Identification of Aeromonas hydrophila hybridization group 1 by PCR assays

Synthetic oligonucleotide primers of 24 and 23 bases were used in a PCR assay to amplify a sequence of the lip gene, which encodes a thermostable extracellular lipase of Aeromonas hydrophila. A DNA fragment of approximately 760 bp was amplified from both sources, i.e., lysed A. hydrophila cells and isolated DNA. The amplified sequence was detected in ethidium bromide-stained agarose gels or by Southern blot analysis with an internal HindIII-BamHI 356-bp fragment as a hybridization probe. With A. hydrophila cells, the sensitivity of the PCR assay was < 10 CFU, and with the isolated target, the lower detection limit was 0.89 pg of DNA. Primer specificity for A. hydrophila was determined by the PCR assay with cells of 50 strains of bacteria, including most of the 14 currently recognized DNA hybridization groups of Aeromonas spp. as well as other human and environmental Aeromonas isolates. Detection of A. hydrophila by PCR amplification of DNA has great potential for rapid identification of this bacterium because it has proved to be highly specific.     

Genes coding for enterotoxins and verotoxins in porcine Escherichia coli strains belonging to different O:K:H serotypes: relationship with toxic phenotypes

Seventy-four E. coli strains isolated from piglets with diarrhea or edema disease in Spain were serotyped and examined for production of heat-labile (LT) and heat-stable (ST) enterotoxins (LT-I, LT-II, STaH, STaP, and STb) and verotoxins (VT1, VT2, and VT2v = VTe) by phenotypic (Vero cell assay and infant mouse test) and genotypic (colony hybridization and PCR) methods. In general, an excellent correlation was found between the results obtained with a PCR approach and those determined with biological assays. DNA probes used in the hybridization also showed a very good agreement with phenotypic results, with the exception of a VT1 probe that initially produced 10 false-positive reactions. The gene coding for STb (58 strains) was the most prevalent gene detected by PCR, followed by those coding for STa (46 strains), LT (19 strains), VT2v (11 strains), and VT1 (1 strain). Apparently, in Spain three seropathotypes are predominant: (i) O149:K91:H10 K88+ LT-I+ STb+, (ii) O141:K85ab:H- P987+ STaP+, and (iii) O138:K81:H14 or H- STaP+ VT2v+. We conclude that PCR is a fast, specific, and practical method for the identification of enterotoxin and VT genes in clinical and epidemiological studies.     

Detection and characterization of the fimA gene of Escherichia coli O157:H7

An expected 850-bp DNA fragment containing fimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains of Escherichia coli using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13 HinP1 and four Sau961 restriction profiles among these 39 E. coli strains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared by E. coli O157:H7, O157:H- and a few O55 serotype strains. DNA sequence analysis of the fimA region demonstrated that E. coli O157:H7 strain 933 and O157:H- strain E32511 contained identical DNA sequences that were distinct from other E. coli strains, especially a 16-bp sequence 5' to fimA that was conspicuously absent only in E. coli O157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from all E. coli O157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detect E. coli strains of the O157:H7 serotype.     

Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR

Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.     

Antibacterial action of vinegar against food-borne pathogenic bacteria including Escherichia coli O157:H7

The bacteriostatic and bactericidal actions of vinegar on food-borne pathogenic bacteria including enterohemorrhagic E. coli (EHEC) O157:H7 were examined. The growth of all strains evaluated was inhibited with a 0.1% concentration of acetic acid in the vinegar. This inhibition was generally increased in the presence of sodium chloride or glucose. There was almost no difference in sensitivity to the bacteriostatic action of vinegar among the strains of pathogenic E. coli. Vinegar had a bactericidal effect on food-borne pathogenic bacteria including EHEC O157:H7. This action against EHEC O157:H7 was synergically enhanced by sodium chloride but was attenuated with glucose. For EHEC strains (O157:H7, O26:H11, O111:HNM) the difference in the inactivation rate due to vinegar among strains used was small, although an enteropathogenic E. coli (EPEC) O111:K58:H- strain was more sensitive, being more quickly killed compared with EHEC strains. The inactivation rate due to vinegar was constant irrespective of inoculum size. However, it differed greatly depending on growth phase of the cells, where logarithmic growth phase cells were more sensitive and easily killed than stationary phase cells. The bactericidal activity of vinegar increased with the temperature. Various conditions for bactericidal effects on EHEC O157:H7 were examined by the multiparametric analysis of five factors: acetic acid concentration in the vinegar, sodium chloride concentration, temperature, incubation time, and viable cell number. The combined use of vinegar and sodium chloride, with use of an appropriate treatment temperature, was found to be markedly effective for the prevention of bacterial food poisoning.     

Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.     

Radiation inactivation of human prostate cancer cells: the role of apoptosis

DNA extracted from all Brucella species, reference and vaccine strains were amplified by PCR using primers specific for the genes encoding a 31-kDa Brucella protein, the heat shock proteins (DnaJ, DnaK, HtrA and GroEL) and 16S RNA. No difference was found between Brucella species and biovars with all primer pairs used, even after restriction enzyme analysis of the amplified fragments. The specificity of the amplified products was confirmed by hybridization with a digoxigenin 3'-labelled specific probe and by PCR using 98 non-Brucella micro-organisms' DNA. Only Ochrobactrum anthropi and Phyllobacterium spp. yielded a PCR product by using 31-kDa DnaK, DnaJ, GroEL and 16S RNA primers. After hybridization and restriction analysis, 16S RNA fragments of 3301 and 3331 O. anthropi strains showed a total similarity to those from Brucella. A similar result was shown with DnaJ fragments obtained with 3301 strain of O. anthropi after EcoRI digestion.     

The novel heat-stable enterotoxin subtype gene (ystB) of Yersinia enterocolitica: nucleotide sequence and distribution of the yst genes

The gene (ystB) encoding the novel subtype of the heat-stable enterotoxin (Y-STb) was cloned from the chromosome of a clinical isolate of Yersinia enterocolitica 84-50 (serotype O:5, biotype 1A) and the nucleotide sequence was determined. The ystB contained 216 base pairs that encoded a protein of 71 amino acid residues. The C-terminal 30 residues of the precursor protein exactly corresponded to the amino acid sequence of the Y-STb toxin, purified from the culture supernatant of the wild strain. Homology search revealed that there are 76.9% nucleotide sequence similarity between ystB and the Yersinia kristensenii ST gene, and 73.5% with the Y. enterocolitica prototype sequence of yst (ystA). When tested with the PCR generated ystB specific probe, 36 of 304 Y. enterocolitica strains from 18 countries hybridized with the probe. All the ystB probe positive strains belonged to biotype 1A and mostly to the so-called non-pathogenic serotype O:5, O:6, O:7,8 O:7,13 and O:10, while ystA was predominantly found among the pathogenic serotypes (78.5%). Out of 36 ystB gene positive strains, 18 were clinical origin from six countries, which were also positive in the suckling mice assay suggesting that ystB may play an important role in the pathogenesis, and the so-called non-pathogenic serotypes could be virulent for human.     

Immunochemical characterization of O polysaccharides composing the alpha-D-rhamnose backbone of lipopolysaccharide of Pseudomonas syringae and classification of bacteria into serogroups O1 and O2 with monoclonal antibodies

Murine monoclonal antibodies (MAbs) reacting with Pseudomonas syringae lipopolysaccharide (LPS) O polysaccharides (OPS) composed of tetra- and tri-alpha-D-rhamnose repeats in the backbone [3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1-2)D-Rha(alpha1] and [3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1] were generated and used for immunochemical analysis and for serological classification of the bacteria. A total of 195 of 358 P. syringae strains tested representing 21 pathovars were shown to share a common epitope, 1a, and were classified into serogroup O1. All strains with pathovars aptata, glycinea, japonica, phaseolicola, and pisi, most of the strains with pathovars atrofaciens and striafaciens, and half of the strains with pathovar syringae were classified into serotypes O1a', O1b, O1c, and O1d within serogroup O1. Serogroup-specific epitope 1a was inferred to be related to the (alpha1-2)D-Rha(alpha1-3) site of the OPS backbone. The serotype-specific epitopes 1b, 1c, 1d, and 1a' were inferred as relating to the immunodominant lateral (alpha1-3)D-Rha, (beta1-4)D-GlcNAc, and (alpha1-4)D-Fuc substituents and backbone-located site (alpha1-3)D-Rha(alpha1-2), respectively, of OPSs that share the common tetra-D-rhamnose repeats in the backbone. A total of 7.3% of the strains studied, all with pathovars morsprunorum and lapsa, were classified as serotypes O2a and O2d within serogroup 02. Serotype-specific epitope 2a was inferred as being related to the backbone-located site D-Rha(alpha1-3)D-Rha and epitope 2d to the immunodominant lateral (alpha1-4)D-Fuc residue of OPS consisting of tri-D-rhamnose repeats in the backbone. Epitope 2d alternated with 2a within the same LPS molecule and did not cross-react with epitope 1d. Serotypes O2a and O2d were observed in some strains correlating with the coexpression of the two chemotypes of OPS by the same strain. The serogroup O1-specific MAb Ps1a reacted weakly but definitely with all strains from serogroup 02. We propose serological formulas for serogroups O1 and 02 as well as for individual strains within these serogroups.     

Virulence properties and clonal structures of strains of Escherichia coli O119 serotypes

A total of 110 Escherichia coli strains of serogroup O119 were examined for the presence of virulence properties characteristic of enteropathogenic E. coli (EPEC). Three virulence patterns were distinguished based on the detection of a chromosomal gene mediating intimate attachment (eaeA) and plasmid DNA involved in localized adherence (EAF and bfpA). The first pattern, represented by strains which hybridized with three gene probes, was the most common (68%) and, with a single exception, included only O119:H6 strains. Of these strains, 90% showed a typical localized adherence (LA) pattern in HEp-2 cells and 96% were positive for intimate attachment in a fluorescent-actin staining test with a 3-h incubation period. The second pattern was represented by strains which hybridized with the eaeA gene only. Most (89.5%) of these strains showed the LA phenotype but only after 6 h of incubation (LA-like phenotype). The third pattern consisted of strains which were positive for eaeA and bfpA but did not hybridize with the EAF probe. Most (80%) of these strains exhibited the LA-like phenotype. Analysis of several eaeA+ bfpA+ strains for the expression of the pilin subunit (BfpA) of the bundle-forming pili demonstrated that all LA strains expressed BfpA whereas the LA-like strains did not. The study of the clonal relationships, carried out by multilocus enzyme electrophoresis in 79 representative strains, defined 11 distinct electrophoretic types (ETs). ET1 included 66% of the strains, most of which displayed the eaeA+ bfpA+ EAF+ pattern and were serotyped as O119:H6 or O119:H-. The remaining 10 ETs were each represented by no more than five strains and, with the exception of ET8, included strains of a single serotype. The genetic relatedness of the ETs revealed two main clusters, with most strains in cluster A having the eaeA+ bfpA+ EAF+ combination and a O119:H6 serotype. Cluster B was represented by atypical EPEC strains with only the eaeA+ and the eaeA+ bfpA+ virulence pattern.     

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.     

Characterization of lipopolysaccharide-deficient mutants of Pseudomonas aeruginosa derived from serotypes O3, O5, and O6

Well-characterized rough mutants are important for the understanding of structures, functions, and biosynthesis of lipopolysaccharide (LPS) in gram-negative organisms. In this study, three series of Pseudomonas aeruginosa LPS-deficient mutants, namely PAC strains derived from serotype O3, AK strains derived from strain PAO1 (serotype O5), and serotype O6-derived mutants were subjected to biochemical analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining as well as immunochemical characterization using LPS-specific monoclonal antibodies. The O-side-chain deficiency among the O6-derived mutants was also examined, and three mutants, A28, R5, and H4, were subsequently chosen for the elucidation of component sugars of the core structure of serotype O6 LPS. LPS of strain A28 has L-rhamnose and proportionally higher amounts of D-glucose, a feature shared by the O5-derived mutant, strain AK1401 (previously demonstrated as a mutant with a core-plus-one O repeat). In contrast strains R5 and H4 were shown to be devoid of L-rhamnose and have low and undetectable amounts of D-glucose, respectively, which indicated their core deficiency. The LPS-deficient or -sufficient characteristics of the P. aeruginosa strains examined correlated will with serum sensitivity data. This report represents a comprehensive analysis of rough mutants derived from O3 and O5 strains that have been used by others in many studies and a first look at the core oligosaccharide region of serotype O6 LPS obtained with the O6-derived mutants generated in this study.     

Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs

We examined the taxonomic position of seven Aeromonas isolates, recovered from Flemish and Scottish drinking water production plants and reservoirs, which were previously recognized by numerical analysis of genomic AFLP fingerprints as members of an unknown Aeromonas taxon that most closely resembled the species Aeromonas bestiarum (DNA hybridization group [HG] 2). The new phenotypic and DNA-DNA hybridization data obtained in this study show that the A. bestiarum-like strains constitute a separate Aeromonas species, for which the name Aeromonas popoffii sp. nov. is being proposed. The new species exhibited an internal DNA relatedness ranging from 79 to 100% and was 22 to 63% related to the type or reference strains of other Aeromonas spp. The highest DNA binding values were determined with A. bestiarum (51 to 63%), followed by Aeromonas hydrophila sensu stricto (HG1; 50 to 60%) and Aeromonas salmonicida (HG3; 39 to 55%). Although fingerprints generated by ribotyping and cellular fatty acid analysis often were highly similar, minor differences between the respective fingerprints were of significance for the differentiation of A. popoffii from its closest taxonomic neighbors, HG1, HG2, and HG3. Phenotypically, all seven strains of A. popoffii were positive for acid and gas production from D-glucose and glycerol, growth in KCN broth, arginine dihydrolase, DNase, Voges-Proskauer reaction, and resistance to vibriostatic agent O/129 and ampicillin but displayed negative reactions for production of urease, tryptophan deaminase, ornithine decarboxylase, and lysine decarboxylase (LDC). None of the strains displayed strong hemolytic activity. The lack of D-sucrose fermentation and LDC production and the ability to utilize DL-lactate as the sole energy and carbon source were useful characteristics for the biochemical separation of A. popoffii from A. bestiarum. Other Aeromonas spp. could be differentiated phenotypically from the new species by at least two features. The chromosomal G+C content of A. popoffii ranges from 57.7 to 59.6 mol%. Strain LMG 17541 is proposed as the type strain.     

Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes

A polymerase chain reaction (PCR) technique for the identification of Shiga-like toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.     

Strain-specific identification of probiotic Lactobacillus rhamnosus with randomly amplified polymorphic DNA-derived PCR primers

In the present work, strain-specific PCR primers for Lactobacillus rhamnosus Lc 1/3 are described. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. They were screened for specificity by hybridization with DNA from 11 L. rhamnosus strains. A 613-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific to L. rhamnosus Lc 1/3 was constructed based on the sequence. The primer pair was tested with 11 Lactobacillus species and 11 L. rhamnosus strains and was found to be strain specific. The nucleotide sequence of the specific RAPD marker was found to contain part of a protein encoding region which showed significant similarity to several transposases for insertion sequence elements of various bacteria, including other lactic acid bacterium species.     

Differentiation of Campylobacter jejuni serotype O19 strains from non-O19 strains by PCR

Guillain-Barré syndrome (GBS), a neurologic disease characterized by acute paralysis, is frequently preceded by Campylobacter jejuni infection. Serotype O19 strains are overrepresented among GBS-associated C. jejuni isolates. We previously showed that all O19 strains tested were closely related to one another by randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism analyses. RAPD analysis demonstrated a 1.4-kb band in all O19 strains tested but in no non-O19 strains. We cloned this O19-specific band; nucleotide sequence analysis revealed a truncated open reading frame with significant homology to DNA gyrase subunit B (gyrB) of Helicobacter pylori. PCR using the random primer and a primer specific for gyrB showed that in non-O19 strains, the random primer did not recognize the downstream gyrB binding site. The regions flanking each of the random primer binding sites were amplified by degenerate PCR for further sequencing. Although the random primer had several mismatches with the downstream gyrB binding site, a single nucleotide polymorphism 6 bp upstream from the 3' terminus was found to distinguish O19 and non-O19 strains. PCR using 3'-mismatched primers based on this polymorphism was designed to differentiate O19 strains from non-O19 strains. When a total of 42 (18 O19 and 24 non-O19) strains from five different countries were examined, O19 strains were distinguishable from non-O19 strains in each case. This PCR method should permit identification of O19 C. jejuni strains.     

Sequencing of Escherichia coli O111 O-antigen gene cluster and identification of O111-specific genes

Shiga toxin (Stx)-producing Escherichia coli strains of serogroup O111 are the most frequently isolated non-O157 strains causing outbreaks of gastroenteritis with hemolytic-uremic syndrome. The O111 O-antigen gene cluster had been cloned and about half of it has been sequenced; we have now sequenced the remainder of the gene cluster, which is 12.5 kb in length and which comprises 11 genes. On the basis of sequence similarity, we have identified all the O-antigen genes expected, including five sugar biosynthetic pathway genes, three transferase genes, the O-unit flippase gene, and the O-antigen polymerase gene. By PCR testing with E. coli strains representing all 166 O-antigen forms, some randomly selected gram-negative bacteria, and Salmonella enterica serovar Adelaide, we showed that four O-antigen genes are highly specific to O111. This work provides the basis for a sensitive test for the rapid detection of E. coli O111. This is important both for decisions related to patient care, because early treatment may reduce the risk of life-threatening complications, and for the detection of sources of contamination.     

Detection of Yersinia enterocolitica serogroup O:3 by a PCR method

Yersinia enterocolitica is the etiologic agent of a range of clinical situations in humans, but only a small number of serotypes are involved. Among these, Y. enterocolitica O:3 is the most frequently implicated. A PCR method was developed to detect Y. enterocolitica O:3. For this purpose, two pairs of primers were designed to amplify two fragments of the rfb cluster of Y. enterocolitica O:3: a 253-bp fragment of the rfbB gene and a 405-bp fragment of the rfbC gene. A specific detection was obtained only with rfbC primers, which yielded a PCR product of the expected size exclusively with pathogenic Y. enterocolitica of serotype O:3. This pair of primers was combined with the ail, inv, and virF primers previously described (H. Nakajima, M. Inoue, T. Mori, K.-I. Itoh, E. Arakawa, and H. Watanabe, J. Clin. Microbiol. 30:2484-2486, 1992) to allow both the detection and the differentiation between Y. pseudotuberculosis, pathogenic Y. enterocolitica of serotype O:3 and other pathogenic Y. enterocolitica.     

Genetic heterogeneity of clinical strains of Yersinia enterocolitica traced by ribotyping and relationships between ribotypes, serotypes, and biotypes

A series of 74 Yersinia enterocolitica clinical strains collected in a Spanish region and 10 reference strains, assigned to nine serotypes and five biotypes, were analyzed by ribotyping procedures. Riboprobing, performed separately with HindIII and BglI and using an rrn operon as the probe, generated 13 and 11 ribotypes (discrimination index [DI] = 0.56 and 0.55), respectively. PCR ribotyping, performed with primers complementary to conserved regions of 16S and 23S rRNA genes, generated 13 ribotypes (DI = 0. 56). A combination of data from the three procedures allowed for further discrimination into 17 combined ribotypes (DI = 0.83). The dendrogram obtained by cluster analysis of data from riboprobing indicated a high heterogeneity of the ribosomal DNA regions of the strains under study (similarities between 10 and 92%), which were grouped into three clusters at a similarity level of 0.32. The major cluster included 10 branches, and 7 of these formed a subcluster (similarity coefficient, >83%) represented by strains of serotype O:3 and biotype 2, 3, or 4. The second cluster included four branches, represented by strains belonging to seven non-O:3 serotypes, biotypes 1A and 2, and two of these branches included pyrazinamidase-positive as well as pyrazinamidase-negative strains. The remaining three branches, represented by O:3-biotype 4 strains, formed a third cluster weakly related to the others. Data from this study showed that Y. enterocolitica O:3 organisms assigned to a prevalent and endemic lineage and non-O:3 organisms assigned to three other less-frequent lineages are circulating and causing human disease in the Spanish region under study.