Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry

Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level.     

A cluster of atypical Yersinia strains with a distinctive 16S rRNA signature

Thirty-eight bacterial isolates from raw milk samples in Queensland, Australia were identified as members of the genus Yersinia on the basis of biochemical profile, ability to hybridize with a genus-specific DNA probe, comparative 16S rDNA sequence analysis, and the presence of characteristic 16S rDNA signature nucleotides which occur in all Yersinia spp. Twenty-five of these isolates reacted with typing sera (O:22 or O:58) of Y. enterocolitica; the remainder were non-typable. None of the isolates displayed any of the phenotypic or genetic virulence-associated characteristics of Y. enterocolitica. Comparative 16S rDNA sequence analysis revealed that members of this group appear to represent a new sub-line within the genus Yersinia, most closely related to Y. frederiksenii hybridization group 2 (unnamed genomospecies 2). This finding was confirmed by DNA hybridization studies which indicated that the strains belonged to the unnamed genomospecies, Yersinia frederiksenii genomospecies 2, which is biochemically indistinguishable from Y. frederiksenii (Y. frederiksenii genomospecies 1). A 23-nucleotide 16S rDNA signature stretch which characterised these strains was identified.     

Genotyping of Human and Porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri Strains from Switzerland by Amplified Fragment Length Polymorphism Analysis

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.     

Prevalence of Very Low Numbers of Potential Pathogenic Isolates of <Emphasis Type="Italic">Yersinia</Emphasis><Emphasis Type="Italic">enterocolitica</Emphasis> and <Emphasis Type="Italic">Yersinia intermedia</Emphasis> in Traditional Fast Foods of India

In this study, an incidence pattern of 1.7% for Yersinia enterocolitica and 2.5% for Y. intermedia were observed in an analysis of 120 diversified food samples collected from the local market of Mysore, Southern India. Two native isolates characterized as Y. enterocolitica belonged to biotype 1B and revealed the presence of major virulence related traits such as regulator of virulence, mucoid Yersinia factor regulator, attachment invasion locus, heat stable enterotoxin, Yersinia type II secretory system and phospholipase A in PCR. Force type neighbor-joining phylograms generated for Y. enterocolitica based on PCR amplicons of rovA and ypl showed 100% homology with two to three strains of Y. enterocolitica and about 75% homology with several strains of Y. pestis.     

Effect of Flagellar Mutations on Yersinia enterocolitica Biofilm Formation

Yersinia enterocolitica biovar 1B is one of a number of strains pathogenic to humans in the genus Yersinia. It has three different type III secretion systems, Ysc, Ysa, and the flagella. In this study, the effect of flagella on biofilm formation was evaluated. In a panel of 31 mutant Y. enterocolitica strains, we observed that mutations that abolish the structure or rotation of the flagella greatly reduce biofilm formation when the bacteria are grown under static conditions. These results were further evaluated by assessing biofilm formation under continuous culture using a flow cell chamber. The results confirmed the important contribution of flagella to the initiation of biofilm production but indicated that there are differences in the progression of biofilm development between static growth and flow conditions. Our results suggest that flagella play a critical role in biofilm formation in Y. enterocolitica.     

A simplified method for detecting pathogenic Yersinia enterocolitica in slaughtered pig tonsils

The aim of this study was to collect preliminary data on the carriage of pathogenic Yersinia enterocolitica in slaughtered pigs in France and to test a simplified method for detecting these strains from tonsils. From January to March 2009, 900 tonsil swabs were taken from pigs at one slaughterhouse in Brittany, France. The swabs were vortexed in 10 ml PSB broth, then 1 ml was added to 9 ml ITC broth. The media were incubated for 48 h at 25 °C. The PSB enrichment broth was streaked on CIN plates and the ITC enrichment broth on SSDC plates. In addition to the ISO 10273 method, we also streaked ITC enrichment broth on CIN plates. The plates were incubated for 24 h at 30 °C, and we then streaked a maximum of four typical colonies per plate onto a plate containing chromogenic medium (YeCM), for the isolation of pathogenic Y. enterocolitica isolates. In parallel, biochemical assays were carried out to confirm the identification of the isolates as Yersinia and to determine biotype.After passage on a YeCM plate and biochemical tests, 380 strains were confirmed to be pathogenic Y. enterocolitica. Finally, with the ISO 10273 method, 9.1% (CI_95% [5.8-12.4]) of tonsil swabs and 60% (CI_95% [45.4-74.6]) of the batches were positive. With the ITC-CIN method, 14.0% (CI_95% [10.7-17.3]) of the tonsil swabs and 68.9% (CI_95% [54.3-83.5]) of the batches were positive. Identification as pathogenic Y. enterocolitica was confirmed for 97.0% of the typical colonies obtained on the chromogenic medium, YeCM. The most prevalent biotype was biotype 4 (80.5% of the isolates), followed by biotype 3.This study demonstrates that the ITC-CIN method, followed by streaking on YeCM, may be an effective approach to the isolation of pathogenic Y. enterocolitica from tonsil swabs and the recovery of positive samples. This method is less time-consuming than the ISO 10273 method and reduces the number of biochemical tests required for the confirmation of Yersinia identification, through the use of YeCM.     

Evaluation of a Modified Cefsulodin-Irgasan-Novobiocin Agar for Isolation of Yersinia spp

Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the presence of competitive microbiota makes the isolation of Y. enterocolitica and Y. pseudotuberculosis from naturally contaminated foods difficult. We attempted to evaluate the performance of a modified Cefsulodin-Irgasan-Novobiocin (CIN) agar in the differentiation of Y. enterocolitica from non-Yersinia species, particularly the natural intestinal microbiota. The modified CIN enabled the growth of Y. enterocolitica colonies with the same efficiency as CIN and Luria-Bertani agar. The detection limits of the modified CIN for Y. enterocolitica in culture medium (10 cfu/ml) and in artificially contaminated pork (10⁴ cfu/ml) were also comparable to those of CIN. However, the modified CIN provided a better discrimination of Yersinia colonies from other bacteria exhibiting Yersinia-like colonies on CIN (H₂S-producing Citrobacter freundii, C. braakii, Enterobacter cloacae, Aeromonas hydrophila, Providencia rettgeri, and Morganella morganii). The modified CIN exhibited a higher recovery rate of Y. enterocolitica from artificially prepared bacterial cultures and naturally contaminated samples compared with CIN. Our results thus demonstrated that the use of modified CIN may be a valuable means to increase the recovery rate of food borne Yersinia from natural samples, which are usually contaminated by multiple types of bacteria.     

Oligomeric coiled-coil adhesin YadA is a double-edged sword

Yersinia adhesin A (YadA) is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Suprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt) Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs) upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadA_V98D,N99A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions.     

Characterization of enterotoxin produced by four Yersinia enterocolitica strains of pig origin

All four isolates of Yersinia enterocolitica and one isolate of Y. frederiksenii from pigs were found to be enterotoxigenic. Whole-cell preparations of Y. enterocolitica isolates did not induce any change in the rabbit ligated gut test after 6 and 18 h of inoculation, but Y. frederiksenii on the other hand showed a positive gut response at 18 h. Cell-free supernatant (CFS) of all five isolates induced dilatation in the rabbit gut up to 6 h, after which Y. enterocolitica became negative, while Y. frederiksenii continued to show a reaction up to 18 h. CFS of all five isolates were also found positive with the infant mouse test. Of the five isolates of Yersinia, three gave a positive reaction for the permeability factor on rabbit skin. Yersinia enterotoxin could be concentrated by methanol extraction. It was stable at 100°C for 20 min and at 120°C for 15 min. However, its activity was lost at low (2.0) and high pH (10.0). Enterotoxic preparations of Y. enterocolitica lost part of their enterotoxic activity upon dialysis.     

Behavior of Yersinia enterocolitica in the Presence of the Bacterivorous Acanthamoeba castellanii

Free-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction between Yersinia enterocolitica, an important food-borne pathogen, and the free-living amoeba Acanthamoeba castellanii was studied. Several cocultivation assays were set up to assess the resistance of Y. enterocolitica to A. castellanii predation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that all Y. enterocolitica strains persist in association with A. castellanii for at least 14 days, and associations with A. castellanii enhanced survival of Yersinia under nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of one Yersinia strain showed temperature- and time-dependent permeabilization. Intraprotozoan survival of Y. enterocolitica depended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate the Yersinia cells inside the amoebae. As Yersinia and Acanthamoeba share similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology of Y. enterocolitica.     

Identification of Yersinia enterocolitica at the Species and Subspecies Levels by Fourier Transform Infrared Spectroscopy

Yersinia enterocolitica and other Yersinia species, such as Y. pseudotuberculosis, Y. bercovieri, and Y. intermedia, were differentiated using Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. A set of well defined Yersinia strains from Switzerland and Germany was used to create a method for FT-IR-based differentiation of Yersinia isolates at the species level. The isolates of Y. enterocolitica were also differentiated by FT-IR into the main biotypes (biotypes 1A, 2, and 4) and serotypes (serotypes O:3, O:5, O:9, and “non-O:3, O:5, and O:9”). For external validation of the constructed methods, independently obtained isolates of different Yersinia species were used. A total of 79.9% of Y. enterocolitica sensu stricto isolates were identified correctly at the species level. The FT-IR analysis allowed the separation of all Y. bercovieri, Y. intermedia, and Y. rohdei strains from Y. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification of Y. enterocolitica isolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of the ail gene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning the ail gene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case of Y. enterocolitica also subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.The genus Yersinia belongs to the bacterial family Enterobacteriaceae and encompasses three well-known human pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Pathogenic strains of Y. enterocolitica cause yersiniosis, an acute enteric disease. In Germany and Switzerland, strains of Y. enterocolitica belong to the most frequently isolated pathogens connected with bacterial gastroenteritis (27, 31). Y. enterocolitica also causes other clinical syndromes, such as enterocolitis, acute mesenteric lymphadenitis, mimicking appendicitis, postinfectious arthritis, and systemic infections (7, 21). It is assumed that the main contamination source is food of animal origin, especially pork meat or raw milk (8, 21, 27). Therefore, the focus of diagnosis for these bacteria as food-borne pathogens includes the examination of food samples in food inspection and veterinary controls of livestock.The species Y. enterocolitica sensu lato as described by Frederiksen (9) was recently subdivided into several species: Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii, Y. kristensenii, Y. aldovae, Y. mollaretii, Y. rohdei, and Y. bercovieri (20). The identification of Y. enterocolitica sensu stricto by traditional agar plate techniques (ISO standard 10273:2003) is complicated by the fact that on the commonly used selective agar plates, especially the cefsulodin-irgasan-novobiocin (CIN) agar, several unrelated bacteria also grow (1, 20). In addition, some Yersinia strains are inhibited by CIN agar (10). The differentiation of putative Yersinia strains isolated from the CIN agar is additionally impeded because the commonly used commercial identification systems (for example, API 20E or API Rapid 32IDE) do not include all Yersinia strains in their databases and usually misidentify them as Y. enterocolitica (12). Nevertheless, the biochemical test system API 20E is still used as an affordable tool for the identification of Y. enterocolitica. This probably results in a constant misidentification of certain Yersinia species, particularly Y. bercovieri, Y. rohdei, and Y. intermedia, as Y. enterocolitica (1, 12, 15).Y. enterocolitica sensu stricto comprises pathogenic and nonpathogenic members. The species can be grouped into various biotypes by biochemical tests and independently into different serotypes by immunological tests. Both types are connected with different pathogenic potential. The most common biotype-serotype combinations associated with human diseases were biotype 1B/serotype O:8, 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3 (7). Biotype 1A is deemed to be non- or less pathogenic for humans. Biotype 1B is widespread in the United States and only rarely detected in Europe and Japan (11, 14, 26, 28). Based on different DNA-DNA hybridization values and 16S rRNA gene sequences, it was proposed to name the “American” strains Y. enterocolitica subsp. enterocolitica (19). Biotypes 2 and 4 are often isolated from yersiniosis patients, and biotype 3 seems to be pathogenic but rare (6, 21).Pathogenic strains of Y. enterocolitica harbor certain virulence factors, such as the plasmid-encoded yadA gene and the chromosomally encoded ail gene (17, 32). In contrast, apathogenic strains of Y. enterocolitica do not contain these two genes. However, the plasmid harboring the yadA gene can be lost under certain cultivation conditions in the laboratory (4). This may lead to false-negative results in any test system based on the presence of this plasmid. Therefore, the ail gene appears to be the best-suited marker for the detection of pathogenic Y. enterocolitica strains. The product of the ail gene is an adhesion and invasion factor (17). Therefore, the detection of the ail gene by PCR is used as an indication of the presence of pathogenic strains of Y. enterocolitica in selective enrichments or isolated pure cultures (33).Recently, Fourier transform infrared spectroscopy (FT-IR) has been established as a new method for identification of bacteria, yeasts, and other microorganisms (3, 16, 22, 24, 38). This method analyzes the total composition of all components of the cell using infrared spectroscopy (13, 18). The FT-IR method is rapid and reliable and therefore can be easily adapted to routine analysis. Furthermore, there accrue almost no costs for consumables during sample preparation and measurements. The technique offers a wide range of applications for differentiation at the species and subspecies levels. It has already been used for the differentiation of several food-borne pathogens, like Listeria monocytogenes (25), Escherichia coli (13), and Bacillus cereus (23, 29). Recently, promising results were obtained by combination of FT-IR and multivariate methods for data processing, in particular artificial neural networks (ANN) (25, 35).In the present work, FT-IR combined with ANN analysis was applied for classification of Yersinia strains at the species level and of Y. enterocolitica at the subspecies level. Furthermore, differentiation between pathogenic and apathogenic strains of Y. enterocolitica by FT-IR was attempted.     

Development of a Fluorogenic 5′ Nuclease PCR Assay for Detection of the ail Gene of Pathogenic Yersinia enterocolitica

In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the Y. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica strains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect ≤4 Y. enterocolitica CFU/ml in pure culture and 10 Y. enterocolitica CFU/ml independent of the presence of 10⁸ CFU of contaminating bacteria per ml. This set was also capable of detecting ≤1 CFU of Y. enterocolitica per g of ground pork or feces after a 24-h enrichment in a Yersinia selective broth.     

Detection by using monoclonal antibodies of Yersinia enterocolitica in artificially-contaminated pork

Monoclonal antibodies against Yersinia enterocolitica were produced by fusion of NS‐1 mouse myeloma cells with spleen cells of ICR mice immunized with heat‐killed and heat‐killed plus SDS‐mercaptoethanol treated forms of Y. enterocolitica ATCC 27729 alone or mixed with Y. enterocolitica MU. The twenty‐five MAbs obtained from five fusions were divided into nine groups according to their specificities to different bacterial strains and species, as determined by dot blotting. The first five groups of MAbs were specific only to Y. enterocolitica, but did not recognize all of the isolates tested. MAbs in groups 6 and 7 reacted with all isolates of Y. enterocolitica tested but showed cross‐reaction with some Yersinia spp. and Edwardsiella tarda, especially in the case of group 7. MAbs in groups 8 and 9 reacted with all isolates of Y. enterocolitica and Yersinia spp., as well as other Gram‐negative bacteria that belong to the family Enterobacteriaceae. These MAbs recognized Y. enterocolitica antigens with apparent molecular weights ranging from 10 – 43 kDa by Western blotting, and could detect Y. enterocolitica from ～10³– 10⁵ colony forming units (CFUs) by dot blotting. The hybridoma clone YE38 was selected for detection of Y. enterocolitica in pork samples which had been artificially‐contaminated by inoculation with Y. enterocolitica ATCC 27729 at concentrations of ～10⁴– 10⁶ CFUs/g and incubation in peptone sorbitol bile broth at 4°C. Samples were collected and applied on a nitrocellulose membrane for dot blotting with trypticase soy and cefsulodin‐Irgasan‐novobiocin agars. After 48 hr of incubation, the detection limit was ～10²– 10³ CFU/g by dot blotting.     

Identification of Yersinia enterocolitica using a random genomic DNA microarray chip

Aims: To fabricate a DNA chip containing random fragments of genomic DNA of Yersinia enterocolitica and to verify its diagnostic ability. Methods and Results: A DNA microarray chip was fabricated using randomly fragmented DNA of Y. enterocolitica. Chips were hybridized with genomic DNA extracted from other Y. enterocolitica strains, other Yersinia spp. and bacteria in different genera. Genomic DNA extracted from Y. enterocolitica showed a significantly higher hybridization rate compared with DNA of other Yersinia spp. or bacterial genera, thereby distinguishing it from other bacteria. Conclusions: A DNA chip containing randomly fragmented genomic DNA from Y. enterocolitica can detect Y. enterocolitica and clearly distinguish it from other Yersinia spp. and bacteria in different genera. Significance and Impact of the Study: A microarray chip containing randomly fragmented genomic DNA of Y. enterocolitica was fabricated without sequence information, and its diagnostic ability to identify Y. enterocolitica was verified.     

Genomic characterization of the Yersinia genus

Background

New DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Yersinia that cause invasive human diseases (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments.

Results

We used high-throughput sequencing-by-synthesis instruments to obtain 25- to 42-fold average redundancy, whole-genome shotgun data from the type strains of eight species: Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. rohdei, and Y. ruckeri. The deepest branching species in the genus, Y. ruckeri, causative agent of red mouth disease in fish, has the smallest genome (3.7 Mb), although it shares the same core set of approximately 2,500 genes as the other members of the species, whose genomes range in size from 4.3 to 4.8 Mb. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of Cluster of Orthologous Groups families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogeneases and B-12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats.

Conclusions

Rapid high-quality draft sequencing was used successfully to compare pathogenic and non-pathogenic members of the Yersinia genus. This work underscores the importance of the acquisition of horizontally transferred genes in the evolution of Y. pestis and points to virulence determinants that have been gained and lost on multiple occasions in the history of the genus.     

Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081

The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.     

Biochemical,serological, and phage typing characteristics of 459Yersinia strains isolated from a terrestrial ecosystem

The origins of human contamination withYersinia enterocolitica are still unknown. We have investigated the major components of a terrestrial ecosystem (soil, earthworms, field voles, shrews, crops, hares, rabbits, and birds) for the presence ofYersinia. Four hundred fifty-nine strains ofYersinia were isolated. We report the first isolations of typicalY. enterocolitica belonging to classical or new biotypes and ofY. enterocolitica-like organisms (sucrose negative; rhamnose positive; melibiose and rhamnose positive) from soil samples, earthworms, crops, and birds. Sucrose-negativeY. enterocolitica strains and biotypes 1, 2, and 3, usually associated with human nonmesenteric syndromes, are predominant in soil, which can be considered as a reservoir for these biotypes.Y. enterocolitica serogroups O∶3 and O∶9, strains of which are responsible in Europe for human mesenteric syndromes, were not found in this study. The epidemiology ofY. enterocolitica infections is discussed.     

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Prevalence of ColE1-Like Plasmids and Colicin K Production among Uropathogenic Escherichia coli Strains and Quantification of Inhibitory Activity of Colicin K

Colicin K exhibited pronounced inhibitory activity against uropathogenic Escherichia coli (UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS2.