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PubMed2005

Evaluation of ribotyping as a tool for molecular typing of Yersinia pseudotuberculosis strains of worldwide origin.

Yersinia pseudotuberculosis is a gram-negative bacterium that infects a wide range of animals, including humans, and is transmitted by the fecal-oral route. This species is found globally and is responsible for human outbreaks, mainly in cold countries. The aim of this study was to evaluate the potential of ribotyping for the molecular typing of worldwide isolates. For this purpose, 80 strains of Y. pseudotuberculosis belonging to the six classical serotypes and nine subserotypes and isolated from various countries and different hosts were analyzed. Combination of the EcoRI and EcoRV ribopatterns allowed the delineation of 27 ribotypes. In most instances, ribotypes were associated with specific subserotypes and allowed their subdivision. No association between the ribotype and the geographical origin of the strains was observed, arguing for a global spread of this organism. Similarly, no marked association between the ribotype and the type of host was noted, confirming the circulation of this pathogen in the environment, different animal species, and human hosts. Y. pseudotuberculosis exhibited ribopatterns very close to those of Y. pestis, although not completely identical. Altogether, the present study demonstrates that ribotyping may be a useful tool for molecular typing of global isolates of Y. pseudotuberculosis but that it has some limitations due to the small number of hybridizing bands that generate the diversity of the profiles.

Animals↗
PubMed2006

Automated ribotyping to distinguish the different non Sau/ non Sep staphylococcal emerging pathogens in orthopedic implant infections.

Several species belonging to Staphylococcus genus, other than Staphylococcus aureus and Staphylococcus epidermidis (non Sau/ non Sep species), exhibit increasing abilities as opportunistic pathogens in the colonisation of periprosthetic tissues. Consequently, the availability of means for accurate identification is crucial to assess the pathogenic characteristics and to clarify clinical relevance of the individual species. Here, 146 clinical staphylococcal isolates belonging to non Sau/ non Sep species from prosthesis-associated orthopedic infections were analyzed by conventional enzymatic galleries and by automated ribotyping. Twelve different species were recognised: S. capitis, S. caprae, S. cohnii, S. equorum, S. haemolyticus, S. hominis, S. lugdunensis, S. pasteuri, S. sciuri, S. simulans, S. warneri, S. xylosus. Ribotype identifications were compared with the phenotypes obtained by the Api 20 Staph system and/or ID 32 Staph system. ID 32 Staph profiles were more consistent with ribotyping results than Api Staph profiles. Across the different staphylococcal species investigated, correct identifications with Api Staph were 45%, while with ID 32 Staph they were 59%. It has, however, to be mentioned that ID 32 Staph was mostly applied to discriminate unmatched ribotyping and Api Staph identifications, thus to a subpopulation of strains with ""atypical"" metabolic profile. Automated ribotyping provided a correct identification for 91% of the isolates. These results confirm automated ribotyping as a convenient rapid technique, still subject to improvements, which will accurately and rapidly recognise the newly emerging staphylococcal pathogens in implant-related orthopedic infections.

Automation↗
PubMed2004

Automated ribotyping, a rapid typing method for analysis of Erysipelothrix spp. strains.

Automated ribotyping classified 70 Erysipelothrix species strains, previously classified into 14 RAPD patterns and into 63 PFGE patterns, into 27 ribogroups. Twenty-three strains of the 70 analyzed and classified into 13 ribogroups were previously classified into six ribotypes by the traditional ribotyping method. Moreover, automated ribotyping differentiated seven strains that were not differentiated by PFGE. Therefore, automated ribotyping was more sensitive than RAPD and traditional ribotyping, and it might be a useful method for a rapid screening in epidemiological study of strains of this genus, and more accurate results can be obtained when this method is used together with PFGE.

Automation↗
PubMedDate not supplied

Geographic variability of Escherichia coli ribotypes from animals in Idaho and Georgia.

Several genotypic methods have been developed for determining the host origin of fecal bacteria in contaminated waters. Some of these methods rely on a host origin database to identify environmental isolates. It is not well understood to what degree these host origin isolates are geographically variable (i.e., cosmopolitan or endemic). This is important because a geographically limited host origin database may or may not be universally applicable. The objective of our study was to use one genotypic method, ribotyping, to determine the geographic variability of the fecal bacterium, Escherichia coli, from one location in Idaho and three locations in Georgia for cattle (Bos taurus), horse (Equus caballus), swine (Sus scrofa), and chicken (Gallus gallus domesticus). A total of 568 fecal E. coli isolates from Kimberly, ID (125 isolates), Athens, GA (210 isolates), Brunswick, GA (102 isolates), and Tifton, GA (131 isolates), yielded 213 ribotypes. The percentage of ribotype sharing within an animal species increased with decreased distance between geographic locations for cattle and horses, but not for swine and chicken. When the E. coli ribotypes among the four host species were compared at one location, the percent of unshared ribotypes was 86, 89, 81, and 79% for Kimberly, Athens, Brunswick, and Tifton, respectively. These data suggest that there is good ribotype separation among host animal species at each location. The ability to match environmental isolates to a host origin database may depend on a large number of environmental and host origin isolates that ideally are not geographically separated.

Animals↗
PubMed1995

Comparison of arbitrarily primed polymerase chain reaction and ribotyping for subtyping Actinobacillus actinomycetemcomitans.

This study investigated the compatibility of arbitrarily primed polymerase chain reaction (AP-PCR) and ribotyping in the characterization of Actinobacillus actinomycetemcomitans , a major pathogen in the mixed anaerobic microflora of human periodontitis. AP-PCR was performed directly on lysed bacterial colonies using a random-sequence 10-base oligonucleotide primer. Ribotyping was carried out by using purified bacterial chromosomal DNA digested with BglI. DNA fragments were separated electrophoretically, blotted onto a nylon membrane and hybridized with the plasmid pKK3535 containing the rRNA operon of Escherichia coli. The two genetic methods were evaluated on isolates from single individuals and from family members. Twelve AP-PCR types and 47 ribotypes were distinguished among 76 A. actinomycetemcomitans isolates of different serotypes. AP-PCR typing and ribotyping gave compatible results in 18 of 20 comparisons. Although AP-PCR detected less genetic heterogeneity in A. actinomycetemcomitans than ribotyping, the rapid and relatively simple AP-PCR technique seems to be sufficiently discriminative to be used in large scale epidemiological studies which preclude the application of the more laborious ribotyping technique.

Journal Article↗
PubMed1996

Discriminatory power and application of ribotyping of Yersinia enterocolitica O:3 in an epidemiological study.

Ribotyping performed with five restriction endonucleases was used in an attempt to subtype Yersinia enterocolitica serotype O:3 and also as a tool for clonal analysis. DNA from organisms under study (48 isolates from diarrheic human feces, 24 from food, and 5 reference strains) was tested by Southern hybridization using a DNA probe carrying an rRNA operon from Escherichia coli. Strains were grouped into seven ribotypes by the HindIII restriction endonuclease, into five ribotypes by Ncil, Bg/l, and Sa/l; and into two ribotypes by EcoRI, resulting in a discrimination index (DI) of 0.37, 0.17, 0.43, 0.13, and 0.03 for the five endonucleases. By combining the results obtained with two or more restriction endonucleases, a further discrimination was registered, the most efficient combination (in terms of discriminatory power vs. cost in work, time, and money) for routine typing being HindIII-Bg/l (9 types, DI=0.58). In the clonal analysis, results obtained with the five restriction endonucleases allowed us to define 11 groupings or clonal lines, which showed a remarkable degree of genetic heterogeneity (genetic distance coefficients between 0.03 and 0.73) and were grouped into two major clusters. One cluster included 93% of the strains and eight lines. At least two of the most frequent lines can be considered endemic in Asturias, Spain, because organisms belonging to these lines have been circulating and causing human yersiniosis in recent years and have also been isolated from commercial raw meat products. Two Ncil ribotypes from the series under study (92.2% of strains included in the prevalent cluster) were similar but not identical to ribotypes of 0.3 organisms from other geographic areas described in the literature, indicating that the genetic structure of prevalent human pathogens of this serotype is basically clonal.

Animals↗
PubMed1992

Genome fingerprinting by pulsed-field gel electrophoresis and ribotyping to differentiate Pseudomonas aeruginosa serotype O11 strains.

The performance of ribotyping and pulsed-field gel electrophoresis was compared in the differentiation of a collection of 44 Pseudomonas aeruginosa serotype O11 strains isolated in seven hospitals in Singapore. Digestion of genomic DNA by EcoRI and SacI followed by Southern hybridization with the Pseudomonas aeruginosa 16S and 23S rRNA gene revealed seven distinct ribotypes. Ribotyping using a combination of both enzymes revealed 11 ribotypes. In contrast, electrophoretic analysis differentiated 41 different strain types among the 44 clinical isolates using either SpeI or DraI. Pulsed-field gel electrophoresis demonstrated greater sensitivity than ribotyping in the differentiation of Pseudomonas aeruginosa strains of the same ribotype and could thus be used alone in epidemiological investigations of hospital outbreaks of Pseudomonas aeruginosa serotype O11 infection.

DNA Fingerprinting↗
PubMed1994

Development of a ribotyping scheme for Haemophilus influenzae type b.

Ribotyping and outer-membrane protein subtyping were used to characterise 283 consecutive isolates of Haemophilus influenzae type b. These isolates were obtained primarily from patients with invasive disease in the UK and were received by the Public Health Laboratory Service Haemophilus Reference Laboratory prior to the implementation of Haemophilus influenzae serotype b vaccine in the UK. A subtyping scheme using the ribotyping method is suggested. Twenty-two ribotypes are described, 14 of which were found amongst the 283 clinical isolates characterised in this study. In contrast, only four outer-membrane protein subtypes were found amongst the 283 isolates. The ribotyping profiles were further used to estimate the relatedness of isolates. The resulting dendrogram suggested a population genetic structure different from that previously described for Haemophilus influenzae type b using multi-locus enzyme electrophoresis. This study shows the value of ribotyping as a subtyping method for epidemiological studies of Haemophilus influenzae type b. However, the further use of ribotyping for population genetic structure analysis of Haemophilus influenzae type b may be misleading and therefore inappropriate.

Adolescent↗
PubMed1995

Subspecies discrimination of staphylococci from revision arthroplasties by ribotyping.

Sixty-six cultures of Staphylococcus spp. were obtained from bone and tissue samples collected from 37 patients during revision arthroplasties and were speciated and ribotyped to assess strain diversity in each species. There were 10 ribotypes among 51 isolates of S. epidermidis, three among three isolates of S. capitis, two among four isolates of S. aureus and two among two isolates of S. simulans. One ribotype was found among each of: two isolates of S. warneri; two isolates of S. haemolyticus and single isolates of S. cohnii and S. saprophyticus. Low molecular weight bands of ribotype patterns characterized one or two related species whereas high molecular weight bands were useful for distinguishing types within species. Specimens from 17 patients yielded more than one isolate of Staphylococcus spp. In 13 of these patients the isolates were representatives of a single species but in only eight did ribotyping show the isolates to be identical. The findings of multiple species and ribotypes from samples taken from the same patient may have implications for understanding the nature of infection in revision arthroplasty and for antibiotic therapy.

Bacterial Typing Techniques↗
PubMed1999

Identification of enterococci by ribotyping with horseradish-peroxidase-labelled 16S rDNA probes.

Enterococci are frequently associated with hospital-acquired infection. Identification of enterococci using conventional biochemical tests are often tedious to perform in a routine diagnostic laboratory and may give equivocal results. This study evaluates the usefulness of ribotyping by DNA hybridisation to identify 68 members of the bacterial genus Enterococcus characterised by a conventional test scheme. DNA probes (830 bp in size) were derived from the 16S rRNA gene of E. coli or E. faecalis by PCR, labelled with horseradish peroxidase and used in Southern blot hybridisations of enterococcal DNA digested with EcoRI. Unique ribotypes were obtained for 11 different species using 12 Enterococcus type strains. Ribotyping identified 44 E. faecalis isolates, 19 E. faecium isolates, two E. durans isolates and one E. avium isolate in concordance with results of the biochemistry tests. Two isolates that had ribotype patterns identical to the E. faecium type strain were unable to be definitively identified by biochemical tests. The results show that ribotyping is able to differentiate between E. faecium and E. faecalis and may be useful for identifying other enterococci in the hospital setting. In addition, ribotyping using DNA probes and enhanced chemiluminescence is a safe and more reproducible alternative to radiolabelling RNA in a clinical microbiology laboratory.

Bacterial Typing Techniques↗
PubMed1999

Ribotyping as an identification tool for Clostridium botulinum strains causing human botulism.

Ribotyping was used for characterisation of 68 Clostridium botulinum strains and five related Clostridium species to determine the applicability of this method for identification of species causing human botulism. Thirteen restriction enzymes were initially tested for suitability for ribotyping of C. botulinum, of which EcoRI and HindIII were selected. Both enzymes clearly differentiated between proteolytic (group I) and a nonproteolytic (group II) strains of C. botulinum, and can be recommended for Group/species identification. Using a commercial software package (GelCompar), a numerical analysis of the discriminatory abilities of EcoRI and HindIII ribotyping within and between the two C. botulinum groups was performed. EcoRI had the higher discriminatory index (0.982), but the ribopatterns generated with group II strains were partly muddled and difficult to interpret. All HindIII ribopatterns were easy to analyse and the discriminatory index for all strains was almost equally high (0.954), whereas this enzyme did not discriminate well between group I isolates. The Clostridium strains diverged at 35+/-13% (mean+/-standard deviation) Dice similarity in dendrograms based on cluster analysis of the ribotyping results. These findings are in good agreement with taxonomical ribotyping studies with other bacterial genera, indicating that ribotyping is a highly suitable method for C. botulinum species identification.

Blotting, Southern↗
PubMed2003

Comparison of AP-PCR typing and PCR-ribotyping for estimation of nosocomial transmission of Clostridium difficile.

We recently attempted to clarify an increased incidence of Clostridium difficile-associated diarrhoea (CDAD) in our hospital by arbitrarily primed polymerase chain reaction (AP-PCR) typing of isolates from 147 consecutive patients collected during a 12 month period (Wullt et al. J Hosp Infect 1999;43:265-273). In the present study we compared the results based on previous AP-PCR data with those based on recent PCR ribotyping of the same isolates and re-analysis of a subset of isolates by AP-PCR typing. The pattern of PCR ribotypes was similar among inpatients and outpatients. A cluster of three closely related PCR ribotypes, related to those of the serogroup H and A8 type strains, dominated and comprised 31% of inpatient and 28% of outpatient C. difficile isolates. The apparent nosocomial transmission rate among inpatients with CDAD was only 9% by AP-PCR typing compared with 18 or 36% by PCR ribotyping depending on the definition used (proportion of patients sharing C. difficile type and ward within two or 12 months). Corresponding rates for all CDAD patients were 5% by AP-PCR and 11 or 21% by PCR ribotyping. Thus, most CDAD patients apparently became ill due to their endogenous strain of C. difficile. Because of the low concordance between the two typing methods the proportion of patients fulfilling the criteria for nosocomial transmission by both methods was only 1%. Re-examination of isolates from patients with recurrences revealed a reproducibility problem with AP-PCR typing. We conclude, that of these two PCR-based options for typing of C. difficile PCR ribotyping offers a superior experimental robustness compared with AP-PCR typing.

Clostridioides difficile↗
PubMed1997

Universal ribotyping method using a chemically labelled oligonucleotide probe mixture.

Some of the present problems in ribotyping are associated with a lack of uniform reactivity of probes when bacterial DNAs are of phylogenetically diverse origins. To overcome these problems, a set of five oligonucleotides (referred to as OligoMix5) was selected to react with conserved sequences located near both extremities of rrs (16S rRNA gene) and near both extremities and the middle of rrl (23S rRNA gene). DNA samples from 13 bacterial species selected to represent various phylogenetic branches within the Eubacteria were cleaved by a restriction endonuclease and electrophoresed in 0.8% agarose, and the fragments were vacuum-transferred to nylon membranes and hybridized with digoxigenin-labelled OligoMix5, plasmid DNA from pKK3535 (cloned rrn operon from Escherichia coli) or pBA2 (cloned rrs from Bacillus subtilis), or acetylamino-fluorene-labelled E. coli 16 + 23S rRNA. The results showed OligoMix5 to visualize patterns in DNA from phylogenetically diverse bacteria with comparable intensity. Banding patterns (not band intensity) obtained with OligoMix5 were identical with those obtained with 16 + 23S rRNA or plasmid pKK3535 for each strain studied and represented complete ribotypes. For DNA from Gram-positive bacteria, complete ribotypes were observed after prolonged enzymatic detection of bands when probes were either E. coli 16 + 23S rRNA or pKK3535. Patterns given by plasmid pBA2 were subsets of the complete ribotypes for 9/13 strains. Each oligonucleotide of the OligoMix5 set was used as a probe to determine its contribution to the complete ribotype. The five oligonucleotide probes, used individually, visualized one to four patterns per DNA sample. Use of DNA from Xenorhabdus sp. CIP 105189 cleaved by EcoRI is suggested to control the quality of the oligonucleotide probes composing OligoMix5. Probe OligoMix5 was found to be an essential tool for ribotyping phylogenetically diverse eubacteria.

2-Acetylaminofluorene↗
PubMed1997

Genotypic relationships among strains classified under the (Pasteurella) haemolytica-complex as indicated by ribotyping and multilocus enzyme electrophoresis.

Two-hundred and one strains classified under the (Pasteurella) haemolytica-complex isolated from cattle, sheep, deer, pigs, hares and rabbits were investigated by ribotyping. Fifty-nine of these strains were selected for further studies using multilocus enzyme electrophoresis (MEE). A correlation between the clusters identified by ribotyping and MEE was demonstrated and the results furthermore indicated that a genetic basis exists for most clusters previously outlined by the use of quantitative evaluation of phenotypic data. The taxonomic relevance of ornithine decarboxylase and fermentation of L-arabinose, D-sorbitol and glucosides for taxonomic delineation within the (P.) haemolytica-complex was supported. A taxonomic importance was further indicated for ONPG, ONPX, ONPF, meso-inositol, D-xylose, maltose, dextrine and NPG in relation to some of the taxa. Within the porcine taxon 15, however, differences in ornithine decarboxylase did not correspond to genetic clusters. Six lineages were revealed by MEE. Lineage A contained electrophoretic types (ETs) representing biogroups 1, 3A-3H, 8A and 9, indicating a genetic relationship between these groups--an observation which was supported by ribotyping. Lineage B included biogroup 8D, 3 strains from biogroup 10 and a single strain from biogroup 1 and taxon 18/biovar 1. Lineage C contained strains allocated to biogroup 6 from ruminants and the porcine taxon 15. The similarity between these two groups was accentuated by ribotyping. Lineage D and the single isolate in lineage E contained strains allocated to biogroups 7, 10, 8B and 8C, in addition to single strains from biogroups 6 and 9. The same strains were found in the heterogenous ribotype cluster 17. Lineage F contained strains representing the leprine taxon 20 and the ruminant (P.) granulomatis. Ribotyping indicated that the ruminant biogroup 3J was affiliated with both taxon 20 and (P.) granulomatis.

Alleles↗
PubMed1998

Ribotypes of clinical Vibrio cholerae non-O1 non-O139 strains in relation to O-serotypes.

The emergence of Vibrio cholerae O139 in 1992 and reports of an increasing number of other non-O1 serogroups being associated with diarrhoea, stimulated us to characterize V. cholerae non-O1 non-O139 strains received at the National Institute of Infectious Diseases, Japan for serotyping. Ribotyping with the restriction enzyme BglI of 103 epidemiological unrelated mainly clinical strains representing 10 O-serotypes yielded 67 different typing patterns. Ribotype similarity within each serotype was compared by using the Dice coefficient (Sd) and different levels of homogeneity were observed (serotypes O5, O41 and O17, Sd between 82 and 90%: serotypes O13 and O141 Sd of 72; and O2, O6, O7, O11, O24 Sd of 62-66%). By cluster analysis, the strains were divided into several clusters of low similarity suggesting a high level of genetic diversity. A low degree of similarity between serotypes and ribotypes was found as strains within a specific serotypes often did not cluster but clustered with strains from other serotypes. However, epidemiological unrelated O5 strains showed identical or closely related ribotypes suggesting that these strains have undergone few genetic changes and may correspond to a clonal line. Surprisingly, 10 of 16 O141 strains studied contained a cholera toxin (CT) gene, including 7 strains recovered from stool and water samples in the United States. This is to our knowledge the first report of CT-positive clinical O141 strains. The closely related ribotypes shown by eight CT-positive strains is disturbing and suggest that these strains may be of a clonal origin and have the potential to cause cholera-like disease. Despite the low degree of correlation found between ribotypes and serotypes, both methods appears to be valuable techniques in studying the epidemiology of emerging serotypes of V. cholerae.

Bacterial Toxins↗
PubMed1998

Ribotyping of strains of Moraxella (Branhamella) catarrhalis cultured from the nasopharynx and middle ear of children with otitis media.

Moraxella (Branhaomella) catarrhalis is frequently present in the nasopharyngeal microflora of small children, especially during episodes of acute otitis media . By means of ribotyping (restriction endonuclease analysis of chromosomal DNA combined with rRNA probing), we studied the genetic heterogeneity of 78 cultures of M. catarrhalis obtained from different localities in the nasopharynx of nine young children with secretory otitis media. Using HindIII and PstI as endonucleases, five different ribotypes were recognized, representing at least five different genotypes of M. catarrhalis. The distribution of these types was found to be almost identical to the distribution among 16 M. catarrhalis strains cultured from middle ear exudates of 16 children with acute otitis media. Ribotype HAPA was found in two-thirds of all the cultures investigated, and 44% of the children harboured more than one ribotype in the nasopharynx at the same time. The vast majority of the nasopharyngeal M. catarrhalis cultures were beta-lactamase positive. One child had both a HAPA ribotype, beta-lactamase-negative strain in the nasopharyngeal secretions, and HAPA ribotype, beta-lactamase-positive strains at the entrance of the eustachian tube, the nasopharyngeal tonsils, the folds of the nasopharyngeal tonsils and the oropharynx. All except one of the M. catarrhalis strains cultured from middle ear exudates were beta-lactamase positive.

Child↗
PubMed1997

Studies on the distribution of high-level gentamicin-resistant Enterococcus faecalis using ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences.

Twenty-five high-level gentamicin resistant (HLGR) Enterococcus faecalis strains were isolated from three different University laboratories in Italy. The resistant strains were variously distributed in the three centers with percentages of prevalence ranging from about 3% up to 14%. Almost all strains shared high-level resistance to streptomycin (23 out of 25). Ribotyping and restriction analysis of the 16S-23S rRNA intergenic spacer sequences were used to genetically differentiate the various strains and to study their spreading in the university hospitals serviced by the three laboratories. At least three ribotypes were identified, which showed a peculiar distribution in the various centers. Only the ribotype B was isolated from the University of Padua. In Cagliari, most strains belonged to ribotype A (4/6), whereas in Genoa there was an equal distribution of the ribotypes A and B. A clonal spreading of some HLGR strains is suggested by these findings. The restriction analysis of the 16S-23S rRNA intergenic-spacer sequences gave comparable results with classical ribotyping and, in addition, was quicker and easier to perform than the latter.

Drug Resistance, Microbial↗
PubMed1997

Ribotype differences between clinical and environmental isolates of Burkholderia pseudomallei.

Burkholderia pseudomallei is isolated frequently from the soil in regions where the disease melioidosis occurs. However, recent surveys in Thailand have shown that the frequency of isolation of the organism from soil samples is not directly related to the incidence of melioidosis in an area. To determine whether strain populations of B. pseudomallei prevalent in soil are gentypically related to strains causing clinical disease, rRNA BamHI restriction fragment length polymorphisms (RFLP) of 139 soil environmental isolates and 228 human isolates were compared. Two groups of ribotype patterns were found. Group I comprised 37 different ribotype patterns which were characterised by five to eight hybridisation bands of 2.8- > 23 kb. All of these ribotypes were identified among the clinical isolates, and 18 of them were also found in 59 environmental isolates. Group II was represented by 12 ribotypes found only in environmental strains. These ribotype patterns comprised one to five bands in the size range 9- > 23 kb. All but one of the 73 isolates in this group grew on a minimal medium supplemented with L-arabinose. In contrast, only 3% of the 66 isolates from the environment with group I ribotype patterns could utilise this sugar as their sole energy source. These findings suggest that B. pseudomallei strains that utilise arabinose constitute a population that is genetically distinct from other environmental and clinical strains.

Arabinose↗