Biovar 1A Strains Research Articles

Characterisation of Yersinia Enterocolitica Strains Isolated from Wildlife in the Northwestern Italian Alps.

IntroductionYersiniosis is a zoonosis causing gastroenteritis, diarrhoea, and occasionally reactive arthritis and septicaemia. Cases are often linked to meat consumption and the most common aetiological agent is the Gram-negative bacilliform Yersinia enterocolitica bacterium. The occurrence of Yersinia spp. among wild animals has mostly been studied in wild boar, but it has seldom been in other species.Material and MethodsA total of 1,868 faecal samples from animals found dead or hunted were collected between 2015 and 2018 in the Valle d’Aosta region of the northwestern Italian Alps. Alpine ibex faecal samples were collected during a health monitoring program in 2018. Bacteria were isolated via PCR and confirmed as Y. enterocolitica biochemically. Strain antimicrobial susceptibility was tested by Kirby–Bauer disc diffusion, and the presence of virulence factors and antimicrobial resistance genes was investigated using whole-genome sequencing.ResultsYersinia enterocolitica strains of biotype 1A were detected in six faecal samples from red deer (0.93%), roe deer (0.49%) and red foxes (0.7%). Strains found in beech martens (3.57%) and Alpine ibex (2.77%) belonged to biotypes 1B and 5, respectively and harboured the pYPTS01 plasmid that had only been detected in Y. pseudotuberculosis PB1/+. All the isolates were resistant to ampicillin and erythromycin.ConclusionThe biovar 1A strains exhibited different virulence factors and behaved like non-pathogenic commensals. The strain from an Alpine ibex also harboured the self-transmissible pYE854 plasmid that can mobilise itself and the pYPTS01 plasmid to other strains. The beech marten could be considered a sentinel animal for Y. enterocolitica. Phenotypic resistance may account for the ability of all the strains to resist β-lactams.

Lon Protease- and Temperature-Dependent Activity of a Lysis Cassette Located in the Insecticidal Island of Yersinia enterocolitica.

The Yersinia genus comprises pathogens that can adapt to an environmental life cycle stage as well as to mammals. Yersinia enterocolitica strain W22703 exhibits both insecticidal and nematocidal activity conferred by the tripartite toxin complex (Tc) that is encoded on the 19-kb pathogenicity island Tc-PAI Ye All tc genes follow a strict temperature regulation in that they are silenced at 37°C but activated at lower temperatures. Four highly conserved phage-related genes, located within the Tc-PAI Ye , were recently demonstrated to encode a biologically functional holin-endolysin gene cassette that lyses its own host W22703 at 37°C. Conditions transcriptionally activating the cassette are not yet known. In contrast to Escherichia coli, the overproduction of holin and endolysin did not result in cell lysis of strain W22703 at 15°C. When the holin-endolysin genes were overexpressed at 15°C in four Y. enterocolitica biovars and in four other Yersinia spp., a heterogenous pattern of phenotypes was observed, ranging from lysis resistance of a biovar 1A strain to the complete growth arrest of a Y. kristensenii strain. To decipher the molecular mechanism underlying this temperature-dependent lysis, we constructed a Lon protease-negative mutant of W22703 in which the overexpression of the lysis cassette leads to cell death at 15°C. Overexpressed endolysin exhibited a high proteolytic susceptibility in strain W22703 but remained stable in the W22703 Δlon strain or in Y. pseudotuberculosis Although artificial overexpression was applied here, the data indicate that Lon protease plays a role in the control of the temperature-dependent lysis in Y. enterocolitica W22703.IMPORTANCE The investigation of the mechanisms that help pathogens survive in the environment is a prerequisite to understanding their evolution and their virulence capacities. In members of the genus Yersinia, many factors involved in virulence, metabolism, motility, or biofilm formation follow a strict temperature-dependent regulation. While the molecular mechanisms underlying the activation of determinants at body temperature have been analyzed in detail, the molecular basis of low-temperature-dependent phenotypes is largely unknown. Here, we demonstrate that a novel phage-related lysis cassette, which is part of the insecticidal and nematocidal pathogenicity island of Y. enterocolitica, does not lyse its own host following overexpression at 15°C and that the Lon protease is involved in this phenotype.

Analysis of iron acquisition and storage-related genes in clinical and non-clinical strains of Yersinia enterocolitica biovar 1A.

Possession of mechanisms for iron acquisition and its storage enhances the ability of the bacteria to survive in the iron-limiting environment of the host. In this study, 81 strains of Yersinia enterocolitica biovar 1A isolated from various clinical (n=51) and non-clinical (n=30) sources were investigated for the presence of the genes related to iron acquisition and storage. Important genes which were present in more than 85% of the strains included hasA, foxA, bfr, bfd, ftnA, and hmsT as well as the fhuCDB, fepBDGCfesfepA, feoAB, yfuABCD, hemPRSTUV, and hmsHFRS gene clusters. Majority of these genes is being reported for the first time in biovar 1A strains and showed significant homology with genes present in the known pathogenic biovars of Y.enterocolitica. However, no significant difference was observed in the distribution of iron acquisition and storage-related genes among clinical and non-clinical biovar 1A strains. Thus, it may be suggested that the presence of iron acquisition and storage-related genes per se might not be responsible for the supposedly better ability of clinical biovar 1A strains to cause infections in humans. However, in the backdrop of this data, the need to undertake functional studies are highly recommended.

Proteomic analysis of Yersinia enterocolitica biovar 1A under iron-rich and iron-poor conditions indicate existence of efficiently regulated mechanisms of iron homeostasis

The pathogenicity of Yersinia enterocolitica biovar 1A strains is controversial as these lack most of the known virulence factors. Acquisition of iron and presence of well-regulated iron homeostasis in bacteria represents an important virulence trait. Differential abundance of proteins was examined under iron-rich and iron-poor conditions in a clinical Y. enterocolitica biovar 1A strain IP27407. Whole cell protein profiles were analysed by 2D gel electrophoresis (2D-GE). Following statistical and MALDI-TOF MS analyses, 28 differentially abundant proteins were identified. Significant iron-responsive changes were observed in the proteins involved in iron acquisition or storage namely, hemin receptor (HemR), periplasmic Fe2+ transport protein (Tpd), periplasmic chelated iron-binding protein (YfeA) and bacterioferritin (Bfr). Quantitative real-time PCR (qRT-PCR) of eight mRNA transcripts revalidated the differential protein abundance. In silico analysis of iron homeostasis mediated by the bacterioferritin and bacterioferritin-associated ferredoxin (Bfr–Bfd) complex suggested two pathways for the release of reserve iron which might be operating under conditions of different iron availability. The study, for the first time, showed the existence of highly competent iron homeostasis mechanisms in Y. enterocolitica biovar 1A and identified the key proteins involved thereof. Such mechanisms might have implications for the pathogenicity of Y. enterocolitica biovar 1A strains. Biological significanceAlthough, a few studies have identified the differentially abundant bacterial proteins in response to iron starvation, little information is available in this regard for Y. enterocolitica (especially, the biovar 1A strains). In the present study, differential abundance of several proteins was identified under iron-rich and iron-poor conditions by 2D-GE and MALDI-TOF/MS analysis. These included proteins which may not only be directly implicated in iron acquisition or storage but also play crucial role in cellular metabolism. Given the absence of most known virulence factors in Y. enterocolitica biovar 1A strains, demonstration of well-regulated mechanisms for efficient iron homeostasis constitutes an important observation. The proteins, as identified in the present study, provide useful insights to further unravel the potential pathogenicity of the biovar 1A strains.

Yersinia enterocolitica: Revisitation of an Enduring Human Pathogen

Yersinia enterocolitica remains a charismatic microorganism. The research findings outlined in this article have uncovered new virulence factors, such as type III secretion systems, and have advanced our understanding of the pathogenesis of gastrointestinal infections caused by Y. enterocolitica. Furthermore, a clarification of the potential capability of biovar 1A strains, long regarded as avirulent, has now proven that they cause symptomatic infections in appropriate patient settings, such as immunosuppression or hemochromatosis. Researchers have also expanded the spectra of non-gastrointestinal clinical manifestations of disease to include cutaneous infections, endocarditis, pneumonia, pharyngitis, and meningitis.

Interaction of Yersinia enterocolitica biovar 1A with cultured cells in vitro does not reflect the two previously identified clonal groups

Yersinia enterocolitica biovar 1A strains have been delineated into two clonal groups (A and B) based on repetitive extragenic palindrome- and enterobacterial repetitive intergenic consensus-PCR genotyping. The present study investigated the interaction of Y. enterocolitica biovar 1A strains with cultured cells in vitro by their ability to adhere, invade and survive within these cells. The response of macrophages to these strains was also studied by quantifying the expression of inducible nitric oxide synthase, production of nitric oxide and cytokines, and activation of NFκB. The survival rate of clonal group B strains inside macrophages was significantly higher than that of clonal group A strains. In addition, strains harbouring the fepA gene showed better survival inside macrophages. However, the production of nitric oxide and cytokines and activation of NFκB did not show any significant differences between the two clonal groups. In this study, interaction of Y. enterocolitica biovar 1A with cultured cells in vitro did not reflect the previously identified clonal groups, but was more dependent on the characteristics of the individual strains. Therefore, a combination of genotype and phenotype data must be used to characterize this extremely heterogeneous organism.

Identification and distribution of putative virulence genes in clinical strains ofYersinia enterocoliticabiovar 1A by suppression subtractive hybridization

To detect putative virulence genes in clinical strains of Yersinia enterocolitica biovar 1A by suppression subtractive hybridization between two closely related strains of clinical and nonclinical origin having the same serotype (O:6,30-6,31). Suppression Subtractive Hybridization (SSH) was used to identify genomic differences between clinical (serotype O:6,30-6,31, from diarrhoeic human stools) and nonclinical (serotype O:6,30-6,31, from wastewater) strains of Y. enterocolitica biovar 1A. Following genomic subtraction and DNA sequencing, nine DNA sequences that were present only in clinical biovar 1A strains were identified. The sequences identified using SSH showed similarity to conserved hypothetical proteins, proteins related to iron acquisition and haemin storage, type 1 secretion proteins, flagellar hook proteins, exported protein and ABC transport system. All these sequences showed high similarity with Y. enterocolitica 8081 (biovar 1B). The distribution of these genes was further analysed using PCR in 26 clinical strains of Y. enterocolitica biovar 1A. The results revealed that the distribution of these genes was not uniform. Genes related to iron acquisition and storage, and flagellar proteins might be responsible for virulence of some of the clinical strains of Y. enterocolitica biovar 1A. Genes identified in this study might be useful in understanding the pathogenic potential of clinical strains of Y. enterocolitica biovar 1A.

Whole cell protein profiling reiterate phylogenetic relationships among strains of Yersinia enterocolitica biovar 1A as discerned earlier by different genotyping methods

To evaluate whole cell protein profiling vis-à-vis genotyping to discern phylogenetic relationships among strains of Y. enterocolitica biovar 1A. Whole cell protein profiling of Y. enterocolitica biovar 1A strains was performed using SDS-PAGE. Twenty-one distinct protein profile types were obtained among a collection of 81 strains isolated from clinical and nonclinical sources. Whole cell protein profiling exhibited discriminatory index (DI) of 0·80 and clustered the strains into two distinct clonal groups. The clinical and the aquatic serotype O:6,30-6,31 strains were clustered into two discrete subgroups. Whole cell protein profiling displayed sufficient diversity among strains of Y. enterocolitica biovar 1A, and the phylogenetic relationships obtained were in good agreement with those established earlier by genotyping techniques. Whole cell protein profiling was as discriminatory as some of the genotyping methods and has the potentiality to be used as an adjunct tool to study epidemiology of Y. enterocolitica.

Genetic relationships between clinical and non-clinical strains of Yersinia enterocolitica biovar 1A as revealed by multilocus enzyme electrophoresis and multilocus restriction typing

BackgroundGenetic relationships among 81 strains of Y. enterocolitica biovar 1A isolated from clinical and non-clinical sources were discerned by multilocus enzyme electrophoresis (MLEE) and multilocus restriction typing (MLRT) using six loci each. Such studies may reveal associations between the genotypes of the strains and their sources of isolation.ResultsAll loci were polymorphic and generated 62 electrophoretic types (ETs) and 12 restriction types (RTs). The mean genetic diversity (H) of the strains by MLEE and MLRT was 0.566 and 0.441 respectively. MLEE (DI = 0.98) was more discriminatory and clustered Y. enterocolitica biovar 1A strains into four groups, while MLRT (DI = 0.77) identified two distinct groups. BURST (Based Upon Related Sequence Types) analysis of the MLRT data suggested aquatic serotype O:6,30-6,31 isolates to be the ancestral strains from which, clinical O:6,30-6,31 strains might have originated by host adaptation and genetic change.ConclusionMLEE revealed greater genetic diversity among strains of Y. enterocolitica biovar 1A and clustered strains in four groups, while MLRT grouped the strains into two groups. BURST analysis of MLRT data nevertheless provided newer insights into the probable evolution of clinical strains from aquatic strains.

Molecular and biochemical characterization of urease and survival of Yersinia enterocolitica biovar 1A in acidic pH in vitro

BackgroundYersinia enterocolitica, an important food- and water-borne enteric pathogen is represented by six biovars viz. 1A, 1B, 2, 3, 4 and 5. Despite the lack of recognized virulence determinants, some biovar 1A strains have been reported to produce disease symptoms resembling that produced by known pathogenic biovars (1B, 2-5). It is therefore imperative to identify determinants that might contribute to the pathogenicity of Y. enterocolitica biovar 1A strains. Y. enterocolitica invariably produces urease and the role of this enzyme in the virulence of biovar 1B and biovar 4 strains has been reported recently. The objective of this work was to study genetic organization of the urease (ure) gene complex of Y. enterocolitica biovar 1A, biochemical characterization of the urease, and the survival of these strains under acidic conditions in vitro.ResultsThe ure gene complex (ureABCEFGD) of Y. enterocolitica biovar 1A included three structural and four accessory genes, which were contiguous and was flanked by a urea transport (yut) gene on the 3' side. Differences were identified in ure gene complex of biovar 1A strain compared to biovar 1B and 4 strains. This included a smaller ureB gene and larger intergenic regions between the structural genes. The crude urease preparation exhibited optimal pH and temperature of 5.5 and 65°C respectively, and Michaelis-Menten kinetics with a Km of 1.7 ± 0.4 mM urea and Vmax of 7.29 ± 0.42 μmol of ammonia released/min/mg protein. The urease activity was dependent on growth temperature and growth phase of Y. enterocolitica biovar 1A, and the presence of nickel in the medium. The molecular mass of the enzyme was > 545 kDa and an isoelectric point of 5.2. The number of viable Y. enterocolitica biovar 1A decreased significantly when incubated at pH 2.5 for 2 h. However, no such decrease was observed at this pH in the presence of urea.ConclusionsThe ure gene cluster of biovar 1A strains though similar to biovar 1B and 4 strains, exhibited important differences. The study also showed the ability of biovar 1A strains of Y. enterocolitica to survive at highly acidic pH in vitro in the presence of urea.

Distribution of virulence-associated genes inYersinia enterocoliticabiovar 1A correlates with clonal groups and not the source of isolation

Yersinia enterocolitica, an important food- and water-borne enteric pathogen, is represented by six biovars viz. 1A, 1B and 2-5. Some biovar 1A strains, despite lacking virulence plasmid (pYV) and chromosomal virulence genes, have been reported to cause symptoms similar to that produced by isolates belonging to known pathogenic biovars. Virulence-associated genes viz. ail, virF, inv, myfA, ystA, ystB, ystC, tccC, hreP, fepA, fepD, fes, ymoA and sat were studied in 81 clinical and nonclinical strains of Y. enterocolitica biovar 1A by PCR amplification. All strains lacked ail, virF, ystA and ystC genes. The distribution of other genes with respect to clonal groups revealed that four genes viz. ystB, hreP, myfA and sat were associated exclusively with strains belonging to clonal group A. The clonal groups A and B were differentiated previously based on rep (REP-/ERIC) - PCR genomic fingerprinting. The distribution of virulence-associated genes, however, did not differ significantly between clinical and nonclinical strains. In strains of Y. enterocolitica biovar 1A, clonal groups seem to reflect virulence potential better than the source (clinical vs. nonclinical) of isolation.

Molecular characterization of β-lactamase genesblaAandblaBofYersinia enterocoliticabiovar 1A

The beta-lactamase genes blaA and blaB were detected by PCR amplification in strains of Yersinia enterocolitica biovar 1A isolated from India, Germany, France and the USA. Both genes were detected in all strains. Polymerase chain reaction-restriction fragment length polymorphism revealed genetic heterogeneity in blaA but not in blaB. Cluster analysis of blaA restriction profiles grouped the strains into three groups. The blaA gene of Y. enterocolitica biovar 1A showed a high degree of sequence homology to that of Y. enterocolitica 8081 (biovar 1B) and Y. enterocolitica Y-56 (biovar 4), whereas homology was low with class A beta-lactamase genes of other members of the family Enterobacteriaceae. The pI 8.7 of enzyme Bla-A of Y. enterocolitica biovar 1A was similar to that of biovars 2, 3 and 4. The enzyme Bla-B focused at 6.8 and 7.1, indicating that biovar 1A strains produced a 'B-like' enzyme. This is the first study to have investigated the genetic heterogeneity of the beta-lactamase genes of Y. enterocolitica.

Beta-lactamase expression in Yersinia enterocolitica biovars 1A, 1B, and 3.

Characteristic patterns of beta-lactam susceptibility are associated with different biovars of Yersinia enterocolitica. In a previous study differences in beta-lactam susceptibility among biovar 2, 4 and 5 strains were largely attributed to differences in expression of beta-lactamase A (BlaA) and beta-lactamase B (BlaB). The basis for differences in beta-lactam susceptibility of strains of biovars 1A, 1B and 3 is now considered. All the strains examined had blaB; nine of 31 biovar 3 strains and two of 13 biovar 1B strains had blaA, but PCR did not amplify blaA from biovar 1A strains. Nevertheless, inhibition data indicated that the majority of uninduced biovar 1A strains expressed BlaA and BlaB in similar amounts. Strong inducibility was seen in all these strains. Biovar 1B strains (which were less inducible than strains of biovar 1A) predominantly produced BlaA without induction; ticarcillin-sensitive strains of biovar 3 produced only BlaB but were not inducible; without induction biovar 3 strains resistant to ticarcillin and amoxycillin/clavulanate produced either predominantly BlaA, predominantly BlaB or exclusively BlaB and induction was demonstrated except for strains producing BlaB alone; biovar 3 strains resistant to ticarcillin but sensitive to amoxycillin/clavulanate predominantly produced BlaA without induction and were inducible for beta-lactamase activity. After induction, nearly all strains predominantly or exclusively produced BlaB. Although PCR amplification fragments with primers specific for blaA were obtained only from some strains, the induction and inhibition data suggest that all Y. enterocolitica strains possess enzymes related to BlaA- as well as BlaB. Nevertheless, expression of the beta-lactamase is regulated differently in different biovars and varies within most biovars. Failure to predict beta-lactamase expression profiles from MIC data indicates the presence of additional mechanisms contributing to differences in susceptibility.

Is natural genetic transformation a mechanism of horizontal gene transfer in Yersinia?

We investigated the capacity of different Yersinia strains with emphasis to Yersinia enterocolitica to take up and incorporate DNA by natural genetic transformation. Our studies were initiated by the observation of partial homology between the virulence plasmid of a pathogenic Y. enterocolitica strain (O: 3, biovar 4) and plasmids indiginous to different a pathogenic Y. enterocolitica biovar 1A strains revealed by hybridization studies. Furthermore, an observation of natural genetic transformation in a strain of Y. enterocolitica has been published by CALLAHAN and KOROMA (1979). To detect an uptake and incorporation of DNA, we incubated potential recipient strains with naked DNA under varying experimental conditions. The parameters tested were--the recipient strain,--the markers used to detect a DNA transfer,--the condition of the transforming DNA,--the nutrient availability,--the temperature,--the growth phase, and--the influence of stress. In our experiments, we could not identify conditions under which Y. enterocolitica could be naturally transformed. We thus conclude that natural transformation is unlikely to be an important mechanism for horizontal gene transfer in Yersinia.

Comparison of Plasmids of Strains of Yersinia enterocolitica Biovar 1A with the virulence Plasmid of a Pathogenic Y. enterocolitica Strain

The plasmid content of apathogenic Y. enterocolitica biovar 1A strains was determined and the plasmids were compared with the virulence plasmid of a pathogenic Y. enterocolitica strain. About 38% of the selected biovar 1A strains contained plasmids of different sizes ranging from 2.7 kb to more than 70 kb. Some of the larger plasmids had a size similar to that of the virulence plasmid of a pathogenic reference strain. The restriction patterns of these plasmids were different from the restriction pattern of the virulence plasmid of the pathogenic reference strain. Differences were also observed in hybridization studies with the virulence plasmid. The plasmids from 15 out of 16 biovar 1A strains showed no homology, whereas the plasmid of one biovar 1A strain partially hybridized to the virulence plasmid.