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PubMed1995

Ribotyping and plasmid profiling of Vibrio anguillarum serovar O2 and Vibrio ordalii.

One hundred and twenty-nine strains of Vibrio anguillarum serovar O2 and 14 strains of Vibrio ordalii were ribotyped and examined for plasmid contents. A total of 35 different ribotypes were detected. The V. anguillarum serovar O2 strains were divided into 32 different ribotypes. The V. ordalii strains showed three different ribotypes, clearly distinct from those of the V. anguillarum strains. Ribotypes were separated into seven clusters, of which one comprised the V. ordalii strains. Clustering of the strains indicated a genetic difference between North European and South European V. anguillarum O2 strains. Sero-subgroups O2a and O2b shared ribotypes; however, three of the clusters did not include O2a strains. All V. ordalii strains had a plasmid of 32 kb. This plasmid was not detected in any of the V. anguillarum strains. Seventeen different plasmid profiles with 17 different sized plasmids were detected among the V. anguillarum strains. Most of the plasmids were small (< 6 kb) and found in several strains. Except for one South European strain, plasmids were detected only in the North European strains of V. anguillarum O2.

Cluster Analysis↗
PubMed1998

Characterization of coagulase-negative staphylococci by primer-specific polymerase chain reaction and ribotyping.

OBJECTIVE: To analyze, by primer-specific polymerase chain reaction (PCR) and ribotyping, coagulase-negative staphylococci (CNS). METHODS: Forty-five clinical isolates of CNS were identified by the API ID32 STAPH system and ribotyping. Additionally, primer-specific PCR was evaluated for identification of clinical strains of Staphylococcus epidermidis. RESULTS: Forty-five isolates of CNS from neonates with nosocomial bacteremia were studied. The results of the S. epidermidis-specific PCR were compared with those obtained using ribotyping and the API ID32 STAPH system. Excellent congruence was found between primer-specific PCR and ribotyping. Primer-specific PCR proved to be a fast and reliable method for the identification of S. epidermidis strains. According to the primer-specific PCR and ribotyping analysis, a few CNS isolates were found to be incorrectly identified by the API ID32 STAPH system. CONCLUSIONS: Primer-specific PCR is a fast and reliable method for the identification of S. epidermidis. Primer-specific PCR in combination with ribotyping is a promising approach for studying the epidemiology of S. epidermidis and other CNS species in hospital.

Journal Article↗
PubMed2000

Pathovars of Pseudomonas syringae causing bacterial brown spot and halo blight in phaseolus vulgaris L. are distinguishable by ribotyping.

Ribotyping was evaluated as a method to differentiate between Pseudomonas syringae pv. phaseolicola and pv. syringae strains causing bacterial brown spot and halo blight diseases in Phaseolus vulgaris L. Ribotyping, with restriction enzymes BglI and SalI and using the Escherichia coli rrnB operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures yielded 19 combined ribotypes. Cluster analysis of the combined ribotypes differentiated the pathovars phaseolicola and syringae, as well as different clonal lineages within these pathovars. The potential of ribotyping to screen for correlations between lineages and factors such as geographical region and/or bean varieties is also reported.

Cluster Analysis↗
PubMed1995

Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Acinetobacter isolates.

Seventy-three isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, including 26 isolates from 10 hospital outbreaks, were typed by ribotyping with EcoRI and ClaI and by pulsed-field gel electrophoresis (PFGE) of genomic DNA after digestion with ApaI. Ribotyping with EcoRI distinguished 31 ribopatterns. Digestion with ClaI generated another eight ribotypes. PFGE, in contrast, identified 49 distinct patterns with seven variants. Both methods detected all outbreak-related isolates. By ribotyping, nine epidemiologically unrelated strains could not be differentiated from outbreak strains, in contrast to only one isolate not identified by PFGE. Thus, PFGE was more discriminating than ribotyping. However, ribotyping is known to generate banding patterns specific to each DNA group in the A. calcoaceticus-A. baumannii complex that may be used for taxonomic identification of the strains. PFGE was shown to lack this property. Both methods are therefore useful for strain differentiation in epidemiological studies of Acinetobacter isolates.

Acinetobacter↗
PubMed1999

Pulsed-field gel electrophoresis is more efficient than ribotyping and random amplified polymorphic DNA analysis in discrimination of Pasteurella haemolytica strains.

One hundred thirty-three strains of Pasteurella haemolytica of both biotypes (90 and 43 strains of biotypes A and T, respectively) and almost all the serotypes were subjected to ribotyping, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE) analysis for epidemiological purposes. A total of 15 patterns recorded as ribotypes HA to HO were found for the P. haemolytica biotype A strains, with ribotypes HA, HC, and HD being encountered most often (66 strains [74%]); and 20 ribotypes, designated HA' to HT', that were clearly distinct from those observed for biotype A strains were observed for strains of biotype T. RAPD analysis generated a total of 44 (designated Rp1 to Rp44) and 15 (designated Rp1' to Rp 15') unique RAPD patterns for biogroup A and biogroup T, respectively. Analysis of the data indicated that a given combined ribotype-RAPD pattern could be observed for biotype A strains of different serotypes, whatever the zoological or geographic origin, whereas this was not the case for biotype T strains. PFGE appeared to be more efficient in strain discrimination since selected strains from various zoological or geographical origins harboring the same ribotype-RAPD group were further separated into unique entities.

Animals↗
PubMed2000

Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile.

Clostridium difficile is now recognized as the major agent responsible for nosocomial diarrhea in adults. Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of C. difficile infections. However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated. We compared the results of three methods-AP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with SmaI endonuclease)-to differentiate 99 strains of C. difficile that had been previously serogrouped. Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G. Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5. Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C. PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE. We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for C. difficile.

Bacterial Typing Techniques↗
PubMed2004

Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping.

Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A(-)/B(+)) strains. Thirty-nine clinical toxin A(-)/B(+) isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands. The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods. Furthermore, the toxin profile and clindamycin resistance were determined. Reference strains of C. difficile representing 30 known serogroups were also included in the analysis. AFLP discriminated 29 types among the reference strains, whereas the two PCR ribotyping methods distinguished 25 and 26 types. The discriminatory power of AFLP and PCR ribotyping among 12 different unrelated isolates was similar. Typing of 39 toxin A(-)/B(+) isolates revealed 2 AFLP types and 2 and 3 PCR ribotypes. Of 39 toxin A(-)/B(+) isolates, 37 had PCR ribotype 017/20 and AFLP type 20 (95%). A deletion of 1.8 kb was seen in 38 isolates, and 1 isolate had a deletion of approximately 1.7 kb in the tcdA gene, which encodes toxin A. Clindamycin resistance encoded by the erm(B) gene was found in 33 of 39 toxin A(-)/B(+) isolates, and in 2 of the 12 unrelated isolates (P < 0.001, chi-square test). We conclude that clindamycin-resistant C. difficile toxin A(-)/B(+) strain (PCR ribotype 017/20, AFLP type 20, serogroup F) has a clonal worldwide spread.

Bacterial Proteins↗
PubMed1999

The diversity and distribution of the predominant ribotypes of Actinomyces naeslundii genospecies 1 and 2 in samples from enamel and from healthy and carious root surfaces of teeth.

The bacterial communities associated with root caries are highly diverse and undergo succession during lesion formation. Consequently, root caries is said to have a polymicrobic etiology, typified by variation in the predominant species among samples from different lesions. Despite the polymicrobic etiology, A. naeslundii genospecies 1 and 2 (previously A. viscosus) have consistently been shown to be associated with root caries in humans; they predominate in some lesions and have been suggested to play a significant role in the disease. Several genetic variants of A. naeslundii are known to be present among the oral A. naeslundii population of an individual. The current study was initiated to explore the possibility that a variant in these A. naeslundii populations had characteristics which made it best fitted to colonize or promote root-surface caries lesions. Using ribotyping to detect variants, we tested the hypothesis that 'a ribotype of A. naeslundii best fitted to the environment would be selected and predominate in the A. naeslundii population of lesions'. Samples of plaque from enamel, normal root surfaces, plaque overlying the lesion, and material from within the lesion were taken from nine patients with soft root caries. The flora from 14 lesions and 9 enamel sites was analyzed on selective and non-selective media, and A. naeslundii genospecies were identified by serology. We ribotyped 972 isolates, showing 54 different patterns. Between 6 and 20 ribotypes were isolated from eight of nine patients. In general, each site from a patient showed a similar distribution of ribotypes. These results do not support the hypothesis and suggest that any phenotypic characters that allow A. naeslundii genospecies 1 and 2 to colonize or contribute to the formation of root-caries lesions are common among strains identified by ribotyping.

Actinomyces↗
PubMed1998

Ribotyping of Pseudomonas aeruginosa from infected patients: evidence of common strain types.

Sixty-nine strains of Pseudomonas aeruginosa isolated from infected patients at three hospitals in Valencia were serotyped and analyzed by comparison of restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping). Genomic Southern blots of EcoRI restriction digests were hybridized with a universal rRNA gene probe from Escherichia coli 16S and 23S rRNA. Strains were genetically diverse and 12 different ribotypes of 2 to 7 bands between 5 and 21.5 kb were defined. All strains shared a common band of 6.0 kb. The predominant ribotypes were R1 and R2, representing 25% and 41% of all isolates, respectively. Ribotypes were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype. This study demonstrated the prevalence of certain strain types associated with infected patients at Valencia hospitals, confirming the high typability and reproducibility of a single enzyme ribotyping for epidemiological studies of Pseudomonas aeruginosa. Ribotyping could be particularly useful if used in conjunction with serotyping.

Bacterial Typing Techniques↗
PubMed1995

Ribotyping of clinical Vibrio vulnificus isolates.

Restriction fragment length polymorphism analysis of rRNA genes (ribotyping) was used to differentiate Vibrio vulnificus isolates. Among the 10 restriction enzymes tested, HindIII was shown to provide the most discriminatory patterns. Stul was used for further analysis of strains that were indistinguishable with HindIII. Thirteen clinical V. vulnificus strains were analyzed for their ribotypes with HindIII, as well as Stul when necessary. Four of the clinical strains were isolated from different samples collected from the same patient, and were shown to have identical ribotypes. All the others gave unique ribotypes, indicating the large genetic divergence in V. vulnificus clinical isolates. The ribotype of V. vulnificus by HindIII remained unchanged after successive in vitro and in vivo passages. HindIII gave rise to five bands which were shown in every V. vulnificus strain but not in other vibrio species tested, suggesting that ribotyping with this restriction enzyme may be useful for confirming the identification of this bacterium.

Bacterial Typing Techniques↗
PubMed2004

Phage types, ribotypes and tetracycline resistance genes of Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from different origins in Italy.

The tetracycline resistance (tet) gene patterns of 52 tetracycline resistant Salmonella enterica subsp. enterica (S.) serovar Typhimurium isolates collected from animals, food of animal origin, and humans in Italy, were investigated to evaluate whether the tet gene patterns could be used for strain differentiation in addition to phage typing and ribotyping. The detection of tet genes was performed by specific PCR assays. Ribotyping was performed automatically using PvuII as restriction enzyme. Ten different ribotyping patterns were detected. All isolates were positive for at least one of the tet genes studied and six different tet gene patterns were observed. Ribotyping and tet gene patterns showed discriminatory indices of 0.741 and 0.812, respectively. Multiple tet genes were commonly found among tetracycline resistant S. typhimurium isolates from various sources. The resulting tet gene patterns allowed further discrimination of strains which were otherwise indistinguishable by their phage type, ribotype and origin. Thus, the analysis of tet gene patterns might represent an additional tool for the differentiation of S. typhimurium isolates.

Animals↗
PubMed2001

Ribotyping of Burkholderia pseudomallei from clinical and soil isolates in Thailand.

Burkholderia pseudomallei is a soil saprophyte that causes melioidosis in humans and animals. Restriction fragment length polymorphism of the ribosomal DNA regions (ribotyping) were analyzed in 577 isolates comprising 371 clinical and 206 soil isolates collected throughout Thailand in 1997. A total of 77 distinct ribotype patterns consisting of 2-9 bands with sizes ranging from 2.8 to >23 kb were found. Twelve major ribotypes were identified of which types 3, 8 and 23 were commonly found (278/577, 48.2%) in both clinical (217/371, 58.5%) and environmental isolates (61/206, 29.6%). Three unique environmental types were found whereas a unique clinical type was not observed. Even though ribotypes show high heterogeneity in the rDNA region, the unique environmental patterns were clearly different from the clinical patterns as clearly seen by UPGMA dendrogram. Moreover, the three major types (types 3, 8 and 23) were discovered in nearly half of B. pseudomallei isolates. Subtyping of these major ribotypes in correlation with clinical profiles may help researchers to identify the virulence factor of the organism.

Arabinose↗
PubMed2000

Ribotyping of vibrio populations associated with cultured oysters (Ostrea edulis).

The intraspecific variability of Vibrio splendidus, V. harveyi and V. tubiashii recovered from oysters (Ostrea edulis) collected at the Mediterranean coast near Valencia, Spain, was analyzed by ribotyping. The two former species represented the most abundant ones, and the third one was the only species described as pathogenic for oysters. A total of 115 environmental strains were studied, 84 of V. splendidus, 23 of V. harveyi and 8 of V. tubiashii. Chromosomal DNA was digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Ribotyping among natural populations of the three species rendered 5 to 9 bands, and showed a high genetic diversity, with a ratio no. of strains/no. of ribotypes between 1.1 and 1.5. Cluster analysis of V. splendidus ribotypes suggests a seasonal pattern of incidence, with those ribotypes corresponding to winter and spring samples being maintained in the oysters over the year.

Animals↗
PubMed2000

Strain differentiation in Bacteroides fragilis by ribotyping and computer-assisted gel analysis.

It is important to distinguish between Bacteroides fragilis strains as their virulence may vary and as B. fragilis seems to be a heterogeneous species. The aim of our study was to evaluate ribotyping for differentiation of 46 strains of B. fragilis and for assessment of strain heterogeneity within and between the two DNA-DNA homology strain groups established in this species. Twenty-seven strains belonged to Johnson's DNA homology group I and eight to group II. Eleven strains had not been assigned to any group (NI group). DNA from all strains was cut with BglI, EcoRI and HindIII. Restriction fragment length polymorphisms were investigated using a non-radioactive digoxigenin-labelled cDNA probe transcribed from Escherichia coli 16S+23S rRNA. Ribotyping with BglI was most discriminatory, revealing a total of 26 different patterns by visual inspection of gels. EcoRI followed with 20 patterns and HindIII with 13 patterns. The gels from ribotyping were processed using the Dendron computer-assisted program. Strain clusters established using Dendron were not always in agreement with homology-based strain groups. Strains of the NI group fell into both homology groups. Ribotyping, as it is based on a relatively small portion of the genome, is useful for strain distinction in epidemiological studies with B. fragilis, whereas DNA-DNA homology, using the entire genome, is more reliable for taxonomy. The Dendron computer-assisted program, which enabled objective assessment of multiple banding patterns, increased the reliability of ribotyping.

Bacteroides fragilis↗
PubMed2000

Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains.

PCR-RFLP analysis of the vrrA gene and cerAB gene was used to investigate the genomic diversity in 21 strains of Bacillus anthracis and 28 strains of Bacillus cereus, and was compared with results obtained by ribotyping and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis. VrrA-typing divided the B. anthracis into four groups. Except for one Pasteur vaccine strain, the vrrA PCR-RFLP profiles of the B. anthracis were separated into three groups, which were different from those of the B. cereus strains. Ribotyping separated the B. anthracis isolates into seven ribotypes, and a common fragment of an approximately 850 bp band from the ERIC-PCR fingerprints separated most B. anthracis strains into two groups. VrrA/cerAB PCR-RFLP, ribotyping and ERIC-PCR generated 18, 22 and 23 types, respectively, from B. cereus strains. The results suggest that a combination of all three methods provides a high resolution typing method for B. anthracis and B. cereus. Compared with ribotyping and ERIC-PCR, PCR-RFLP is simple to perform and has potential as a rapid method for typing and discriminating B. anthracis strains from other B. cereus group bacteria.

Bacillus anthracis↗
PubMed2002

Molecular diversity of Campylobacter coli and C. jejuni isolated from pigs at slaughter by flaA-RFLP analysis and ribotyping.

A population of porcine isolates of Camplobacter jejuni (n = 11) and C. coli (n = 17) were examined for genotypic relatedness employing ribotyping, as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin (fla)A gene locus. PCR was employed to amplify a 533 bp fragment from the flaA gene, including the previously described short variable region (SVR), employing the novel primers, A2 and Al and successfully generated this amplicon for all wild-type strains examined (n = 28) of both C. jejuni and C. coli, as well as with both type strains, i.e. C. jejuni NCTC 11351 and C. coli NCTC 11366. Individual genotypes were assigned to each isolate typed employing the four typing methods (flaA-RFLP(Hae) III, flaA-RFLP(Pst) I ribotyping(Hae) III and ribotyping(Pst) I) and were assigned an arbitrary genotype code in ascending alphabetical order in comparison with a database of established genotypes for each of the methods employed. This study showed that several flaA-RFLP and ribopatterns existed within C. jejuni and C. coli, and demonstrated a heterogeneous diversity of strains occurring in the pigs examined. Ribotyping of strains with 16S and 23S rRNA with Pst I and Hae III digested chromosomal DNA allowed subdivision of strains into nine and eight groups, respectively. RFLP analyses with Pst I and Hae III digests probed with the flaA gene probe allowed subdivision of strains into eight and eleven subtypes, respectively. Employment of RFLP with the flaA nucleic acid probe and Hae III digests produced the greatest amount of variation of any genotyping scheme employed. Although there was a high degree of variability demonstrated by both typing methods, most isolates ( > 60%) clustered into four main genotypes, i.e. genotypes A-D. FlaA-PCR-RFLP typing demonstrated that the majority of isolates, 67.9 and 60.7%, were included in these four main genotypes for Pst I and Hae III restriction digests, respectively, although there was a high prevalence (7/11; 63.6%) of fla(Hae) III genotype A occurring within the C. jejuni isolates. Likewise, ribotyping studies demonstrated that most isolates were clustered into these four main genotypes, accounting for 81.5 and 60.7% of isolates for Pst I and Hae III restriction digests, respectively. This may indicate that the clonal population of campylobacters within this pig population is largely composed of persistent and dominant types, with a smaller number of hypervariable subtypes. Such data may useful in determining epidemiological routes of transmission of campylobacters from animal to animal, as well as helping to identify virulence determinants in persistent subtype populations.

Animals↗
PubMed2002

Genotypic heterogeneity of Pasteurella gallinarum as shown by ribotyping and 16S rRNA sequencing.

Forty-five strains mainly isolated from chickens in Zimbabwe and Denmark, two pig and three rat isolates all identified as Pasteurella gallinarum by conventional phenotypic tests were characterized by ribotyping, and selected strains were subsequently analysed by 16S rRNA gene sequencing. High genotypic diversity was observed, the number of ribotypes totalling 24. A major group of 47 isolates including the type strain of P. gallinarum clustered at 56% similarity and included 21 ribotypes. Ribotyping showed that some genotypes of P. gallinarum seem to be globally distributed. The three isolates from rodents did not share even a single common ribotype fragment with strains from birds and the pig isolates. Two avian isolates from Denmark and Zimbabwe and the pig strain showed from 97.6 to 99.8% 16S rRNA sequence similarity with the type strain of P. gallinarum and with type strains of Pasteurella volantium and Pasteurella avium. Two rat strains showed 98.6% 16S rRNA gene sequence similarity with each other, but were only related with P. gallinarum at 93% similarity. These isolates showed the highest similarity with [Actinobacillus] muris at 96.4 to 95.0% similarity. We suggest that conventional identification of P. gallinarum consequently should consider the source of isolation to obtain a correct diagnosis, and that isolation from animals other than fowl should be confirmed by genotypic analysis such as 16S rRNA gene sequence comparison.

Animals↗
PubMed2001

PCR ribotyping of clinically important Clostridium difficile strains from Hungary.

Isolates of Clostridium difficile from different hospital wards at the University Hospital of Szeged in Hungary were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping). A total of 15 different ribotypes was detected among the 65 isolates tested. The predominant type, PCR ribotype 087, accounted for 39% of all isolates, in contrast with an international typing study where ribotype 001 was the most common. Two non-toxigenic C. difficile strains were found to exhibit the same pattern, which was distinct from those of all the ribotypes described previously, suggesting that this is a new type.

Animals↗