Insights into the genomic traits of Yersinia frederiksenii, Yersinia intermedia and Yersinia kristensenii isolated from diverse sources in Brazil.

Yersinia is an important genus comprising foodborne, zoonotic and pathogenic bacteria. On the other hand, species of the so-called group Yersinia enterocolitica-like are understudied and mostly characterized as non-pathogenic, despite of some reports of human infections. The present study aimed to provide genomic insights of Yersinia frederiksenii (YF), Yersinia intermedia (YI) and Yersinia kristensenii (YK) isolated worldwide. A total of 22 YF, 20 YI and 14 YK genomes were searched for antimicrobial resistance genes, plasmids, prophages, and virulence factors. Their phylogenomic relatedness was analyzed by Gegenees and core-genome multi-locus sequence typing. Beta-lactam resistance gene blaTEM-116 and five plasmids replicons (pYE854, ColRNAI, ColE10, Col(pHAD28) and IncN3) were detected in less than five genomes. A total of 59 prophages, 106 virulence markers of the Yersinia genus, associated to adherence, antiphagocytosis, exoenzymes, invasion, iron uptake, proteases, secretion systems and the O-antigen, and virulence factors associated to other 20 bacterial genera were detected. Phylogenomic analysis revealed high inter-species distinction and four highly diverse YF clusters. In conclusion, the results obtained through the analyses of YF, YI and YK genomes suggest the virulence potential of these strains due to the broad diversity and high frequency of prophages and virulence factors found. Phylogenetic analyses were able to correctly distinguish these closely related species and show the presence of different genetic subgroups. These data contributed for a better understanding of YF, YI and YK virulence-associated features and global genetic diversity, and reinforced the need for better characterization of these Y. enterocolitica-like species considered non-pathogenic.

Identification of microbial pathogens in Neolithic Scandinavian humans.

With the Neolithic transition, human lifestyle shifted from hunting and gathering to farming. This change altered subsistence patterns, cultural expression, and population structures as shown by the archaeological/zooarchaeological record, as well as by stable isotope and ancient DNA data. Here, we used metagenomic data to analyse if the transitions also impacted the microbiome composition in 25 Mesolithic and Neolithic hunter-gatherers and 13 Neolithic farmers from several Scandinavian Stone Age cultural contexts. Salmonella enterica, a bacterium that may have been the cause of death for the infected individuals, was found in two Neolithic samples from Battle Axe culture contexts. Several species of the bacterial genus Yersinia were found in Neolithic individuals from Funnel Beaker culture contexts as well as from later Neolithic context. Transmission of e.g. Y. enterocolitica may have been facilitated by the denser populations in agricultural contexts.

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.

Susceptibility of Yersinia enterocolitica to the novel fluoroquinolone delafloxacin.

Minimum inhibitory concentration to delafloxacin and ciprofloxacin were performed with Y. enterocolitica, Y. frederiksenii and Y. kristensenii. All organisms were sensitive to delafloxacin and ciprofloxacin. Our study indicates that delafloxacin may have a reasonable in vitro susceptibility profile against Yersinia among the species studied, which has not been previously reported.

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).

Comparative genomic insights into Yersinia hibernica - a commonly misidentified Yersinia enterocolitica-like organism.

Food-associated outbreaks linked to enteropathogenic Yersinia enterocolitica are of concern to public health. Pigs and their meat are recognized risk factors for transmission of Y. enterocolitica. This study aimed to describe the comparative genomics of Y. enterocolitica along with a number of misclassified Yersinia isolates, now constituting the recently described Yersinia hibernica. The latter was originally cultured from an environmental sample taken at a pig slaughterhouse. Unique features were identified in the genome of Y. hibernica, including a novel integrative conjugative element (ICE), denoted as ICEYh-1 contained within a 255 kbp region of plasticity. In addition, a zebrafish embryo infection model was adapted and applied to assess the virulence potential among Yersinia isolates including Y. hibernica.

Yersinia canariae sp. nov., isolated from a human yersiniosis case.

A Gram-negative rod from the Yersinia genus was isolated from a clinical case of yersiniosis in the United Kingdom. Long read sequencing data from an Oxford Nanopore Technologies (ONT) MinION in conjunction with Illumina HiSeq reads were used to generate a finished quality genome of this strain. Overall Genome Related Index (OGRI) of the strain was used to determine that it was a novel species within Yersinia, despite biochemical similarities to Yersinia enterocolitica. The 16S ribosomal RNA gene accessions are MN434982-MN434987 and the accession number for the complete and closed chromosome is CP043727. The type strain is SRR7544370T (=NCTC 14382T/=LMG 31573T).

PREVALENCE OF YERSINIA AMONG WILD SIKA DEER (CERVUS NIPPON) AND BOARS (SUS SCROFA) IN JAPAN.

We examined the prevalence of Yersinia, including pathogenic species such as Yersinia enterocolitica and Yersinia pseudotuberculosis, among wild sika deer (Cervus nippon) and boars (Sus scrofa) captured in Japan. The prevalence of Yersinia in the wild deer was 75% (207/277) and in the boars was 74% (40/54). A total of 417 isolates of nine Yersinia species were isolated from the animals examined: the largest number of isolates (48%, 200/417) were Y. enterocolitica biotype 1A. Pathogenic Y. enterocolitica 1B/O:8 were also isolated from two deer, and Y. pseudotuberculosis serogroups 3 and 4 were isolated from two boars and a deer, respectively. The pathogenic Y. enterocolitica 1B/O:8 isolates carried four virulence genes (ail, ystA, yadA, and virF), and Y. pseudotuberculosis serogroups 3 and 4 isolates carried three virulence genes (inv, yadA, and lcrF). Although the Y. enterocolitica 1B/O:8 and Y. pseudotuberculosis isolates were sensitive to almost all the antimicrobials tested, the two Y. enterocolitica 1B/O:8 isolates were resistant to azithromycin and ampicillin, and the three Y. pseudotuberculosis isolates were resistant only to azithromycin. These findings suggested that wild deer and boars might be important reservoirs for the agent causing human yersiniosis.

Untargeted accurate identification of highly pathogenic bacteria directly from blood culture flasks.

To improve the preparedness against exposure to highly pathogenic bacteria and to anticipate the wide variety of bacteria that can cause bloodstream infections (BSIs), a safe, unbiased and highly accurate identification method was developed. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method can identify highly pathogenic bacteria, their near-neighbors and bacteria that are common causes of BSIs directly from positive blood culture flasks. The developed Peptide-Based Microbe Detection Engine (http://proteome2pathogen.com) relies on a two-step workflow: a genus-level search followed by a species-level search. This strategy enables the rapid identification of microorganisms based on the analyzed proteome. This method was successfully used to identify strains of Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia pseudomallei, Burkholderia mallei, Francisella tularensis, Yersinia pestis and closely related species from simulated blood culture flasks. This newly developed LC-MS/MS method is a safe and rapid method for accurately identifying bacteria directly from positive blood culture flasks.

Genus-wide Yersinia core-genome multilocus sequence typing for species identification and strain characterization.

The genus Yersinia comprises species that differ widely in their pathogenic potential and public-health significance. Yersinia pestis is responsible for plague, while Yersinia enterocolitica is a prominent enteropathogen. Strains within some species, including Y. enterocolitica, also vary in their pathogenic properties. Phenotypic identification of Yersinia species is time-consuming, labour-intensive and may lead to incorrect identifications. Here, we developed a method to automatically identify and subtype all Yersinia isolates from their genomic sequence. A phylogenetic analysis of Yersinia isolates based on a core subset of 500 shared genes clearly demarcated all existing Yersinia species and uncovered novel, yet undefined Yersinia taxa. An automated taxonomic assignment procedure was developed using species-specific thresholds based on core-genome multilocus sequence typing (cgMLST). The performance of this method was assessed on 1843 isolates prospectively collected by the French National Surveillance System and analysed in parallel using phenotypic reference methods, leading to nearly complete (1814; 98.4 %) agreement at species and infra-specific (biotype and serotype) levels. For 29 isolates, incorrect phenotypic assignments resulted from atypical biochemical characteristics or lack of phenotypic resolution. To provide an identification tool, a database of cgMLST profiles and reference taxonomic information has been made publicly accessible (https://bigsdb.pasteur.fr/yersinia). Genomic sequencing-based identification and subtyping of any Yersinia is a powerful and reliable novel approach to define the pathogenic potential of isolates of this medically important genus.

Enhanced Binding Efficiency of Microcantilever Biosensor for the Detection of Yersinia.

A novel microcantilever sensor was batch fabricated for Yersinia detection. The microcantilever surface modification method was optimized by introducing a secondary antibody to increase the number of binding sites. A novel microfluidic platform was designed and fabricated successfully. A 30 µL solution could fully react with the microcantilever surface. Those routines enhanced the binding efficiency between the target and receptor on the microcantilever. With this novel designed microfluidic platform, the specific adsorption of 107 Yersinia on the beam surface with modified F1 antibody was significantly enhanced.

Yersinia infection in two captive guereza colobus monkeys (Colobus guereza).

Two guereza colobus monkeys (Colobus guereza) reared in a zoological garden in Japan suddenly died of multifocal fibrinonecrotic gastroenteritis and septicemia associated with infection by Yersinia spp. It was necessary to microbiologically differentiate Yersinia frederiksenii and Y. enterocolitica. We described the pathological findings and discuss the causal agent to emphasize the need to revert to using a combination of multiple examinations for diagnosis.

Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using ß-Lactamase Fusions.

Development of the TEM-CCF2/4-AM FRET-based system has enabled investigators to track translocation of effector proteins into mammalian cells during infection. This allows for separation of translocated and non-translocated cell populations for further study. Yersinia strains expressing translational Yop-TEM fusions, containing the secretion and translocation signals of a Yop with the TEM-1 portion of ß-lactamase, are used to infect mice, tissues isolated from mice, or mammalian cells in culture. Infected and harvested mammalian cells are treated with either CCF2-AM or CCF4-AM, and cleavage of this fluorescent compound by TEM is detected by fluorescence-activated cell sorting (FACS) analysis. A shift from green to blue emission spectra of individual cells is indicative of translocation of a given Yop-TEM fusion protein into the host cell during Yersinia infection due to a disruption in FRET between the two fluors of the compound. In Yersinia, this method has been used to understand Type III secretion dynamics and Yop functions in cells translocated by effectors during infection. Here, we describe how to generate Yop-TEM constructs, and how to detect, quantify, isolate, and study Yop-TEM containing cells in murine tissues during infection and in ex vivo tissues by cell sorting and flow cytometry analysis. In addition, we provide guidance for analyzing TEM-positive cells via a plate reader and fluorescent microscopy.

Analysis of Inflammasome Activation in Response to Yersinia Infection by Fluorescence Microscopy Detection of Active Caspase-1 Puncta.

The type of cell death triggered by a particular environmental stimulus influences the outcome of infection or inflammatory disease processes. The ability to identify the cell death pathway that is activated in response to infection is essential for understanding the pathogenesis and host response to infection. Activation of the cysteine protease caspase-1 in various inflammasome complexes indicates that cells are undergoing pyroptosis, a regulated, proinflammatory cell death. Inflammasome assembly and caspase activation can be measured by various methods ranging from detection of inflammasome-dependent cell death, cytokine secretion, cleavage of caspase-1, or the formation of "puncta" within the cell that contain inflammasome components, such as caspase-1 or the adapter protein ASC. Here we describe a method for detecting caspase-1 activation on a single cell level in the context of infection by the Gram-negative pathogen Yersinia using immunofluorescence microscopy. We previously used this approach to quantify caspase-1 puncta formation in cells containing Yersinia translocon components (Zwack et al., MBio 6:e02095-14, 2015). This is a modification of methods used previously by Broz et al. (Cell Host Microbe 8:471-483, 2010) and Case and Roy (MBio 2:e00117-11, 2011). By taking a microscopy-based approach that allows us to quantify puncta as well as other cell-biological features of infection (i.e., number of bacteria associated with a particular cell; levels of bacterial effector or translocon proteins in caspase-1 puncta-containing cells; or levels or localization of host cellular proteins), we can better quantify the heterogeneity between cells undergoing pyroptosis and cells that are not under the same infection conditions. These approaches have the potential to generate hypotheses that can enable further mechanistic insight into activation of pyroptosis in response to bacterial infection.

Yersinia hibernica sp. nov., isolated from pig-production environments.

A Gram-stain-negative, rod-shaped strain isolated from pig-production environments was identified as a new species within the genus Yersinia using multifaceted genomic and biochemical approaches. The genome of this strain was closed using a hybrid assembly approach combining both high accuracy short read sequencing data with long read sequencing technology. Phylogenetic analysis of the 16S rRNA gene showed ~98 % similarity to Yersinia kristensenii and ~98 % similarity to Yersinia enterocolitica. Average nucleotide identity (OrthoANI) values were calculated as 85.79 % to Y. kristensenii ATCC 33638T and 85.73 % to Y. enterocolitica ATCC 9610T thereby providing evidence that this isolate should be considered as a novel species. The type strain is CFS1934T (=NCTC 14222T=LMG 31076T).

Yersinia kristensenii subsp. rochesterensis subsp. nov., isolated from human feces.

A single bacterial isolate, EPLC-04T, was isolated from human feces and identified as representing a member of the genus Yersinia on the basis of phenotypic characteristics, matrix assisted laser desorption ionization time-of-flight mass spectrometry and partial 16S rRNA gene sequencing. The isolate's phenotypic profile differed from that described for the most closely related species, Yersinia kristensenii, by exhibiting lipase production and lacking pyrazinamidase activity. Multiple genetic targets, including the complete (1465 bp) 16S rRNA gene sequence and partial sequences of groEL (539 bp), gyrB (935 bp), glnA (525 bp) and recA (535 bp) indicated that the isolate exhibited 98.91, 92.16, 90.81, 92.78 and 89.01 % identity with Yersinia aldovae, 98.98, 91.99, 90.17, 89.77 and 89.55 % identity with Yersinia intermedia, and 99.66, 98.11, 98.50, 98.49 and 98.51 % identity with Y. kristensenii, respectively. Phylogenetic reconstructions based on the combination of the four housekeeping genes indicated that the isolate formed a unique branch, supported by a bootstrap value of 100 %. Digital DNA-DNA homology and 16S rRNA gene sequencing identified EPLC-04T as representing Y. kristensenii. However, the unique phenotypic traits and results of phylogenetic analysis indicate that it represents a novel subspecies of Y. kristensenii. The name Yersinia kristenseniisubsp. rochesterensis subsp. nov. is proposed for this novel taxon (type strain EPLC-04T=ATCC BAA-2637T, DSMZ 28595T).

High seroprevalence of pathogenic Yersinia spp. in sheep and goats across nine government farms in the Pakistani Punjab.

INTRODUCTION: Seroprevalence of Y. enterocolitica and Y. pseudotuberculosis infections in animals and humans is not established in Pakistan. There are only a few reports on the prevalence of pathogenic Yersinia spp. and infections in small ruminants, however, the role of sheep and goats in the transmission of pathogenic Yersinia remains unclear. METHODOLOGY: A primary survey investigated the presence of anti-Yersinia antibodies among a small population of ruminants detected by recombinant antigen targets in nine government farms dispersed throughout the Punjab province of Pakistan. RESULTS: Antibodies specific for Y. enterocolitica were detected in 7/9 sheep flocks and in 4/4 goat flocks. Antibodies specific for Y. pseudotuberculosis were detected in 4/9 sheep flocks. Two sheep flocks revealed the presence of both Y. enterocolitica and Y. pseudotuberculosis specific antibodies. CONCLUSION: Due to the high number of the population involved in raising small ruminants the risk to veterinary and public health must be rapidly determined.

Prevalence of Yersinia Species in the Ileum of Crohn's Disease Patients and Controls.

Yersinia are common contaminants of food products, but their prevalence in the human gut is poorly documented. Yersinia have been implicated in Crohn's Disease (CD, an inflammatory bowel disease) however their role in CD is controversial. We performed highly sensitive PCR assays of specific sequences for the gyrB gene of Y. aldovae, Y. bercovieri, Y. enterocolitica, Y. intermedia, Y. mollaretii and the inv gene of Y. pseudotuberculosis. We analyzed a total of 470 ileal samples taken from 338 participants (262 CD patients and 76 controls) belonging to three independent cohorts. All patients and controls were phenotyped and genotyped for the main CD susceptibility variants: NOD2, ATG16L1, and IRGM. Yersinia were found in 7.7% of ileal samples (respectively 7.9 and 7.6% in controls and CD patients) corresponding to 10% of participants (respectively 11.8 and 9.5% in controls and CD patients). Y. enterocolitica, Y. pseudotuberculosis and Y. intermedia were the most frequently identified species. The bacteria were more frequent in resected specimens, lymph nodes and Peyer's patches. Yersinia were no more likely to be detected in CD tissues than tissues from inflammatory and non-inflammatory controls. CD patients treated with immunosuppressants were less likely to be Yersinia carriers. In conclusion, this work shows that Yersinia species are frequently found at low levels in the human ileum in health and disease. The role of Yersinia species in this ecosystem should now be explored.

Rapid detection of Yersinia enterocolitica serotype O:3 using a duplex PCR assay.

Yersinia enterocolitica, a member of the Enterobacteriaceae family, is a zoonotic agent that causes gastrointestinal diseases and some extraintestinal disorders in humans. Y. enterocolitica ssp. palearctica bioserotype 4/O:3 is the primary pathogenic bioserotype in Europe, where it has a high public health relevance. The isolation and identification of Y. enterocolitica from various sources on selective media have been seldom successful due to several reasons. In an attempt to overcome the problems associated with traditional culture-based methods, we developed a single duplex PCR assay for the detection of Y. enterocolitica ssp. palearctica bioserotype 4/O:3 using DNA extracted from a source. We combined the primer for tufA (elongation factor Tu) with the primer for rfbC (the biosynthesis of the O side chain) in one single reaction, which showed good results when we analyzed 88 Yersinia strains and when it was tested in the DNA from stool samples of two groups of pregnant women, one comprising HIV-positive women and the other comprising of HIV-negative women. Furthermore, the duplex PCR assay was found to be 16 times better in detecting Yersinia spp. in stool samples than the culture-based method. In addition, it was found to be a rapid screening method for the detection of Y. enterocolitica serotype O:3, and it could still detect other Y. enterocolitica serotypes and Yersinia species as well. We anticipate that the duplex PCR assay could be a useful tool for hospital and veterinary surveillance studies on Yersinia worldwide.

Isothermal DNA amplification combined with lateral flow dipsticks for detection of biothreat agents.

The recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis, and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis, medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results.