Genome-Based Characterization of Listeria monocytogenes, Costa Rica.

Genomic data on the foodborne pathogen Listeria monocytogenes from Central America are scarce. We analyzed 92 isolates collected during 2009-2019 from different regions in Costa Rica, compared those to publicly available genomes, and identified unrecognized outbreaks. Our findings suggest mandatory reporting of listeriosis in Costa Rica would improve pathogen surveillance.

Neonatal Listeriosis Presentation and Outcome: A Prospective Study of 189 Cases.

BACKGROUND: Listeriosis is caused by the foodborne pathogen Listeria monocytogenes. It can present as a maternal-neonatal infection. We implemented a nationwide prospective cohort and analyzed the features of neonatal listeriosis. METHODS: We studied all neonates born alive from mothers with microbiologically proven maternal-neonatal listeriosis enrolled from November 2009 to December 2017. We analyzed presentation, neonatal outcome at discharge, and predictors of severe presentation and outcome. RESULTS: We studied 189 infants; 133 of 189 (70%) had abnormal clinical status at birth, including acute respiratory distress in 106 of 189 (56%). There were 132 of 189 (70%) infants who developed early-onset listeriosis and 12 of 189 (6%) who developed late-onset listeriosis; all presented with acute meningitis. There were 17 of 189 (9%) infants who had major adverse outcomes: 3%, (5 of 189) death; 6% (12 of 189), severe brain injury; and 2% (3 of 189), severe bronchopulmonary dysplasia. Fifteen of 17 infants were born <34 weeks of gestation (P < .0001 vs infants born ≥34 weeks of gestation). Maternal antimicrobial treatment ≥1 day before delivery was associated with a significant decrease in presentation severity for the infant, resulting in significantly fewer inotropic drugs, fluid resuscitation, and mechanical ventilation requirement (odds ratio, 0.23; 95% confidence interval, 0.09-0.51; P < .0001). CONCLUSIONS: Antenatal maternal antimicrobial treatment is associated with reduced neonatal listeriosis severity, justifying the prescription of preemptive maternal antimicrobial therapy when maternal-fetal listeriosis is suspected. Neonatal outcome is better than reported earlier, and its major determinant is gestational age at birth. CLINICAL TRIALS REGISTRATION: NCT01520597.

In vitro and in silico parameters for precise cgMLST typing of Listeria monocytogenes.

BACKGROUND: Whole genome sequencing analyzed by core genome multi-locus sequence typing (cgMLST) is widely used in surveillance of the pathogenic bacteria Listeria monocytogenes. Given the heterogeneity of available bioinformatics tools to define cgMLST alleles, our aim was to identify parameters influencing the precision of cgMLST profiles. METHODS: We used three L. monocytogenes reference genomes from different phylogenetic lineages and assessed the impact of in vitro (i.e. tested genomes, successive platings, replicates of DNA extraction and sequencing) and in silico parameters (i.e. targeted depth of coverage, depth of coverage, breadth of coverage, assembly metrics, cgMLST workflows, cgMLST completeness) on cgMLST precision made of 1748 core loci. Six cgMLST workflows were tested, comprising assembly-based (BIGSdb, INNUENDO, GENPAT, SeqSphere and BioNumerics) and assembly-free (i.e. kmer-based MentaLiST) allele callers. Principal component analyses and generalized linear models were used to identify the most impactful parameters on cgMLST precision. RESULTS: The isolate's genetic background, cgMLST workflows, cgMLST completeness, as well as depth and breadth of coverage were the parameters that impacted most on cgMLST precision (i.e. identical alleles against reference circular genomes). All workflows performed well at ≥40X of depth of coverage, with high loci detection (> 99.54% for all, except for BioNumerics with 97.78%) and showed consistent cluster definitions using the reference cut-off of ≤7 allele differences. CONCLUSIONS: This highlights that bioinformatics workflows dedicated to cgMLST allele calling are largely robust when paired-end reads are of high quality and when the sequencing depth is ≥40X.

Listeria ilorinensis sp. nov., isolated from cow milk cheese in Nigeria.

During microbial assessment of cow milk cheese products in the city of Ilorin, Nigeria, a Listeria-like isolate was detected that could not be assigned to any known species. Whole-genome sequence analyses against all currently known 26 Listeria species confirmed that this isolate constitutes a new taxon within the genus Listeria, with highest similarity to Listeria costaricensis (average nucleotide identity blast of 82.66%, in silico DNA-DNA hybridization of 28.3%). Phenotypically, it differs from L. costaricensis by the inability to ferment sucrose, l-fucose and starch. The absence of haemolysis and Listeria pathogenic islands suggest that this novel species is not pathogenic for humans and animals. The name Listeria ilorinensis sp. nov. is proposed, with the type strain CLIP 2019/01311T (=CIP 111875T=DSM 111566T).

Ruminant-associated Listeria monocytogenes isolates belong preferentially to dairy-associated hypervirulent clones: a longitudinal study in 19 farms.

Studies have shown that ruminants constitute reservoirs of Listeria monocytogenes, but little is known about the epidemiology and genetic diversity of this pathogen within farms. Here we conducted a large-scale longitudinal study to monitor Listeria spp. in 19 dairy farms during three consecutive seasons (N = 3251 samples). L. innocua was the most prevalent species, followed by L. monocytogenes. Listeria monocytogenes was detected in 52.6% of farms and more frequently in cattle (4.1%) and sheep (4.5%) than in goat farms (0.2%). Lineage I accounted for 69% of L. monocytogenes isolates. Among animal samples, the most prevalent sublineages (SL) and clonal complexes (CC) were SL1/CC1, SL219/CC4, SL26/CC26 and SL87/CC87, whereas SL666/CC666 was most prevalent in environmental samples. Sixty-one different L. monocytogenes cgMLST types were found, 28% common to different animals and/or surfaces within the same farm and 21% previously reported elsewhere in the context of food and human surveillance. Listeria monocytogenes prevalence was not affected by farm hygiene but by season: higher prevalence was observed during winter in cattle, and during winter and spring in sheep farms. Cows in their second lactation had a higher probability of L. monocytogenes faecal shedding. This study highlights dairy farms as a reservoir for hypervirulent L. monocytogenes.

Genome Typing and Epidemiology of Human Listeriosis in New Zealand, 1999 to 2018.

This study describes the epidemiology of listeriosis in New Zealand between 1999 and 2018 as well as the retrospective whole-genome sequencing (WGS) of 453 Listeria monocytogenes isolates corresponding to 95% of the human cases within this period. The average notified rate of listeriosis was 0.5 cases per 100,000 population, and non-pregnancy-associated cases were more prevalent than pregnancy-associated cases (averages of 19 and 5 cases per annum, respectively). WGS data was assessed using multilocus sequencing typing (MLST), including core-genome and whole-genome MLST (cgMLST and wgMLST, respectively) and single-nucleotide polymorphism (SNP) analysis. Thirty-nine sequence types (STs) were identified, with the most common being ST1 (21.9%), ST4 (13.2%), ST2 (11.3%), ST120 (6.1%), and ST155 (6.4%). A total of 291 different cgMLST types were identified, with the majority (n = 243) of types observed as a single isolate, consistent with the observation that listeriosis is predominately sporadic. Among the 49 cgMLST types containing two or more isolates, 18 cgMLST types were found with 2 to 4 isolates each (50 isolates in total, including three outbreak-associated isolates) that shared low genetic diversity (0 to 2 whole-genome alleles), some of which were dispersed in time or geographical regions. SNP analysis also produced results comparable to those from wgMLST. The low genetic diversity within these clusters suggests a potential common source, but incomplete epidemiological data impaired retrospective epidemiological investigations. Prospective use of WGS analysis together with thorough exposure information from cases could potentially identify future outbreaks more rapidly, including those that may have been undetected for some time over different geographical regions.

Listeria spp. Isolated from Tonsils of Wild Deer and Boars: Genomic Characterization.

Listeria monocytogenes is a major human and animal foodborne pathogen. However, data from environmental reservoirs remain scarce. Here, we used whole-genome sequencing to characterize Listeria species isolates recovered over 1 year from wild animals in their natural habitats in Spain. Three different Listeria spp. (L. monocytogenes [n = 19], Listeria ivanovii subsp. londoniensis [n = 4], and Listeria innocua [n = 3]) were detected in 23 animal tonsils (9 deer, 14 wild boars) and 2 feeding troughs. No Listeria species was detected in feces. L. monocytogenes was detected in tonsils of 44.4% (8 out of 18) of deer and 40.7% (11 out of 27) of wild boars. L. monocytogenes isolates belonged to 3 different core genome multilocus sequence typing (cgMLST) types (CTs) of 3 distinct sublineages (SL1, SL387, and SL155) from lineages I and II. While cgMLST type L1-SL1-ST1-CT5279 (IVb; clonal complex 1 [CC1]) occurred only in one animal, types L1-SL387-ST388-CT5239 (IVb; CC388) and L2-SL155-ST155-CT1170 (IIa; CC155) were retrieved from multiple animals. In addition, L1-SL387-ST388-CT5239 (IVb; CC388) isolates were collected 1 year apart, revealing their long-term occurrence within the animal population and/or environmental reservoir. The presence of identical L. monocytogenes strains in deer and wild boars suggests contamination from a common food or environmental source, although interhost transmission cannot be excluded. Pathogenicity islands LIPI-1, LIPI-3, and LIPI-4 were present in 100%, 5%, and 79% of the L. monocytogenes isolates, respectively, and all L. monocytogenes lineage II isolates (n = 3) carried SSI-1 stress islands. This study highlights the need for monitoring L. monocytogenes environmental contamination and the importance of tonsils as a possible L. monocytogenes intrahost reservoir.IMPORTANCEListeria monocytogenes is a foodborne bacterial pathogen responsible for listeriosis. Whole-genome sequencing has been extensively used in public health and food industries to characterize circulating Listeria isolates, but genomic data on isolates occurring in natural environments and wild animals are still scarce. Here, we show that wild animals carry pathogenic Listeria and that the same genotypes can be found at different time points in different host species. This work highlights the need of Listeria species monitoring of environmental contamination and the importance of tonsils as a possible L. monocytogenes intrahost reservoir.

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.].

Listeria valentina sp. nov., isolated from a water trough and the faeces of healthy sheep.

In the context of a study on the occurrence of Listeria species in an animal farm environment in Valencia, Spain, six Listeria-like isolates could not be assigned to any known species. Phylogenetic analysis based on the 16S rRNA gene and on 231 Listeria core genes grouped these isolates in a monophyletic clade within the genus Listeria, with highest similarity to Listeria thailandensis. Whole-genome sequence analyses based on in silico DNA-DNA hybridization, the average nucleotide blast and the pairwise amino acid identities against all currently known Listeria species confirmed that these isolates constituted a new taxon within the genus Listeria. Phenotypically, these isolates differed from other Listeria species mainly by the production of acid from inositol, the absence of acidification in presence of methyl α-d-glucoside, and the absence of α-mannosidase and nitrate reductase activities. The name Listeria valentina sp. nov. is proposed for this novel species, and the type strain is CLIP 2019/00642T (=CIP 111799T=DSM 110544T).

Atypical Hemolytic Listeria innocua Isolates Are Virulent, albeit Less than Listeria monocytogenes.

Listeria innocua is considered a nonpathogenic Listeria species. Natural atypical hemolytic L. innocua isolates have been reported but have not been characterized in detail. Here, we report the genomic and functional characterization of representative isolates from the two known natural hemolytic L. innocua clades. Whole-genome sequencing confirmed the presence of Listeria pathogenicity islands (LIPI) characteristic of Listeria monocytogenes species. Functional assays showed that LIPI-1 and inlA genes are transcribed, and the corresponding gene products are expressed and functional. Using in vitro and in vivo assays, we show that atypical hemolytic L. innocua is virulent, can actively cross the intestinal epithelium, and spreads systemically to the liver and spleen, albeit to a lesser degree than the reference L. monocytogenes EGDe strain. Although human exposure to hemolytic L. innocua is likely rare, these findings are important for food safety and public health. The presence of virulence traits in some L. innocua clades supports the existence of a common virulent ancestor of L. monocytogenes and L. innocua.

Whole-genome sequencing reveals Listeria monocytogenes diversity and allows identification of long-term persistent strains in Brazil.

Advances in whole-genome sequencing (WGS) technologies have documented genetic diversity and epidemiology of the major foodborne pathogen Listeria monocytogenes (Lm) in Europe and North America, but data concerning South America are scarce. Here, we examined the population structure and genetic diversity of this major foodborne pathogen collected in Brazil. Based on core genome multilocus sequence typing (cgMLST), isolates from lineages I (n = 22; 63%) and II (n = 13; 37%) were distributed into 10 different sublineages (SLs) and represented 31 new cgMLST types (CTs). The most prevalent SLs were SL9 (n = 9; 26%), SL3 (n = 6; 17%) and SL2 and SL218 (n = 5; 14%). Isolates belonging to CTs L2-SL9-ST9-CT4420 and L1-SL315-ST520-CT4429 were collected 3 and 9 years apart, respectively, revealing long-term persistence of Lm in Brazil. Genetic elements associated with stress survival were present in 60% of isolates (57% SSI-1 and 3% SSI-2). Pathogenic islands were present in 100% (LIPI-1), 43% (LIPI-3) and 6% (LIPI-4) of the isolates. Mutations leading to premature stop codons were detected in the prfA and inlA virulence genes. This study is an important contribution to understanding the genomic diversity and epidemiology of Lm in South America. In addition, the results highlight the importance of using WGS to reveal Lm long-term persistence.

Listeria thailandensis sp. nov.

During a screening of Listeria species in food samples in Thailand, a Listeria-like bacterium was recovered from fried chicken and could not be assigned to any known species. Phylogenetic analysis based on the 16S rRNA gene and on 243 Listeria core genes placed the novel taxon within the Listeria aquatica, Listeria floridensis, Listeria fleishmannii and Listeria costaricensis clade (Listeria sensu lato), with highest similarity to L. floridensis (98.9 %) and L. costaricensis (98.8 %). Whole-genome sequence analyses based on the average nucleotide blast identity (ANI<86 %), the pairwise amino acid identity (AAI>64 %) and on the percentage of conserved proteins (POCP>77 %) with currently known Listeria species confirmed that the strain constituted a new taxon within the genus Listeria. At the phenotypical level, it differs from other Listeria species by the production of acid from d-tagatose and inositol. The name Listeria thailandensis sp. nov. is proposed for the novel species, and is represented by the type strain CLIP 2015/00305T (=CIP 111635T=DSM 107638T).

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).

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

Background and aimThe 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.

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes.

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.

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.

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.

Risk factors for mortality in end-stage kidney disease patients under online-hemodiafiltration: three-year follow-up study.

The aim of this work was to evaluate the predictors of mortality in a group of end-stage kidney disease (ESRD) patients under dialysis, by performing a three-year follow-up study. From the 236 patients included in this study, 54 patients died during the three-year follow-up period. Our data showed that the risk of death was higher in patients presenting lower levels of mean cell hemoglobin concentration, transferrin, and albumin. Our study showed that poor nutritional status and an inflammatory-induced iron depleted erythropoiesis are important factors for mortality in these patients.

Predictors of health-related quality of life perceived by end-stage renal disease patients under online hemodiafiltration.

PURPOSE: Patients' perception of health-related quality of life (HRQOL) is a consistent and powerful predictor of the outcome of end-stage renal disease (ESRD) patients under dialysis. This study aims to identify factors that could affect the HRQOL of ESRD patients under online hemodiafiltration (OL-HDF). METHODS: We evaluated 322 ESRD patients under OL-HDF (59.63% males; 64.9 ± 14.3 years old) from five dialysis units in the north of Portugal. Socio-demographic data, comorbidities, hematological data, iron status, dialysis adequacy, nutritional and inflammatory markers were collected from patients records. Patient's reported HRQOL score was assessed by using the Kidney Disease Quality of Life-Short Form (KDQOL-SF). RESULTS: ESRD patients showed a mean (± SD) of 53.17% (± 15.31%) in SF-36 total score, 50.17% (± 9.51%) in the SF-36 mental component summary (MCS) and 49.75% (± 9.44%) in the SF-36 physical component summary (PCS). Red cell distribution width (RDW), feminine gender and diabetes were found as significant predictors of SF-36 total score of HRQOL, which accounts for 12% of the total explained variance. Patient satisfaction, RDW, body mass index and gender were identified as predictors for the PCS, which accounts for 22% of total explained variance. Furthermore, patient satisfaction and dry weight were found as predictors for MCS. These predictors accounted for 28% of the total explained variance. CONCLUSIONS: Our results showed that the coexistence of diabetes, gender and erythropoietic disturbances are predictors of HRQOL in patients under OL-HDF and suggest that more attention should be given to woman patients, to the improvement of anemia and to diabetic patients, who are more prone to perceive a worst HRQOL.