A novel cgMLST for genomic surveillance of Yersinia enterocolitica infections in France allowed the detection and investigation of outbreaks in 2017-2021.

Enteric yersiniosis, the third most common food-borne zoonosis in Europe, is mainly caused by the pathogen Yersinia enterocolitica. In France, the yersiniosis microbiological surveillance is conducted at the Yersinia National Reference Laboratory (YNRL). Since 2017, isolates have been characterized by whole genome sequencing (WGS) followed by a 500-gene Yersinia-cgMLST. We report here the data of the WGS-based surveillance on Y. enterocolitica isolates for the 2017-2021 period. The YNRL characterized 7,642 Y. enterocolitica strains distributed in 2,497 non-pathogenic isolates from lineages 1Aa and 1Ab, and 5,145 specimens belonging to 8 pathogenic lineages. Among pathogenic isolates, lineage 4 was the most common (87.2%) followed by lineages 2/3-9b (10.6%), 2/3-5a (1.2%), 2/3-9a (0.6%), 3-3b, 3-3c, 1B, and 3-3d (0.1% per each). Importantly, we developed a routine surveillance system based on a new typing method consisting of a 1,727-genes core genome Multilocus Sequence Typing (cgMLST) specific to the species Y. enterocolitica followed by isolate clustering. Thresholds of allelic distances (AD) were determined and fixed for the clustering of isolates: AD ≤ 5 for lineages 4, 2/3-5a, and 2/3-9a, and AD ≤ 3 for lineage 2/3-9b. Clustering programs were implemented in 2019 in routine surveillance to detect genomic clusters of pathogenic isolates. In total, 419 clusters with at least 2 isolates were identified, representing 2,504 of the 3,503 isolates characterized between 2019 and 2021. Most clusters (n = 325) comprised 2 to 5 isolates. The new typing method proved to be useful for the molecular investigation of unusual grouping of cases as well as for the detection of genomic clusters in routine surveillance. IMPORTANCE: We describe here the new typing method used for molecular surveillance of Yersinia enterocolitica infections in France based on a novel core genome Multilocus Sequence Typing (cgMLST) specific to Y. enterocolitica species. This method can reliably identify the pathogenic Y. enterocolitica subspecies and compare the isolates with a high discriminatory power. Between 2017 and 2021, 5,145 pathogenic isolates belonging to 8 lineages were characterized and lineage 4 was by far the most common followed by lineage 2/3-9b. A clustering program was implemented, and detection thresholds were cross-validated by the molecular and epidemiological investigation of three unusual groups of Y. enterocolitica infections. The routine molecular surveillance system has been able to detect genomic clusters, leading to epidemiological investigations.

Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017.

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

Multidrug-resistant Corynebacterium diphtheriae in people with travel history from West Africa to France, March to September 2023.

We describe 10 unlinked cases of Corynebacterium diphtheriae infection (nine cutaneous, one respiratory) in France in 2023 in persons travelling from Guinea, Mali, Senegal, Niger or Nigeria and Central African Republic. Four isolates were toxigenic. Seven genomically unrelated isolates were multidrug-resistant, including a toxigenic respiratory isolate with high-level resistance to macrolides and beta-lactams. The high rates of resistance, including against first-line agents, call for further microbiological investigations to guide clinical management and public health response in ongoing West African outbreaks.

Corynebacterium ramonii sp. nov., a novel toxigenic member of the Corynebacterium diphtheriae species complex.

The Corynebacterium diphtheriae species complex comprises seven bacterial species, including Corynebacterium ulcerans, a zoonotic pathogen from multiple animal species. In this work, we characterise phenotypically and genotypically isolates belonging to two C. ulcerans lineages. Results from phylogenetic analyses, in silico DNA-DNA hybridization (DDH) and MALDI-TOF spectra differentiate lineage 2 from C. ulcerans lineage 1, which, together with their distinct transmission dynamics (probable human-to-human vs animal-to-human), indicates that lineage 2 is a separate Corynebacterium species, which we propose to name Corynebacterium ramonii. This species is of particular medical interest considering that its human-to-human transmission is likely, and that some C. ramonii isolates carry the diphtheria toxin gene.

Traceability, virulence and antimicrobial resistance of Yersinia enterocolitica in two industrial cheese-making plants.

Between 2018 and 2019, 309 environmental and food samples were collected from two industrial cheese-making plants located in Sardinia, in order to investigate Y. enterocolitica presence and to characterize the isolates. Y. enterocolitica isolates were further compared with isolates detected during a previous investigation from sheep and goat raw milk samples. Y. enterocolitica was detected in 7.4 % of the samples and the prevalence was higher, even if not significantly (P > 0.05) higher in non-food contact surface samples (10.2 %) than in food contact surface samples (3.8 %). The highest prevalence was detected in floor samples (13.5 %), followed by drain samples (7.2 %), which might serve as main harborage sites for further contamination. Y. enterocolitica was also detected in food contact surfaces, namely shelves of the Ricotta cooling room and packaging room, one cheese cutting machine surface and one raw milk filter sample. The biotype 1A isolates identified in this study were classified into six different serotypes. Additionally, a bioserotype 2/O:5,27 isolate was identified in one goat milk sample. All 1A isolates possessed the virulence genes invA and ystB while the 2/O:5,27 isolate showed the presence of ail, ystA, invA and yadA genes, thus confirming a pathogenic potential. The isolates showed intrinsic resistance to amoxicillin-clavulanic acid, ticarcillin and cefoxitin due to the presence of the blaA gene. Whole genome sequencing allowed to identify seven different sequence types among the 1A isolates, thus showing a high genetic diversity. The same Y. enterocolitica sequence type (ST3) was detected from three different areas of the same cheese-making plant, indicating a possible transfer of the microorganism along the processing lines. Y. enterocolitica contamination in cheese-making plants can pose a risk to human health. Preventive measures include the hygienic design of the plant layout and equipment, in association with proper cleaning and disinfection programmes.

Is Yersinia bercovieri Surpassing Yersinia enterocolitica in Wild Boars (Sus scrofa)?

Yersiniosis was the fourth reported zoonosis in the European Union in 2018. As well-known, pigs are recognized important reservoirs of Yersinia enterocolitica. The study was focused on Y. enterocolitica detection in mesenteric lymph nodes and faeces of 305 wild boars, but Yersinia bercovieri was more common, being isolated from 108 animals (35.4%). Cold season (p = 1.17 × 10-5) and young age (p = 0.004) significantly increased Y. bercovieri detection. Y. enterocolitica 1A belonging to six serotypes (O:4.32-4.33; O:5; O:6.30-6.31; O:7.8-8-8.19; O:10-34; O:12.25-12.26) was isolated from 8.2% (25/305) of the animals. Cold season significantly affected (p = 0.037) Y. enterocolitica detection.

Yersinia artesiana sp. nov., Yersinia proxima sp. nov., Yersinia alsatica sp. nov., Yersina vastinensis sp. nov., Yersinia thracica sp. nov. and Yersinia occitanica sp. nov., isolated from humans and animals.

Thirty-three Yersinia strains previously characterized by the French Yersinia National Reference Laboratory (YNRL) and isolated from humans and animals were suspected to belong to six novel species by a recently described core genome multilocus sequence typing scheme. These strains and five additional strains from the YNRL were characterized using a polyphasic taxonomic approach including a phylogenetic analysis based on 500 core genes, determination of average nucleotide identity (ANI), determination of DNA G+C content and identification of phenotypic features. Phylogenetic analysis confirmed that the 38 studied strains formed six well-demarcated clades. ANI values between these clades and their closest relatives were <94.7 % and ANI values within each putative novel species were >97.5 %. Distinctive biochemical characteristics were identified in five out of the six novel species. All of these data demonstrated that the 38 strains belong to six novel species of the genus Yersinia: Yersinia artesiana sp. nov., type strain IP42281T (=CIP 111845T=DSM 110725T); Yersinia proxima sp. nov., type strain IP37424T (=CIP 111847T=DSM 110727T); Yersinia alsatica sp. nov., type strain IP38850T (=CIP 111848T=DSM 110726T); Yersinia vastinensis sp. nov., type strain IP38594T (=CIP 111844T=DSM 110738T); Yersinia thracica sp. nov., type strain IP34646T (=CIP 111842T=DSM 110736T); and Yersinia occitanica sp. nov., type strain IP35638T (=CIP 111843T=DSM 110739T).

Yersinia enterocolitica, a Neglected Cause of Human Enteric Infections in Côte d'Ivoire.

BACKGROUND: Enteropathogenic Yersinia circulate in the pig reservoir and are the third bacterial cause of human gastrointestinal infections in Europe. In West Africa, reports of human yersiniosis are rare. This study was conducted to determine whether pathogenic Yersinia are circulating in pig farms and are responsible for human infections in the Abidjan District. METHODOLOGY/PRINCIPAL FINDINGS: From June 2012 to December 2013, pig feces were collected monthly in 41 swine farms of the Abidjan district. Of the 781 samples collected, 19 Yersinia strains were isolated in 3 farms: 7 non-pathogenic Yersinia intermedia and 12 pathogenic Yersinia enterocolitica bioserotype 4/O:3. Farm animals other than pigs and wild animals were not found infected. Furthermore, 2 Y. enterocolitica 4/O:3 strains were isolated from 426 fecal samples of patients with digestive disorders. All 14 Y. enterocolitica strains shared the same PFGE and MLVA profile, indicating their close genetic relationship. However, while 6 of them displayed the usual phage type VIII, the other 8 had the highly infrequent phage type XI. Whole genome sequencing and SNP analysis of individual colonies revealed that phage type XI strains had unusually high rates of mutations. These strains displayed a hypermutator phenotype that was attributable to a large deletion in the mutS gene involved in DNA mismatch repair. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that pathogenic Y. enterocolitica circulate in the pig reservoir in Côte d'Ivoire and cause human infections with a prevalence comparable to that of many developed countries. The paucity of reports of yersiniosis in West Africa is most likely attributable to a lack of active detection rather than to an absence of the microorganism. The identification of hypermutator strains in pigs and humans is of concern as these strains can rapidly acquire selective advantages that may increase their fitness, pathogenicity or resistance to commonly used treatments.

First isolation and characterization of Leptospira interrogans serogroup Australis from swine in Brazil / Primeiro isolamento e caracterização da Leptospira interrogans sorogrupo Australis de suíno no Brasil

The purpose of this study was to report the first recovery and characterization of Leptospira interrogans (serogroup Australis) from urine of swine in Brazil. The isolate was studied by serogrouping, MLVA, PGFE, and partial sequencing of rrs and secY. It was serogrouped as serogroup Australis, probably serovar Bratislava (titre 1,600), and sequenced as Leptospira interrogans. The MLVA and PGFE profiles also suggested the isolate as serovar Bratislava, since they were indistinguishable from reference strains Balico and Jez Bratislava. This is the first Leptospira interrogans serogroup Australis isolate, probably serovar Bratislava, obtained in Brazil...

O objetivo deste estudo foi relatar o primeiro isolamento e caracterização de Leptospira interrogans (sorogrupo Australis) a partir da urina de suínos no Brasil. O isolado foi caracterizado por sorogrupagem, MLVA, PGFE, e sequenciamento parcial de rrs e secY. Este foi identificado como pertencente ao sorogurpo Australis, provavelmente sorovar Bratislava (título 1600) e sequenciado como Leptospira interrogans. Os perfis de MLVA e PGFE também sugeriram o isolado como sorovar Bratislava, uma vez que estes foram indistinguíveis das cepas de referência Balico e Jez Bratislva. Este é o primeiro isolado de Leptospira interrogans sorogrupo Australis, provavelmente sorovar Bratislava, obtido no Brasil...

Predominance of Leptospira interrogans serovar Bratislava DNA in vaginal fluid of mares suggests sexual transmission of leptospirosis.

The purpose of the present study was to detect the presence of DNA of pathogenic Leptospira sp. in vaginal fluids of mares regarding a possible role of the sexual transmission. A total of 134 breeding mares from four troops were studied and sampling was conducted from vaginal fluids and urine for culture and PCR; and blood for serology. From the 134 serum samples tested, 59 (44%) were seroreactive, and serovar Bratislava was the most frequent (54.2%). None positive culture was obtained, but leptospiral DNA was detected by PCR (lipL32 gene) in 45 (33.5%) urine samples and 43 (32%) vaginal fluid (VF) samples. By phylogenetic analysis of the sequenced amplicons (secY gene) obtained after urine samples, it was found that 14/23 (60.9%) were of Bratislava and nine (39.1%) of Copenhageni. In contrast, the totality of the sequenced amplicons obtained after VF samples were of Bratislava serovar. This study demonstrated by the first time the presence of leptospiral DNA in the vaginal fluid of mares. Furthermore, the identification of that DNA as belonging to serovar Bratislava suggests that the transmission of leptospirosis in horses may occur by sexual via.

Serovar diversity of pathogenic Leptospira circulating in the French West Indies.

BACKGROUND: Leptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012. METHODS AND FINDINGS: Strains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and secY. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchi, and L. santarosai. We also identified L. kmetyi in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with L. interrogans serovars Icterohaemorrhagiae and Copenhageni, L. kirschneri serovar Bogvere, and L. borgpetersenii serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new secY alleles. CONCLUSIONS: The overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars.

Comparison of real-time PCR assays for detection of pathogenic Leptospira spp. in blood and identification of variations in target sequences.

Leptospirosis is considered an underdiagnosed disease. Although several PCR-based methods are currently in use, there is little information on their comparability. In this study, four quantitative real-time PCR (qPCR) assays (SYBR green and TaqMan chemistries) targeting the secY, lfb1, and lipL32 genes were evaluated as diagnostic assays. In our hands, these assays can detect between 10(2) and 10(3) bacteria/ml of pure culture, whole-blood, plasma, and serum samples. In three independent experiments, we found a slightly higher sensitivity of the PCR assays in plasma than in whole blood and serum. We also evaluated the specificity of the PCR assays on reference Leptospira strains, including newly described Leptospira species, and clinical isolates. No amplification was detected for DNA obtained from saprophytic or intermediate Leptospira species. However, among the pathogens, we identified sequence polymorphisms in target genes that result in primer and probe mismatches and affect qPCR assay performance. In conclusion, most of these assays are sensitive and specific tools for routine diagnosis of leptospirosis. However, it is important to continually evaluate and, if necessary, modify the primers and/or probes used to ensure effective detection of the circulating Leptospira isolates.

Development of ertapenem resistance in a patient with mediastinitis caused by Klebsiella pneumoniae producing an extended-spectrum beta-lactamase.

The aim was to study the clinical and microbiological features associated with a carbapenem-resistant Klebsiella pneumoniae isolate that had been selected in vivo by an ertapenem-containing regimen in a patient with mediastinitis despite high blood and mediastinal levels of ertapenem. Carbapenem resistance was characterized by conjugation, PCR, DNA sequencing and analysis of outer-membrane proteins. The isolates susceptible and resistant to the carbapenems were compared by ribotyping and PFGE. Resistance to all available beta-lactams was most probably due to combined production of extended-spectrum beta-lactamase (ESBL) CTX-M-15 and loss of OmpK36 porin. The results of ribotyping and PFGE suggest that the carbapenem-resistant strain was a derivative of the original mediastinal isolate rather than a superinfecting isolate. This observation stresses the risk of selection of pan-penem resistant strains of enterobacteria when ertapenem is used for the treatment of severe infections due to ESBL-producing enterobacteria.

GES-11, a novel integron-associated GES variant in Acinetobacter baumannii.

New extended-spectrum beta-lactamase GES-11 was detected in Acinetobacter baumannii BM4674. The enzyme conferred resistance to beta-lactams, including aztreonam, and reduced susceptibility to carbapenems. The structural gene was part of a class 1 integron borne by self-transferable plasmid pIP847. GES-type beta-lactamases have not been reported previously in A. baumannii.

AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii.

We have identified a second resistance-nodulation-cell division (RND)-type efflux pump, AdeIJK, in clinical isolate Acinetobacter baumannii BM4454. The adeI, adeJ, and adeK genes encode, respectively, the membrane fusion, RND, and outer membrane components of the pump. AdeJ belongs to the AcrB protein family (57% identity with AcrB from Escherichia coli). mRNA analysis by Northern blotting and reverse transcription-PCR indicated that the genes were cotranscribed. Overexpression of the cloned adeIJK operon was toxic in both E. coli and Acinetobacter. The adeIJK genes were detected in all of the 60 strains of A. baumannii tested. The two latter observations suggest that the AdeIJK complex might contribute to intrinsic but not to acquired antibiotic resistance in Acinetobacter. To characterize the substrate specificity of the pump, we have constructed derivatives of BM4454 in which adeIJK (strain BM4579), adeABC (strain BM4561), or both groups of genes (strain BM4652) were inactivated by deletion-insertion. Determination of the antibiotic susceptibility of these strains and of BM4652 and BM4579, in which the adeIJK operon was provided in trans, indicated that the AdeIJK pump contributes to resistance to beta-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, trimethoprim, acridine, safranin, pyronine, and sodium dodecyl sulfate. The chemical structure of these molecules suggests that amphiphilic compounds are the preferred substrates. The AdeABC and AdeIJK efflux systems contributed in a more than additive fashion to tigecycline resistance.