PCR Detection of Virulence Genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and Investigation of Virulence Gene Distribution

Abstract

PCR-based assays were developed for the detection of plasmid-and chromosome-borne virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis, to investigate the distribution of these genes in isolates from various sources. The results of PCR genotyping, based on 5 virulence-associated genes of 140 strains of Y. enterocolitica, were compared to phenotypic tests, such as biotyping and serotyping, and to virulence plasmid-associated properties such as calcium-dependent growth at 37°C and Congo red uptake. The specificity of the PCR results was validated by hybridization. Genotyping data correlated well with biotype data, and most biotypes resulted in (nearly) homogeneous genotypes for the chromosomal virulence genes (ystA, ystB, and ail); however, plasmid-borne genes (yadA and virF) were detected with variable efficiency, due to heterogeneity within the bacterial population for the presence of the virulence plasmid. Of the virulence genes, only ystB was present in biotype 1A; however, within this biotype, pathogenic and apathogenic isolates could not be distinguished based on the detection of virulence genes. Forty Y. pseudotuberculosis isolates were tested by PCR for the presence of inv, yadA, and lcrF. All isolates were inv positive, and 88% of the isolates contained the virulence plasmid genes yadA and lcrF. In conclusion, this study shows that genotyping of Yersinia spp., based on both chromosome-and plasmid-borne virulence genes, is feasible and informative and can provide a rapid and reliable genotypic characterization of field isolates.Yersinia enterocolitica and Y. pseudotuberculosis, both members of the family Enterobacteriaceae, are comprised of strains with different degrees of pathogenicity. Both pathogenic and nonpathogenic strains are frequently isolated from various animals (birds, mammals, and reptiles) as well as from the environment (water and soil). Rodents (mice and rats), hares, rabbits, and birds serve as reservoirs for Y. pseudotuberculosis (1). Pathogenic strains of Y. enterocolitica and Y. pseudotuberculosis are frequently present in pigs without normally causing disease in these animals. Other food-producing animals, such as cattle, harbor mostly nonpathogenic strains of Y. enterocolitica.In humans, Y. enterocolitica and Y. pseudotuberculosis are well-known food-borne pathogens and are mainly transmitted through ingestion of contaminated pork, milk, or water. Yersiniosis frequently occurs in young children as enterocolitis with fever, diarrhea, and abdominal cramps. Although the disease is usually self-limiting, complications (e.g., septicemia) are not uncommon in immunocompromised hosts. Furthermore, sequelae, such as reactive arthritis, have been reported (21).The identification and further typing of subspecies, aiming at recognition of pathogenic strains of Yersinia spp., are traditionally based on phenotypic tests. Y. enterocolitica can be classified into biotype 1A, generally regarded as nonpathogenic (9), and the pathogenic biotypes 1B, 2, 3, 4, and 5. Both species ca...

Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates

“…For virF, ail, inv, ymoA, ystA, ystB and myfA, the primers and cycling parameters described by Bhagat and Virdi (2007) were used. For rovA the protocols defined by Divya and Varadaraj (2011) and for yadA the protocols by Thoerner et al (2003) were used (Table 1). Two microliters of template was added to 28 µL of reaction mixture consisting of 1 X× PCR buffer (Solis BioDyne, Tartu, Estonia), 2.5 mM MgCl 2 (Solis BioDyne, Tartu, Estonia), 200 µM dNTPS (Roche, Rotkreuz, Switzerland), 0.25 µL FIREPol DNA polymerase (Solis BioDyne, Tartu, Estonia) and primers (Microsynth, Balgach, Switzerland) at a final concentration of 0.25 µM.…”

“…However, several of these studies focused solely on porcine isolates (Bonardi et al, 2013;Paixão et al, 2013a,b) or human strains (Stephan et al, 2013;Fredriksson-Ahomaa et al, 2012). Or only a small number of virulence-associated genes wasere analyzed (Thoerner et al, 2003). To our knowledge, no previous study has used this same approach to analyze BT 4 strains from porcine tonsils, porcine feces and human feces for a complete spectrum of relevant virulence-associated genes.…”

“…The virF gene product is a key factor for transcription of yop genes (Cornelis et al, 1998) and is, therefore, fundamental for the type III secretion system. Because pYV can be lost during the culturing process (Thoerner et al, 2003), chromosomal genes should be analyzed as well. The ail (attachment invasion locus) gene has been described as a target for virulence screening (Fredriksson-Ahomaa and Korkeala, 2003;Thisted-Lambertz et al, 2008), as it is only found in strains associated with human infection (Miller et al, 1989).…”

The Role of Pigs as Pharyngeal Carriers of Human Pathogenic Yersinia Enterocolitica Strains

“…Per l'amplificazione del gene plasmidico yadA (238 bp) e di quello cromosomico ail (315 bp) è stata condotta una simplex-PCR seguendo i protocolli di Blais e Phillippe (17) e Thoerner et al (18), rispettivamente. L'amplificazione dei geni cromosomici inv (183 bp), ystA (79 bp) e ystB (146 bp) è stata ottenuta applicando una multiplex-PCR (18).…”

“…L'amplificazione dei geni cromosomici inv (183 bp), ystA (79 bp) e ystB (146 bp) è stata ottenuta applicando una multiplex-PCR (18).…”