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PubMed1998

Genetic relatedness among environmental, clinical, and diseased-eel Vibrio vulnificus isolates from different geographic regions by ribotyping and randomly amplified polymorphic DNA PCR.

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.

Animals↗
PubMed1993

Ribotyping of Pseudomonas aeruginosa: discriminatory power and usefulness as a tool for epidemiological studies.

Restriction fragment length polymorphism of ribosomal DNA regions (ribotyping) of Pseudomonas aeruginosa was evaluated as a tool for epidemiological purposes. Fifty-five epidemiologically unrelated isolates from three geographic areas of Switzerland and 11 isolates obtained during an outbreak of P. aeruginosa infections in a burn unit were typed by this method. Typeability and reproducibility of the method reached 100%. With four selected restriction enzymes (BamHI, ClaI, EcoRI, and PstI), the 55 unrelated isolates could be classified into 33 ribotypes. To assess the value of this method for the interpretation of epidemiological data, we calculated an index of discrimination (ID) which takes into consideration both the number of types defined by the typing method and their relative frequencies. Our ribotyping system obtained a high ID of 0.958 with only four restriction enzymes, comparing well with other different typing schemes for which ID values could be calculated from published data. All clinical isolates of the outbreak belonged to the same ribotype, whereas environmental isolates, initially thought to be the source of the epidemic, belonged to a different ribotype. Thus, the typeability, reproducibility, and discriminatory power of our method as well as its value established in an epidemiological investigation were found to be appropriate for further epidemiological studies of P. aeruginosa.

Bacterial Typing Techniques↗
PubMed1994

Quantitative antibiogram typing using inhibition zone diameters compared with ribotyping for epidemiological typing of methicillin-resistant Staphylococcus aureus.

Antibiogram typing of methicillin-resistant Staphylococcus aureus with selected antibiotics was evaluated as a primary epidemiological typing tool and compared with ribotyping. Antibiograms were derived with the Kirby-Bauer disk diffusion method by using erythromycin, clindamycin, cotrimoxazole, gentamicin, and ciprofloxacin. For typing, antibiogram data were analyzed by similarity analysis of disk zone diameters (quantitative antibiogram typing). One hundred seventy-two isolates were typed. Reproducibility reached 98% for the quantitative antibiogram and 100% for ribotyping. With three selected restriction enzymes (EcoRV, HindIII, and KpnI), 40 epidemiologically unrelated isolates could be classified into 21 ribotypes, whereas quantitative antibiogram typing classified these isolates into 19 groups. To evaluate the discriminatory power of the methods, we calculated an index of discrimination from data obtained with these 40 isolates. This index takes into consideration both the number of types defined by the typing method and their relative frequencies. With both ribotyping and quantitative antibiogram typing, high discrimination indices (0.972 and 0.954, respectively) were obtained. When epidemiological links between patients (ward, period of hospitalization, and contacts between staff and patients) were compared with the results of ribotyping or the quantitative antibiogram typing method, it appeared that both methods were able to discriminate epidemiological clusters, with only a few discrepancies. In conclusion, quantitative antibiogram typing, although not necessarily based on genomic markers, is a simple method which enables a reliable workup of methicillin-resistant S. aureus epidemic when sophisticated molecular typing methods are not available.

Anti-Bacterial Agents↗
PubMed1994

Ribotyping for differentiating Flavobacterium meningosepticum isolates from clinical and environmental sources.

On the basis of DNA-DNA hybridization data, two main genomic relatedness groups (I and II) have been reported for a geographically varied collection of 52 strains of Flavobacterium meningosepticum. Herein, we have shown that genomic group II can be further divided into four subgroups (II:1 to II:4). To examine the taxonomic relevance of the ribosomal patterns of the 52 F. meningosepticum strains, the patterns were compared with existing DNA-DNA hybridization data with restriction enzymes PstI and HindIII. Ribotyping of the 52 F. meningosepticum strains showed banding patterns that could identify them correctly to one of the five genomic groups or subgroups. To assess the value of ribotyping for the interpretation of epidemiological data, the discriminatory power of the method was investigated for the 52 F. meningosepticum strains. With one to four restriction enzymes (PstI, HindIII, ClaI, EcoRI), a discriminatory index of 0.95 to 0.97 was found. The value of ribotyping in an epidemiological setting was assessed for three clinical isolates of F. meningosepticum from an outbreak of meningitis and bacteremia in the neonatal intensive care unit, Rigshospitalet, Copenhagen, Denmark. The three clinical isolates were shown to belong to the same ribotype, characteristic of genomic subgroup II:1. This ribotyping method will prove to be a useful tool for epidemiological studies concerning F. meningosepticum in the future.

Bacteremia↗
PubMed1995

Molecular epidemiology of Corynebacterium diphtheriae from northwestern Russia and surrounding countries studied by using ribotyping and pulsed-field gel electrophoresis.

A selection of 100 Corynebacterium diphtheriae isolates from asymptomatic carriers and clinical cases from five regions in northwestern Russia were examined. Six additional isolates from patients in Finland and Estonia with epidemiological links to Russia were also examined. All isolates were characterized by biotyping, toxigenicity testing, ribotyping, and pulsed-field gel electrophoresis (PFGE). Hybridization of genomic DNA digested with BstEII revealed five ribotype patterns among the biotype gravis isolates (G1 through G5) and two patterns among the biotype mitis isolates (M1 and M2). PFGE using SfiI was not able to distinguish between ribotypes G1, G2, and G4. The predominant ribotype pattern, G1, found in cases of disease in all the areas studied, appears to be disseminating, in view of the isolates received from imported cases in Finland and Estonia. Among the 106 isolates examined, 68 produced pattern G1 and 24 produced pattern M1. Most of the M1 isolates were from the Leningrad Oblast region. Distinct ribotypes such as G2, G3, G4, G5, and M2 could represent endemic disease.

Bacterial Typing Techniques↗
PubMed1996

Comprehensive ribotyping scheme for heat-stable serotypes of Campylobacter jejuni.

Strains from diverse sources belonging to all 47 heat-stable Penner serotypes of Campylobacter jejuni were examined for polymorphism around the 16S rRNA genes. Penner serotype reference strains and a group of nonserotypeable isolates were included in the study. Complete typeability was obtained; 30 distinct PstI and 42 HaeIII polymorphisms were found. Three bands were detected in almost all strains with these enzymes, confirming that three copies of the 16S rRNA gene are typical for C.jejuni. By combination of the two enzyme polymorphisms, 77 16S ribotypes were defined among the 261 strains analyzed. With two exceptions, no specific association was observed between these ribotypes and heat-stable serotypes. Nine serotypes were homogeneous with respect to the 16S ribotype. Most nonserotypeable strains belonged to ribotypes defined elsewhere in the study. The 16S ribotypes of C.jejuni described here were not found in strains of Campylobacter coli, and vice versa.

Animals↗
PubMed1999

Molecular characterization of the genera Proteus, Morganella, and Providencia by ribotyping.

The so-called Proteus-Providencia group is constituted at present by three genera and 10 species. Several of the recognized species are common opportunistic pathogens for humans and animals. Different methods based on the study of phenotypic characters have been used in the past with variable levels of efficiency for typing some species for epidemiological purposes. We have determined the rRNA gene restriction patterns (ribotypes) for the type strains of the 10 different species of the genera Proteus, Morganella, and Providencia. Visual inspection of EcoRV- and HincII-digested DNA from the type strains showed remarkably different patterns for both enzymes, but EcoRV provided better differentiation. Both endonucleases were retained to study a large number of wild and collection strains belonging to the different species. Clinical isolates of Proteus mirabilis, Proteus penneri, Morganella morganii, and Providencia heimbachae showed patterns identical or very similar to those of the respective type strains, so that groups of related patterns (ribogroups) were found to correspond to the diverse species. On the contrary, distinct ribogroups were detected within Providencia alcalifaciens (two ribogroups with both enzymes), Providencia rettgeri (four ribogroups with EcoRV and five with HincII), Providencia stuartii (two ribogroups with EcoRV), Providencia rustigianii (two ribogroups with HincII), and Proteus vulgaris (two ribogroups with both enzymes). The pattern shown by the ancient P. vulgaris type strain NCTC 4175 differed considerably from both P. vulgaris ribogroups as well as from the newly proposed type strain ATCC 29905 and from any other strain in this study, thus confirming its atypical nature. Minor differences were frequently observed among patterns of strains belonging to the same ribogroup. These differences were assumed to define ribotypes within each ribogroup. No correlation was observed between ribogroups or ribotypes and biogroups of P. vulgaris, P. alcalifaciens, P. stuartii, and P. rettgeri. Since, not only different species showed different rRNA gene restriction patterns, but also different ribogroups and ribotypes have been found in the majority of the species, ribotyping would be a sensitive method for molecular characterization of clinical isolates belonging to the genera Proteus, Morganella, and Providencia.

Bacterial Typing Techniques↗
PubMed1997

Isolation of a new variant of Vibrio cholerae O1: V. cholerae O1 ribotype B27 toxinogenotype TB31 during the last cholera epidemic in Senegal.

A total of 205 Vibrio cholerae O1 isolates from recent cholera epidemic in Senegal were analyzed by conventional methods, polymerase chain reaction (PCR) for genes encoding cholera toxin (ctx A), zonula occludens toxin (zot) and accessory cholera enterotoxin (ace), ribotyping and toxinogenotyping. Ribotyping after Bg1 I digestion of total DNA revealed that ribotype B5a, the predominant ribotype of the seventh pandemic in Africa and Asia, was not isolated. A new ribotype designated B27 in our database is predominant and was associated with a new toxinogenotype designated TB31.

Cholera↗
PubMed2006

[A case of multiple recurrence of Clostridium difficile-associated diarrhea--analysis of isolates from the patient using PCR ribotyping].

Recurrence of Clostridium difficile-associated diarrhea (CDAD) is a serious and still unsolved problem. Little is known about the precise mechanism of the recurrence with CDAD. To elucidate the issue, we analyzed C. difficile strains obtained from the patient with multiple recurrence of CDAD. A 72-year-old female received rectoidectomy was developed CDAD after administration of cefmetazole sodium for 5 days. She was recovered from illness by the administration 2g of vancomycin hydrochloride for 2 weeks. However she was recurred CDAD twice within 2 months thereafter. Six C. difficile isolates recovered from the stool specimens obtained during each episode were typed by polymerase chain reaction amplifying of rRNA intergenic spacer regions 8 PCR ribotyping was carried out. The PCR ribotype of the first episode was identical (ribotype smz) to that of the second episode, and the PCR ribotype of the third were distinct (ribotype hr) from those on previous two episodes. These findings indicate that the second episode was caused by the reactivation of continuously infected C. difficile strain (relapse) and the third episode was by reinfection of the new

Aged↗
PubMed1994

Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 from Quebec by restriction endonuclease fingerprinting and ribotyping.

This study was undertaken to assess the discriminatory value of restriction endonuclease fingerprinting (REF) analysis and ribotyping of 21 Serpulina hyodysenteriae isolates of serotypes 8 and 9. For REF analysis, DNAs were digested with the BglII restriction enzyme and the resultant fragments were separated by polyacrylamide gel electrophoresis. For ribotyping, hybridization of BglII genomic fragments with a probe of rrnB operon using an Escherichia coli rDNA probe was performed on all isolates. Although many isolates shared a common pattern by BglII REF and BglII ribotyping analysis, differences among some S. hyodysenteriae isolates were observed. REF and ribotyping using BglII restriction enzyme, were not specific for serotypes. The predominance of an REF and a ribotype pattern among S. hyodysenteriae isolates from Quebec suggested that epidemiologically important S. hyodysenteriae types occur in different swine herds.

Bacterial Typing Techniques↗
PubMed2004

Identification and genetic characterisation of orthopaedic Staphylococcus isolates collected in Italy by automated EcoRI ribotyping.

The aim of this study was to evaluate the possibility to use automated EcoRI ribotyping to address, during the same analysis, both identification and genetic characterisation of 38 Staphylococcus aureus and 64 coagulase-negative staphylococci collected from surgical injuries. The ribotyping identification results confirmed those obtained using the API Staph system for 96% of the isolates. All strains were successfully genotyped and the ribotyping discriminatory power, calculated using the Simpson's index of discrimination, was very high for both groups of staphylococci tested. The same, as well as different biotypes, were identified among isolates with the identical ribotyping profile.

Bacterial Typing Techniques↗
PubMed2005

Ribotyping of Streptococcus uberis from a dairy's environment, bovine feces and milk.

Streptococcus uberis is a major cause of bovine mastitis and infections commonly result from environmental exposure to the pathogen. To identify specific sources of mastitis-causing S. uberis strains, samples were collected monthly from the environment and feces of dry cows in a grazing herd. Environmental and fecal strains of S. uberis were compared to those found in milk. S. uberis was detected in 63% of 94 environmental samples, including water, soil, plant matter, bedding material, flies, and hay, in 23% of 107 fecal samples, and in 4% of 787 milk samples. Automated PvuII ribotyping revealed 48 ribotypes among 266 isolates. Per sample, up to five ribotypes were detected. The distribution of ribotypes did not differ significantly among environmental, fecal and milk samples. Specific environmental sources or strains of udder-pathogenic S. uberis were not identified. Fecal shedding was not persistent and did not differ between dry-off and calving. The proportion of fecal samples containing S. uberis was highest during the summer grazing season. S. uberis was common in farm soil (31 of 35 samples) but not in non-farm soil (0 of 11 samples). We hypothesize that fecal shedding of S. uberis may play a role in maintenance of S. uberis populations in the dairy ecosystem.

Animals↗
PubMed2002

Characterization of Listeria monocytogenes isolates from the meat, poultry and seafood industries by automated ribotyping.

A total of 564 Listeria monocytogenes isolates were characterized by automated ribotyping. The samples were taken from equipment, personnel and the environment after cleaning procedures and during food processing, as well as from raw materials and products from six meat, two poultry and five seafood processing plants located in the Faroe Islands, Finland, Iceland, Norway and Sweden. Altogether, 25 different ribotypes (RTs) were generated. Two RTs occurred in the samples from all three food sectors--meat, poultry and seafood. Four RTs occurred in meat and poultry plant samples and other four RTs occurred in meat and seafood plant samples. Five RTs occurred only in meat plant samples, five only in poultry plant samples and five only in seafood plant samples. Eight of the thirteen plants had their own in-house L. monocytogenes ribotype. There was geographical differences between the RTs, but no correlation between RTs and food sectors was detected. The discrimination power of automated ribotyping was satisfactory to trace the contamination sources in the food processing plants clearly indicating the sites at which improved cleaning procedures were necessary. In addition, it was possible to screen a large number of isolates with two instruments located at different institutes and to make a reliable combination of the results.

Animals↗
PubMed2003

Real-time application of automated ribotyping and DNA macrorestriction analysis in the setting of a listeriosis outbreak.

A cluster of three cases of listeriosis cases occurred against a background of endemic listeriosis in Western Australia. Human and environmental isolates of Listeria monocytogenes obtained during the outbreak investigation were rapidly subtyped by automated ribotyping using an EcoRI protocol and a RiboPrinter. DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) was used to confirm the relatedness of isolates. Serogroup 1/2 predominated among the food samples and the four clinical isolates from the outbreak cluster were also of this serogroup. All isolates from chicken material were serogroup 1/2 and indistinguishable by ribotype pattern. PFGE subdivided strains of this ribotype into four subtypes. The preliminary analysis had an immediate impact on hypothesis generation, environmental health investigations, environmental specimen collection and initial control measures. Sufficient typing data to guide environmental health and disease control initiatives was generated in less than one week by combining automated ribotyping with PCR-based detection of L. monocytogenes in suspect foodstuffs and an L. monocytogenes DNA probe. There were no further cases of bacteriologically confirmed listeriosis in Western Australia for six months after completion of the investigation.

Automation↗
PubMed2003

Longitudinal study of intrafamilial mutans streptococci ribotypes.

To explore the intrafamilial homology and longitudinal stability of colonization by early acquired mutans streptococci, genomic fingerprinting was performed on 254 strains (192 Streptococcus mutans and 62 Streptococcus sobrinus strains) collected from 16 families (16 mother-child pairs, seven fathers and four siblings). Genomic DNA was digested by the restriction endonuclease HindIII, followed by gel electrophoresis, Southern blotting, and hybridization with a digoxigenin-labeled 16S rDNA probe, and hybrid detection by enhanced chemiluminescence. One to five ribotypes were identified per person, and between two and nine (median five) within each family. Fourteen of the 16 mother-child pairs showed homology for at least one ribotype (range 1-4). Six of the seven father-child pairs had one ribotype in common. Ten of the 13 longitudinally examined children showed persistence of at least one ribotype over a period of up to 16 yr. The results support the notion of intrafamilial transfer of mutans streptococci, and suggest that colonization of early acquired strains persists into young adulthood.

Adolescent↗
PubMed2001

Differentiation of Lactobacillus delbrueckii subspecies by ribotyping and amplified ribosomal DNA restriction analysis (ARDRA).

AIMS: To differentiate the subspecies of Lactobacillus delbrueckii, subsp. delbrueckii, subsp. lactis and subsp. bulgaricus. METHODS AND RESULTS: Amplified ribosomal DNA restriction analysis (ARDRA) and ribotyping were applied to over 30 strains. Both methods analyse the ribosomal genes which carry useful information about the evolutionary and taxonomic relationship among bacteria. The methods proved to be reliable and highly reproducible. ARDRA was applied to 16S rDNA, 23S rDNA and the IGS region, thus covering the whole rrn operon with eight restriction enzymes. Only EcoRI differentiated Lact. delbrueckii subsp. bulgaricus from Lact. delbrueckii subsp. delbrueckii/Lact. delbrueckii subsp. lactis, which confirmed the finding of other authors. Ribotyping with different enzymes under precisely optimized conditions revealed a high level of strain polymorphism. Only ribotyping with EcoRI allowed differentiation of the three subspecies on the basis of typical hybridization patterns. CONCLUSION: The successful differentiation of the three subspecies of Lact. delbrueckii by EcoRI ribotyping offers a new possibility for precise identification and differentiation of strains and new isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for differentiation of Lact. delbrueckii subspecies.

Bacterial Typing Techniques↗
PubMed2001

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy.

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.

Animals↗
PubMed2006

A comparison of Listeria monocytogenes serovar 4b isolates of clinical and food origin in Austria by automated ribotyping and pulsed-field gel electrophoresis.

In this study, two typing methods, automated ribotyping and pulsed-field gel electrophoresis (PFGE), were evaluated for the subtyping of Listeria monocytogenes serotype 4b. The strains originated from patients and food samples collected in Austria during 2001-2005 and from Europe and North America in the World Health Organization collaborative study on the subtyping of this species. The largest group of Austrian clinical isolates was of the same PFGE subtype as those isolated from foodborne outbreaks in Switzerland and in the United States. Another subtype of clinical isolates from Austria was indistinguishable to that obtained from isolates responsible for a foodborne outbreak in the United States in 1985. Although the discriminatory power of PFGE was higher than that of automated ribotyping, some PFGE types were differentiated by ribotyping. Thus, combining data obtained by both automated ribotyping and PFGE increases the strain discrimination. Still, many of the Austrian strains remain indistinguishable from strains of foodborne outbreaks in other countries although there is no known epidemiological relation. This complies to previous studies which show the highly clonal nature of L. monocytogenes 4b strains which are responsible for both large outbreaks and sporadic cases.

Animals↗