceptible to fosfomycin in BHIC, with minimum inhibitory concentrations (MICs) of ≤64mg/L; 13 strains (8.4%) had the epidemic clone (EC) marker. In addition, 27 strains (17.5%) had a three doubling dilutions reduction in the MIC from ≥1024mg/L to 128mg/L (96–128mg/L by Etest). The fosfomycin modal MIC is lower under prfA expression. However, this effect is smaller in terms of clinical categorisation of isolates and can be influenced by the serotype and clonal type. In A549 cells, the reductions in bacterial inocula of the two susceptible isolates studied after 1h and 24h of incubation with fosfomycin at 0.5× the human maximum serum concentration (Cmax) were 45.8% and 46.6%, and 93.8% and 99.1%, respectively. Slightly higher reductions were found with fosfomycin at 1× Cmax. The resistant strain tested showed significantly lower reductions in all assays.

A new hybrid bacteriocin, Ent35–MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria

Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35–MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35–MccV may find important applications in food or pharmaceutical industries.

Distribution and characterization of Listeria monocytogenes clinical isolates in Portugal, 1994–2007

In recent years, the number of cases of listeriosis has increased worldwide. Ninety-five isolates of Listeria monocytogenes recovered from Portuguese human cases of listeriosis have been characterized by biotyping (cadmium and arsenic sensitivity), polymerase chain reaction (PCR) grouping, and by pulsed-field gel electrophoresis (PFGE) applying the enzymes AscI and ApaI. Isolates were classified into one of three PCR groups; IVb (71.6%), IIb (17.9%), and IIa (10.5%). Four biotypes were differentiated: sensitive to arsenic/cadmium (48.4%), arsenic-sensitive and cadmium-resistant (25.3%), resistant to arsenic and sensitive to cadmium (18.9%), and resistant to both heavy metals (7.4%). Combined analyses of AscI and ApaI patterns yielded a total of 58PFGE types with five sets (G, Jb, KKa, Me, and U) of Portuguese strains, each of which were indistinguishable by PFGE typing. In the present study, it was demonstrated that there are recurrent pulsotypes and that some were the same pulsotypes linked to outbreaks in France. In addition, there are some pulsotypes spread throughout the country, while others only appear in a restricted region. This study allowed the assembly of a first large pulsotype database of Portuguese clinical strains.

High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak

BackgroundA large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis (PFGE) revealed two similar but distinct AscI PFGE patterns. High-throughput pyrosequencing of two L. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during PFGE.ResultsThe chromosomes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs) and three indels, including a 33 kbp prophage that accounted for the observed difference in AscI PFGE patterns. The distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes.ConclusionsHigh-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.

Pheno- and genotypic characterization of Listeria monocytogenes clinical isolates from the southwestern region of the State of São Paulo, Brazil

Considering the lack of information available in Brazil concerning listeriosis, the present study aimed to analyze phenotypic and genotypically Listeria monocytogenes clinical isolates from the Southwestern region of the State of São Paulo, Brazil. From January 1995 to May 2005, thirteen isolates of L. monocytogenes from twelve patients with listeriosis were sent to the Adolfo Lutz Institute (Campinas Regional Laboratory, São Paulo, Brazil). The antimicrobial susceptibility of the isolates was evaluated by the microdilution broth method for ampicillin, gentamicin, trimethoprim, sulfamethoxazole and vancomycin. They were also serotyped (Denka Seiken, Japan), and sub-typed by PFGE employing ApaI and AscI and the American CDC protocol. None of the isolates showed resistance to the antibiotics tested; however, seven of them presented increased values for sulfamethoxazole MICs. Ten isolates belonged to serotype 4b, two to serotype 1/2a and only one to serotype 1/2b. After digestion with ApaI and AscI, the strains were distributed in 3 different groups according to their profile. It was observed that the spatial or temporally unrelated strains exhibited similar PFGE profiles, indicating a possible clonal relationship among them.