Alexandra Moura

Whole genome-based population biology and epidemiological surveillance of Listeria monocytogenes

Listeria monocytogenes (Lm) is a major human foodborne pathogen. Numerous Lm outbreaks have been reported worldwide and associated with a high case fatality rate, reinforcing the need for strongly coordinated surveillance and outbreak control. We developed a universally applicable genome-wide strain genotyping approach and investigated the population diversity of Lm using 1,696 isolates from diverse sources and geographical locations. We define, with unprecedented precision, the population structure of Lm, demonstrate the occurrence of international circulation of strains and reveal the extent of heterogeneity in virulence and stress resistance genomic features among clinical and food isolates. Using historical isolates, we show that the evolutionary rate of Lm from lineage I and lineage II is low (∼2.5 × 10−7 substitutions per site per year, as inferred from the core genome) and that major sublineages (corresponding to so-called ‘epidemic clones’) are estimated to be at least 50–150 years old. This work demonstrates the urgent need to monitor Lm strains at the global level and provides the unified approach needed for global harmonization of Lm genome-based typing and population biology.

Real-Time Whole-Genome Sequencing for Surveillance of Listeria monocytogenes, France.

During 2015–2016, we evaluated the performance of whole-genome sequencing (WGS) as a routine typing tool. Its added value for microbiological and epidemiologic surveillance of listeriosis was compared with that for pulsed-field gel electrophoresis (PFGE), the current standard method. A total of 2,743 Listeria monocytogenes isolates collected as part of routine surveillance were characterized in parallel by PFGE and core genome multilocus sequence typing (cgMLST) extracted from WGS. We investigated PFGE and cgMLST clusters containing human isolates. Discrimination of isolates was significantly higher by cgMLST than by PFGE (p<0.001). cgMLST discriminated unrelated isolates that shared identical PFGE profiles and phylogenetically closely related isolates with distinct PFGE profiles. This procedure also refined epidemiologic investigations to include only phylogenetically closely related isolates, improved source identification, and facilitated epidemiologic investigations, enabling identification of more outbreaks at earlier stages. WGS-based typing should replace PFGE as the primary typing method for L. monocytogenes.

MALDI-TOF mass spectrometry-based identification of Listeria species in surveillance: A prospective study

This study aimed to evaluate MALDI-TOF MS for species discrimination of Listeria in the context of routine surveillance. MALDI-TOF MS yielded 100% accuracy for the identification of L. monocytogenes, L. innocua, L. ivanovii, L. fleischmannii, L. grayi, L. seeligeri, L. weihenstephanensis and L. welshimeri, as confirmed by whole genome sequence analyses.

Listeria costaricensis sp. nov.

A bacterial strain isolated from a food processing drainage system in Costa Rica fulfilled the criteria as belonging to the genus Listeria, but could not be assigned to any of the known species. Phylogenetic analysis based on the 16S rRNA gene revealed highest sequence similarity with the type strain of Listeria floridensis (98.7 %). Phylogenetic analysis based on Listeria core genomes placed the novel taxon within the Listeria fleishmannii, L. floridensis and Listeria aquatica clade (Listeria sensu lato). Whole-genome sequence analyses based on the average nucleotide blast identity (ANI<80 %) indicated that this isolate belonged to a novel species. Results of pairwise amino acid identity (AAI>70 %) and percentage of conserved proteins (POCP>68 %) with currently known Listeria species, as well as of biochemical characterization, confirmed that the strain constituted a novel species within the genus Listeria. The name Listeria costaricensis sp. nov. is proposed for the novel species, and is represented by the type strain CLIP 2016/00682T (=CIP 111400T=DSM 105474T).

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes

ABSTRACT The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes. Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA. Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA−/LLO−) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA−/LLO− mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.

Cross-border outbreak of listeriosis caused by cold-smoked salmon, revealed by integrated surveillance and whole genome sequencing (WGS), Denmark and France, 2015 to 2017

In August 2017, an outbreak of six listeriosis cases in Denmark was traced to cold-smoked salmon, using epidemiological investigations and whole-genome sequencing (WGS) analyses. Exchange of genome sequences allowed identification in France of a food isolate from a salmon-derived product and a human isolate from 2016 within the same cgMLST cluster as the Danish isolates (L2-SL8-ST8-CT771). The salmon product came from a third European Union country. WGS can rapidly link human cases and food isolates across Europe.

Epistatic control of intrinsic resistance by virulence genes in Listeria

Elucidating the relationships between antimicrobial resistance and virulence is key to understanding the evolution and population dynamics of resistant pathogens. Here, we show that the susceptibility of the gram-positive bacterium Listeria monocytogenes to the antibiotic fosfomycin is a complex trait involving interactions between resistance and virulence genes and the environment. We found that a FosX enzyme encoded in the listerial core genome confers intrinsic fosfomycin resistance to both pathogenic and non-pathogenic Listeria spp. However, in the genomic context of the pathogenic L. monocytogenes, FosX-mediated resistance is epistatically suppressed by two members of the PrfA virulence regulon, hpt and prfA, which upon activation by host signals induce increased fosfomycin influx into the bacterial cell. Consequently, in infection conditions, most L. monocytogenes isolates become susceptible to fosfomycin despite possessing a gene that confers high-level resistance to the drug. Our study establishes the molecular basis of an epistatic interaction between virulence and resistance genes controlling bacterial susceptibility to an antibiotic. The reported findings provide the rationale for the introduction of fosfomycin in the treatment of Listeria infections even though these bacteria are intrinsically resistant to the antibiotic in vitro.

Correction: Epistatic control of intrinsic resistance by virulence genes in Listeria

[This corrects the article DOI: 10.1371/journal.pgen.1007525.].

Retrospective validation of whole genome sequencing-enhanced surveillance of listeriosis in Europe, 2010 to 2015

Background and aim The trend in reported case counts of invasive Listeria monocytogenes (Lm), a potentially severe food-borne disease, has been increasing since 2008. In 2015, 2,224 cases were reported in the European Union/European Economic Area (EU/EEA). We aimed to validate the microbiological and epidemiological aspects of an envisaged EU/EEA-wide surveillance system enhanced by routine whole genome sequencing (WGS). Methods: WGS and core genome multilocus sequence typing (cgMLST) were performed on isolates from 2,726 cases from 27 EU/EEA countries from 2010–15. Results: Quality controls for contamination, mixed Lm cultures and sequence quality classified nearly all isolates with a minimum average coverage of the genome of 55x as acceptable for analysis. Assessment of the cgMLST variation between six different pipelines revealed slightly less variation associated with assembly-based analysis compared to reads-based analysis. Epidemiological concordance, based on 152 isolates from 19 confirmed outbreaks and a cluster cutoff of seven allelic differences, was good (sensitivity > 95% for two cgMLST schemes of 1,748 and 1,701 loci each; PPV 58‒68%). The proportion of sporadic cases was slightly below 50%. Of remaining isolates, around one third were in clusters involving more than one country, often spanning several years. Detection of multi-country clusters was on average several months earlier when pooling the data at EU/EEA level, compared with first detection at national level. Conclusions: These findings provide a good basis for comprehensive EU/EEA-wide, WGS-enhanced surveillance of listeriosis. Time limits should not be used for hypothesis generation during outbreak investigations, but should be for analytical studies.

Draft Genome Sequences of Listeria monocytogenes , Isolated from Fresh Leaf Vegetables in Owerri City, Nigeria

ABSTRACT Here, we report the draft genome sequences of three Listeria monocytogenes isolates from fresh leaves collected in Nigeria, belonging to sequence types ST5 and ST155 (sublineages SL5 and SL155, respectively).