Molecular identification of Yersinia enterocolitica isolated from pasteurized whole milk using DNA microarray chip hybridization.

A DNA microarray chip of four virulence genes and 16S ribosomal DNA gene conserved region among all Gram negative species, including Yersinia, as a positive control was developed and evaluated using 22 Yersinia enterocolitica isolates. Eight different oligonucleotide probes (oligoprobes) with an average size of 22 bp, complementary to the unique sequences of each gene, were designed and immobilized on the surface of chemically modified slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all five genes, and single stranded DNA (ssDNA) samples for microarray analysis were prepared by using a primer extension of amplicons in the presence of one primer of all genes. The presence of genes in Y. enterocolitica was established by hybridization of the fluorescently labeled ssDNA representing different samples of the microarray gene-specific oligoprobes and confirmed by PCR. Results of the study showed specificity of genotyping Y. enterocolitica using multiple microarray-based assays. Final validation of the chip's ability to identify Y. enterocolitica genes from adulterated pasteurized whole milk was confirmed and successful. The limit of chip detection of virulence genes in pasteurized whole milk was found to be 1000 CFU per hybridization.

[1]  D. Fang,et al.  Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections. , 2004, Journal of microbiological methods.

[2]  E. Bottone.  Yersinia enterocolitica: the charisma continues , 1997, Clinical microbiology reviews.

[3]  S. Ostroff.  Yersinia as an emerging infection: epidemiologic aspects of Yersiniosis. , 1995, Contributions to microbiology and immunology.

[4]  Nucleotide sequence of a new class A β-lactamase gene from the chromosome of Yersinia enterocolitica: Implications for the evolution of class A β-lactamases , 1991, Molecular and General Genetics MGG.

[5]  G. Cornelis,et al.  Nucleotide sequence of yst, the Yersinia enterocolitica gene encoding the heat-stable enterotoxin, and prevalence of the gene among pathogenic and nonpathogenic yersiniae , 1990, Infection and immunity.

[6]  P. Heisig,et al.  Beta-lactamase expression in Yersinia enterocolitica biovars 1A, 1B, and 3. , 2000, Journal of medical microbiology.

[7]  Y. Lin,et al.  Detection of pathogenic Yersinia enterocolitica using the multiplex polymerase chain reaction , 1996, Epidemiology and Infection.

[8]  J. Novak,et al.  Radiation resistance of virulence plasmid-containing and plasmid-less Yersinia enterocolitica. , 2002, Journal of food protection.

[9]  V. Chizhikov,et al.  Detection and Genotyping of Human Group A Rotaviruses by Oligonucleotide Microarray Hybridization , 2002, Journal of Clinical Microbiology.

[10]  S. Al-Khaldi,et al.  Gene and bacterial identification using high-throughput technologies: genomics, proteomics, and phonemics. , 2004, Nutrition.

[11]  W. Liesack,et al.  The phylogeny of the genus Yersinia based on 16S rDNA sequences. , 1993, FEMS microbiology letters.

[12]  J. Nicolet,et al.  Association between clinical presentation, biogroups and virulence attributes of Yersinia enterocolitica strains in human diarrhoeal disease , 1996, Epidemiology and Infection.

[13]  H. Korkeala,et al.  Low Occurrence of Pathogenic Yersinia enterocolitica in Clinical, Food, and Environmental Samples: a Methodological Problem , 2003, Clinical Microbiology Reviews.

[14]  P. Heisig,et al.  Expression of β-lactamases in Yersinia enterocolitica strains of biovars 2, 4 and 5 , 1999 .

[15]  V. Chizhikov,et al.  Identification and characterization of Clostridium perfringens using single target DNA microarray chip. , 2004, International journal of food microbiology.

[16]  G. Cornelis,et al.  Homology between virF, the transcriptional activator of the Yersinia virulence regulon, and AraC, the Escherichia coli arabinose operon regulator , 1989, Journal of bacteriology.

[17]  H. Korkeala,et al.  High prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland. , 1999, Journal of food protection.

[18]  S. Falkow,et al.  The ail locus is found uniquely in Yersinia enterocolitica serotypes commonly associated with disease , 1989, Infection and immunity.

[19]  J. M. García‐Lobo,et al.  Tandem Amplification of a 28-Kilobase Region from the Yersinia enterocolitica Chromosome Containing the blaA Gene , 2003, Antimicrobial Agents and Chemotherapy.

[20]  R. Robins-Browne,et al.  Identification of Virulence-Associated Characteristics in Clinical Isolates of Yersinia enterocolitica Lacking Classical Virulence Markers , 1998, Infection and Immunity.

[21]  A. Rasooly,et al.  Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization. , 2004, Molecular and cellular probes.

[22]  Avraham Rasooly,et al.  Simultaneous Analysis of Multiple Staphylococcal Enterotoxin Genes by an Oligonucleotide Microarray Assay , 2004, Journal of Clinical Microbiology.

[23]  T. Jemmi,et al.  PCR Detection of Virulence Genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and Investigation of Virulence Gene Distribution , 2003, Applied and Environmental Microbiology.

[24]  G. Cornelis,et al.  Regulation of the Yersinia enterocolitica enterotoxin Yst gene. Influence of growth phase, temperature, osmolarity, pH and bacterial host factors , 1994, Molecular microbiology.

[25]  A. Rasooly,et al.  Microarray Analysis of Microbial Virulence Factors , 2001, Applied and Environmental Microbiology.

[26]  H. Maas,et al.  Detection of Pathogenic Yersinia enterocolitica by a Rapid and Sensitive Duplex PCR Assay , 2001, Journal of Clinical Microbiology.

[27]  Avraham Rasooly,et al.  Multipathogen oligonucleotide microarray for environmental and biodefense applications. , 2004, Biosensors & bioelectronics.