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PubMed2003

Niche-specific association of Aeromonas Ribotypes from human and environmental origin.

A total of 88 Aeromonas isolates from distinct locations and sources (39 from extraintestinal infections, 31 from diarrhoeic, ten from non-diarrhoeic faeces, all human, and eight from fresh water) were subjected to phenospecies identification, serotyping, ribotyping and detection of some virulence markers. The strains belonged to four different phenospecies marked by 19 O serogroups and 38 ribotypes. No strong correlation between these parameters was found, and no group, as defined by the typing methods, could be characterized with a particular set of virulence markers. There was a clear association of ribotypes with the source of the strains. Cluster analysis allowed the identification of a complex of ribotypes belonging to distinct but related sources, including clinical and environmental isolates. These results suggest that ribotyping may be an epidemiological tool suitable for the study of Aeromonas infections.

Aeromonas↗
PubMed2003

Geographical variation in ribotype profiles of Escherichia coli isolates from humans, swine, poultry, beef, and dairy cattle in Florida.

Waters impacted by fecal pollution can exact high risks to human health and can result in financial losses due to closures of water systems used for recreation and for harvesting seafood. Identifying the sources of fecal pollution in water is paramount in assessing the potential human health risks involved as well as in assessing necessary remedial action. Recently, various researchers have used the ribotyping method to identify sources of bacterial indicators (Escherichia coli and enterococci) in environmental waters. While these studies have identified genotypic differences between human- and animal-derived indicators that are capable of differentiating organisms isolated from humans and various animal hosts, most have focused on organisms collected from a confined geographic area and have not addressed the question of whether these ribotype profiles are watershed specific or if they can be applied universally to organisms from other geographic locations. In this study, E. coli isolates were obtained from humans, beef cattle, dairy cattle, swine, and poultry from locations in northern, central, and southern Florida and were subjected to ribotyping analysis. The intent was to determine (i) if ribotype profiles are capable of discriminating the source of E. coli at the host species level and (ii) if the resulting fingerprints are uniform over an extended geographic area or if they can be applied only to a specific watershed. Our research indicated that, using a single restriction enzyme (HindIII), the ribotyping procedure is not capable of differentiating E. coli isolates from the different animal species sampled in this study. Results indicate, however, that this procedure can still be used effectively to differentiate E. coli as being either human or animal derived when applied to organisms isolated from a large geographic region.

Animals↗
PubMed2000

Ribotyping and restriction endonuclease analysis reveal a novel clone of Bordetella bronchiseptica in seals.

The goal of the present study was to characterize, by ribotyping and restriction endonuclease analysis (REA), 35 phocine Bordetella bronchiseptica isolates and to ascertain their relationship to one another and to isolates acquired from other host species. Thirty-four isolates were obtained in Scotland during a 10-year period encompassing the 1988 epizootic; the remaining isolate was obtained independently in Denmark. All phocine isolates had an identical Pvu II ribotype unique from the 18 ribotypes previously detected in strains from heterologous hosts. Alternative restriction enzymes, useful for subgrouping strains within Pvu II ribotypes, also failed to discriminate among isolates from seals. The exclusive occurrence of a single ribotype of B. bronchiseptica in a particular host species has not been previously observed. Similarly, REA based on either HinfI or Dde I profiles did not reveal detectable polymorphisms, although unique patterns were readily distinguished among a limited number of isolates from other host species. This is the first report demonstrating the utility of REA using frequently cutting enzymes for discrimination of B. bronchiseptica strains. These data suggest that B. bronchiseptica-induced respiratory disease in seals along the Scottish shore may be due to the circulation of a single, unique clone.

Animals↗
PubMed2002

Serotyping and ribotyping of Salmonella using restriction enzyme PvuII.

The subtyping and identification of bacterial pathogens throughout food processing and production chains is useful to the new hazard analysis critical control point-based food safety plans. Traditional manual serotyping remains the primary means of subtyping Salmonella isolates. Molecular biology techniques, however, offer the promise of more rapid and sensitive subtyping of Salmonella. This study evaluates the potential of restriction enzyme PvuII, followed by probing with the rRNA operon from Escherichia coli, to generate serotype-specific DNA fingerprints. A total of 32 identified serotypes were found with an overall agreement in 208 of the 259 (80%) isolates tested between U.S. Department of Agriculture serotype identification and riboprint serotype identification. Many of the isolates that did not correlate were serotype identified as Salmonella Montevideo, which indicates that for this serotype, there are multiple ribotypes. When Salmonella Montevideo isolates were not included, the ribotype identification agreed with serotyping in 207 of the 231 (90%) isolates. The primary outcome of any ribotyping procedure is to give distinct ribotype patterns. This extensive poultry epidemiological study demonstrates that, in addition to ribotype patterns, the identification of isolates to known serotypes provides the investigator with additional information that can be more useful than traditional epidemiology and isolate identification studies.

Animals↗
PubMed1995

Diversity of nosocomial Xanthomonas maltophilia (Stenotrophomonas maltophilia) as determined by ribotyping.

Seventy-seven clinical isolates of Xanthomonas maltophilia (Stenotrophomonas maltophilia) were consecutively collected from the Rigshospitalet, Copenhagen, ribotyped and compared with the ribotypes of 25 other clinical and reference strains of Xanthomonas maltophilia. Using restriction enzyme EcoRI, 20 different ribotypes were observed, with 78 isolates displaying the five most common types. Using another enzyme, BamHI, these 78 isolates were further subdivided into 16 different ribotypes. Three patients harboured two strains with different ribotypes. No type was found related to only one department and no single-strain outbreak was detected. The origin of this wealth of different strains in hospital patients needs to be established.

Bacterial Typing Techniques↗
PubMed1994

The demonstration by ribotyping of the stability of oral Streptococcus mutans infection over 5 to 7 years in children.

The distribution of serotypes and ribotypes of mutans streptococcal isolates obtained from seven unrelated children at 5 and at 10 or 12 yr of age was investigated. For ribotyping, chromosomal DNA from 5 to 13 isolates per subject was digested with restriction endonucleases EcoRI and HindIII. The DNA fragments were electrophoretically separated, blotted on to nylon membrane and hybridized to the plasmid pKK3535, which contains the rRNA operon of the Escherichia coli chromosome. The ribotypes were unique for each child. In five children only one ribotype and serotype (c, e or f) was found. In one child two serotypes (c and f) were found at baseline and only one (serotype c) in the follow-up sample. In one child the same serotype was not found in the baseline (serotype e) and in the follow-up (serotype c) samples. Every child except one had a ribotype that was identical to one found 5-7 yr later. The results suggest that, at the age of 5 yr, infection by Streptococcus mutans has already stabilized and the colonizing strain remains permanent.

Bacterial Typing Techniques↗
PubMed1996

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals.

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.

Animals↗
PubMed1994

Biotyping, ribotyping and esterase electrophoresis as epidemiological tools for Acinetobacter baumannii.

An epidemiological survey was carried out over a one-week period to assess the spread of Acinetobacter baumannii in a medical intensive care unit. Fifty strains were isolated from patients colonized or infected by the organism and from a hospital environment. These strains belonged to biotypes 9 or 18. The rRNA gene restriction patterns (using EcoRI and PvuII as restriction endonucleases) and the esterase electrophoretic profiles were determined on 31 strains, using as comparison strain isolates from another intensive care unit of our hospital and from two other French hospitals. Four EcoRI ribotypes, four PvuII ribotypes and six esterase profiles were identified. All biotype 9 strains isolated in our hospital presented the same ribotype after EcoRI digestion, the same ribotype after PvuII digestion and the same zymotype. The same observation was made on most of the biotype 18 strains. Biotyping is an appropriate method for screening of strains, and ribotyping and esterase electrophoresis could be used as additional methods to delineate outbreaks of nosocomial infections caused by A. baumannii.

Acinetobacter↗
PubMed1994

Ribotyping as an epidemiologic tool for Escherichia coli.

Restriction fragment length polymorphism of ribosomal RNA genes was analysed among 133 Escherichia coli strains predominantly from blood and urine, including 21 isolates from faeces of healthy persons. The strains had also been characterized for their O:K:H serotypes, for the presence of P, S and type 1C fimbriae, non-P, non-S mannose-resistant haemagglutinins and haemolysin production. Hind III-digested genomic DNA was subjected to Southern blot analysis with either plasmid pKK3535 containing E. coli rRNA operon or purified rRNA as a probe. Among the 133 strains 20 ribotypes were obtained. The distribution of strains into different ribotypes generally correlated with their O:K:H serotype. Ribotype variation within serotypes was mainly seen among strains with the K5 capsule. The origin of the strains or the presence of virulence-associated factors did not correlate with the ribotype. In conclusion, ribotyping appears to be a valuable method in epidemiologic studies especially when the serotyping methods are not available.

Bacterial Adhesion↗
PubMed2000

Comparison of serotyping, ribotyping and pulsed-field gel electrophoresis for distinguishing group A Streptococcus strains isolated in Albania.

Conventional serotyping for T antigens, rRNA gene restriction fragment length polymorphism analysis (ribotyping) and pulsed-field gel electrophoresis were compared for distinguishing among group A streptococci isolated in Albania between 1980-1982 and in 1995. A total of twelve serotypes were identified among seventy GAS strains. Ribotyping revealed eight and eleven distinct patterns after digestion with HindIII and PvuII, respectively. Twenty-three strains of serotype T12 were subdivided in 10 ribotypes and 11 strains of T2 serotype were differentiated in 5 ribotypes. By comparison, PFGE generated 37 patterns after SmaI digestion. The index of discrimination, using the Hunter-Gaston formula, was applied to assess the value of these methods for interpretation of the epidemiological data. For serotyping the value of index was 0.85. The ribotyping system revealed an ID of 0.83 when the combination HindIII and PvuII was used. This index reached 0.97 for PFGE. The methods used were useful to subtype the isolates of GAS.

Albania↗
PubMed1999

Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures.

Twelve Vibrio strains were examined phenotypically in 91 biochemical characters and genotypically by ribotyping. Ten were isolated from sea water and two from diseased turbot (Scophthalmus maximus). All isolates originated from one experimental system located in Ría de Vigo (Galicia, north-west Spain). Different type strains were used for comparative purposes. The taxonomic position was analysed with the NTSYST-pc and similarities among strains were calculated by the Simple Matching coefficient (SSM). rRNA gene restriction patterns were performed with the HindIII enzyme. The SSM coefficient separated the 12 Vibrio strains into two groups which included strains that showed a SSM coefficient quite similar to V. splendidus biovar 1 (ATCC 33125) and V. scophthalmi (CECT 4638). None of 91 phenotypical characters were specific in distinguishing both species. The ribotyping confirmed the taxonomic classification of strains. The pathogenicity of each strain was evaluated; 10 environmental strains were avirulent and two, isolated from diseased turbot, were virulent. Different biotypes and ribotypes were found among the avirulent isolates. This work showed ribotyping to be a valuable tool for identification and confirmed the necessity of extending the ribotype database within closely related Vibrio species in order to clarify the taxonomic position.

Animals↗
PubMed2001

Molecular typing and long-term comparison of clostridium difficile strains by pulsed-field gel electrophoresis and PCR-ribotyping.

Thirty-two related and 68 unrelated isolates of Clostridium difficile, isolated in different Italian hospitals since 1987, were analysed by PFGE and PCR-ribotyping to investigate their genetic relatedness. The isolates were classified into 28 groups by PFGE and 20 ribotypes by PCR-ribotyping. A single clone of C. difficile was recognised as the cause of three geographically and chronologically distant outbreaks. The correlation between PFGE and PCR-ribotyping results was good, with agreement for 77 (84%) of the 92 isolates typed by both methods. However, among sporadic isolates the discriminatory power of PFGE was more evident. Eight isolates that were untypable by PFGE could be analysed by PCR-ribotyping. The dendrograms generated showed that the genetic relatedness of the C. difficile isolates obtained by both techniques was comparable. The majority of the isolates in recent years appeared to be genetically unrelated to isolates from past infections. However, two clonal groups identified in all time periods had a common origin and this seems to indicate that they share some advantageous biological characteristics. The constant monitoring of C. difficile epidemiology will allow acquisition of further important data on this nosocomial pathogen.

Clostridioides difficile↗
PubMed1998

Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae.

Individual rrn operons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone of Vibrio cholerae. The genome of an early isolate of the seventh pandemic clone had nine rrn operons of which two were in tandem with other rrn operons. The site for BglI, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate. This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation. Additional rrn recombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon. Spacers of 431, 509, 607 and 711 bp were found. A total of at least eight rrn recombination events were detected. Three rrn loci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G. In addition there was variation due to deletion of tandem operons. The frequency of recombination between rrn operons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study. This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study. Recombination between rrn operons may be a factor in ribotype variation in all systems. The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved.

Bacterial Typing Techniques↗
PubMed1992

Ribotyping of coagulase-negative staphylococci.

The discriminative capacity of ribotyping was initially assessed without knowledge of results obtained for the same isolates by use of more established typing methods. Forty-eight isolates of coagulase-negative staphylococci (CNS) from peritoneal fluids were studied. They were collected prospectively during 31 consecutive episodes of infection associated with peritoneal dialysis in 17 patients. DNA was digested by the restriction endonucleases EcoRI or HindIII and ribotyped by means of a biotinylated cDNA probe to 16S + 23S staphylococcal ribosomal RNA gene sequences. These methods in combination produced a total of 27 types which compared well with numbers of groups distinguished by other typing methods: limited biotype-antibiotic resistogram (ARB; 28), antibiotic resistogram alone (25), API-Staph (12), phage typing (9) and plasmid analysis (22). Ribotyping was highly reproducible and typed all isolates, including those that were not phage-typable (35) or did not contain plasmids (4). When used in a hierarchical manner with ARB, ribotyping results produced 13 additional types in comparison with the other three methods. When used hierarchically with all other typing systems, a further five types were found among isolates from two patients. However, some of the differences observed as a result of ribotyping could have been due to subtle changes produced by mutation, lysogenisation or gene transposition. Since the method requires additional time, expense and technical expertise, it is likely to be useful only when answers to specific epidemiological problems are required or as an initial screen before using other methods of genetic analysis.

Base Sequence↗
PubMed1997

Comparison of Vibrio cholerae O1 isolates by polymerase chain reaction fingerprinting and ribotyping.

The rRNA gene restriction patterns and the polymerase chain reaction (PCR) fingerprinting types of 53 Vibrio cholerae O1 isolates were studied. Five and eight patterns were observed from 27 toxigenic and 26 non-toxigenic O1 isolates after BglI cleavage. PCR fingerprinting with three primer sets aimed at enterobacterial repetitive intergenic consensus (ERIC) sequences, ERIC-related sequences in V. cholerae, another kind of repeated sequences in V. cholerae (VCR) and arbitrary sequences divided the same strains into seven and 10 PCR types, respectively. Eight ribotypes had unique PCR patterns. PCR fingerprinting identified more than one pattern among isolates within each of the remaining ribotypes. However, ribotyping was able to differentiate the same PCR types in one case. A single ribotype and a single PCR pattern were found in toxigenic O1 strains isolated in Taiwan from imported food and imported cases of cholera between 1993 and 1995. Typing of V. cholerae O1 by PCR fingerprinting correlated well with ribotyping, but was more discriminating. PCR assay provides a rapid and simple means of typing these strains for epidemiological studies.

Cholera↗
PubMed1998

Characterisation of diarrhoeagenic Escherichia coli clones by ribotyping and ERIC-PCR.

The ability of ribotyping and enterobacterial repetitive intergenic concensus-polymerase chain reaction (ERIC-PCR) to discriminate diarrhoeagenic Escherichia coli clones of 122 strains belonging to 26 distinct serotypes was evaluated. The 26 serotypes corresponded to 24 ribotypes and 25 ERIC-types. Correlation between multilocus enzyme electrophoresis, ERIC-PCR and ribotyping was c. 90% for the dominant ribotypes. Related clones such as O55:H7 and O157:H7 presented similar ribotypes and clustered together in a dendrogram, and the two divergent clonal groups of enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) were included in distinct branches. The results suggest the possibility of applying these two simpler techniques as tools to identify clones of diarrhoeagenic E. coli.

Bacterial Typing Techniques↗
PubMed1999

Ribotyping of Vibrio parahaemolyticus isolates obtained from food poisoning outbreaks in Taiwan.

Vibrio parahaemolyticus is a prevalent food-borne pathogen in Taiwan, Japan and other Asian countries. This work presents a novel ribotyping method for the molecular epidemiological examination of this pathogen. Genomic DNA was fragmented by HindIII digestion and hybridized with cDNA probe for Escherichia coli 16S and 23S RNA genes. A total of 121 isolates obtained from outbreaks during 1992 and 1994 in Taiwan were characterized by this ribotyping method. Four to seventeen restricted fragments were visualized in these isolates. After hierarchical cluster analysis, these isolates were grouped into thirty different ribotypes. In addition, A3, A7, E3 and F1 were the major ribotypes, consisting of 22.3, 13.2, 9.1, and 8.3% of the isolates, respectively. A, E, F, G and B were the major groups, consisting of 46.2, 14.0, 9.1, 6.7, and 6.7% of the isolates, respectively. The discriminatory ability of this ribotyping method, as determined by Simpson's index of diversity, was 0.93, which closely resembled that of a previously reported pulsed-field gel electrophoresis method.

Bacterial Typing Techniques↗
PubMed1998

Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences.

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.

Animals↗