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PubMed2005

PCR ribotyping of Clostridium difficile isolates originating from human and animal sources.

Molecular typing of Clostridium difficile isolates from animals and humans may be useful for evaluation of the possibility for interspecies transmission. The objective of this study was to evaluate C. difficile isolates from domestic animals and humans using PCR ribotyping. Isolates were also tested using PCR for the presence of genes encoding toxins A and B. One hundred and thirty-three isolates of C. difficile from dogs (n = 92), horses (n = 21) and humans (n = 20), plus one each from a cat and a calf, were evaluated. Overall, 23 ribotypes were identified. Of these, nine were identified from dogs, 12 from horses, seven from humans and one each from the cat and calf. In dogs, humans and horses, one or two different ribotypes predominated. Overall, 25 % of isolates from humans were indistinguishable from isolates from one or more animal species. Genes encoding C. difficile toxins A and B were detected in all human, equine and bovine isolates, and in 69 % of canine isolates. While different ribotypes appear to predominate in different mammalian species, several indistinguishable strains may be found in multiple species. This suggests that there is a potential for interspecies transmission of C. difficile and epidemiological studies are warranted.

Animals↗
PubMed2006

Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences.

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.

Bifidobacterium↗
PubMed2004

Identification method based on PCR combined with automated ribotyping for tracking probiotic Lactobacillus strains colonizing the human gut and vagina.

AIMS: A molecular methodology based on PCR-associated automated ribotyping was developed to specifically detect the Lactobacillus strains of two probiotic products (an orally administered lyophilized preparation and vaginal tablets) in human faeces and vaginal swabs. METHODS AND RESULTS: The 16S-23S rDNA sequences and the ribotype profiles of the probiotic lactobacilli were characterized and new species-specific primer sets were designed. The identification of faecal and vaginal lactobacilli isolated from subjects administered with the probiotic products was performed by using PCR with species-specific primers followed by strain-specific automated ribotyping. CONCLUSIONS: The PCR-ribotyping identification allowed to study the colonization patterns of the probiotic lactobacilli in the human gut and vagina evidencing the strains with the best survival capability. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed molecular method represents a powerful tool of strain-specific identification, useful for differentiating exogenous from indigenous strains in any microbial ecosystem and for rationally choosing probiotic bacteria with the best chance of survival in the host.

Colony Count, Microbial↗
PubMed2005

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed.

AIMS: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. METHODS AND RESULTS: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. CONCLUSIONS: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.

Animals↗
PubMed2006

Genotyping of Campylobacter spp. from retail meats by pulsed-field gel electrophoresis and ribotyping.

AIMS: To determine the genetic relatedness of Campylobacter spp. from retail meat products, and compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and automatic ribotyping. METHODS AND RESULTS: A total of 378 Campylobacter isolates recovered from 159 raw meats (130 chicken, 25 turkey, three pork and one beef) sampled from 50 retail grocery stores of four supermarket chains in the Maryland suburban area from August 1999 to July 2000 were analysed by PFGE with SmaI, 120 isolates of which were also characterized by ribotyping with PstI using RiboPrinter system. A total of 148 unique PFGE patterns were identified, 91 of which were present in multiple Campylobacter isolates and 24 in multiple meat samples. Nineteen Campylobacter clones with identical PFGE patterns recurred frequently (up to nine times) throughout the sampling period. Comparing ribotyping with PFGE, we identified 44 PFGE patterns and 22 RiboGroups among the 120 isolates tested. Multiple PFGE patterns within one RiboGroup were commonly observed, as well as multiple RiboGroups within one PFGE pattern. CONCLUSIONS: Although Campylobacter present in retail meats were genetically diverse, certain clones persisted in poultry meats. PFGE had a greater discriminatory power than ribotyping, and the two methods were complementary in genotyping Campylobacter. SIGNIFICANCE AND IMPACT OF THE STUDY: Genomic DNA fingerprinting of Campylobacter confirmed diverse and recurrent Campylobacter clones in the retail meats, which provides additional data for a better understanding of the epidemiological aspect of Campylobacter infection.

Animals↗
PubMed2006

Characterization of the brewery spoilage bacterium Obesumbacterium proteus by automated ribotyping and development of PCR methods for its biotype 1.

AIMS: To study the ability of automated ribotyping to characterize Obesumbacterium proteus and Hafnia alvei, to design primers and to evaluate standard end-point and real-time PCR for the detection of O. proteus biotype 1 in beer and in brewers's yeast-containing samples. METHODS AND RESULTS: Automated ribotyping was carried out using the standard method with EcoRI and PvuII. The digestions with both enzymes clearly differentiated O. proteus biotypes 1 and 2 and H. alvei. PCR primers were designed according to the 16S rRNA gene sequence of the O. proteus type strain. Two primer sets (Obs137-Obs558 and Obs137-Obs617) detected O. proteus biotype 1 and H. alvei but not O. proteus biotype 2 or other tested beer spoilage bacteria (40 species) in the end-point and real-time PCR, indicating their high specificity. The detection limit for O. proteus was 160-1600 CFU 100 ml(-1) beer in the end-point PCR reactions and < or =160 CFU 100 ml(-1) beer in the real-time PCR reactions. More cells (from 16 to 3200) were needed for detection in the presence of brewer's yeast cells. CONCLUSIONS: Automated ribotyping is a useful tool to characterize and identify O. proteus and H. alvei isolates. The designed primers are suitable for the rapid detection of O. proteus biotype 1 and H. alvei in brewery samples by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.

Beer↗
PubMed2001

Automated ribotyping using different enzymes to improve discrimination of Listeria monocytogenes isolates, with a particular focus on serotype 4b strains.

To develop improved automated subtyping approaches for Listeria monocytogenes, we characterized the discriminatory power of different restriction enzymes for ribotyping. When 15 different restriction enzymes were used for automated ribotyping of 16 selected L. monocytogenes isolates, the restriction enzymes EcoRI, PvuII, and XhoI showed high discriminatory ability (Simpson's index of discrimination > 0.900) and produced complete and reproducible restriction cut patterns. These three enzymes were thus evaluated for their ability to differentiate among isolates representing the two major serotype 4b epidemic clones, those having ribotype reference pattern DUP-1038 (51 isolates) and those having pattern DUP-1042 (20 isolates). Among these isolates, PvuII provided the highest discrimination for a single enzyme (nine different subtypes; index of discrimination = 0.518). A combination of PvuII and XhoI showed the highest discriminatory ability (index of discrimination = 0.590) for these isolates. A group of 44 DUP-1038 isolates and a group of 12 DUP-1042 isolates were identical to each other even when the combined data for all three enzymes were used. We conclude that automated ribotyping using different enzymes allows improved discrimination of L. monocytogenes isolates, including epidemic serotype 4b strains. We furthermore confirm that most of the isolates representing the genotypes linked to the two major epidemic L. monocytogenes clonal groups form two genetically homogeneous groups.

Bacterial Typing Techniques↗
PubMed2003

Characterization of members of the Legionellaceae family by automated ribotyping.

In order to implement a new and reliable method for characterizing different species of Legionella, a genetic fingerprinting study with an automated ribotyping system (RiboPrinter) was completed with members of this genus which were deposited at the American Type Culture Collection. The RiboPrinter examined the different patterns of EcoRI digestion fragments from the rRNA operons of 110 strains, representing 48 of the 49 described Legionella species as well as 70 serogroups of those species. Distinctive and consistent patterns were obtained for the type strains of the 48 species investigated. Legionella pneumophila subsp. fraseri and L. pneumophila subsp. pascullei each generated a specific pattern, whereas L. pneumophila subsp. pneumophila produced six different fingerprint patterns. No correlation seemed to exist between the ribotypes obtained and the 15 serotypes of L. pneumophila. For the other species, those with two known serogroups presented two distinctive patterns with the RiboPrinter with the exception of L. hackeliae and L. quinlivanii, which yielded only one pattern. We also encountered ribotypes for strains which were not identified to the species level. The ribotypes generated for these strains with the RiboPrinter did not match those generated for known type strains, suggesting the putative description of new serogroups or species. Although the automated system did not have sufficient discriminatory ability to serve as an epidemiological tool in a clinical setting, it appeared to be a powerful tool for general genomic analysis of the Legionella isolates (e.g., determination of new species) and assessment of the interrelationship among Legionella strains through the RiboPrinter database connection.

Automation↗
PubMed2001

Molecular analysis of Klebsiella pneumoniae strains isolated in pediatric wards by ribotyping, pulsed field gel electrophoresis and antimicrobial susceptibilities.

The purpose of this study was to investigate the usefulness of different molecular typing techniques in the surveillance and control of the spread of extended-spectrum-beta-lactamase-(ESBL) producing Klebsiella pneumoniae in the pediatric department of the "Agostino Gemelli" hospital of the Catholic University in Rome, over a period of nine months. The strains were characterized by ribotyping using HindIII as restriction enzyme and pulsed field gel electrophoresis (PFGE) using XbaI as endonuclease. Sixty six K. pneumoniae clinical strains were isolated during this period, the first 32 were isolated in the summer of 1998. Among these first isolates, ribotyping generated 26 different patterns whereas PFGE produced 16 patterns. The remaining 34 strains were isolated during January and April 1999 and all of them were ESBL producers. Ribotyping clustered the strains into 6 patterns whereas PFGE generated only 3 patterns. PCR revealed the presence in 10 isolates of both bla(TEM) and bla(SHV) genes and 24 strains carried only the bla(SHV) gene. In our experience ribotyping revealed a higher power of differentiation with respect to PFGE and was of great help in the surveillance of the infection.

Cross Infection↗
PubMed2002

[Diversity of 16s rDNA ribotypes of the Salmonella typhi strains isolated in Guizhou province].

OBJECTIVE: To analyses the genetic diversity and relationship of Salmonella typhi strains isolated from different years and districts in Guizhou province. METHODS: Ribotyping with 16s rDNA probe was used to describe the diversity of the 209 strains which were isolated in 26 counties of Guizhou province, from 1959 to 1999. The antibiotics resistance was also studied. RESULTS: Twenty-six ribotypes were found in all 209 strains, with two dominant types. The strains isolated from local typhoid epidemics belonged to the unique Ribotypes. The major ribotypes of the resistant strains were RT7 and RT1. CONCLUSION: The Salmonella typhi isolates from Guizhou diverged obviously. The abundant clones and multi-resistance of the strains might serve the major reasons of the high morbidity of typhoid in Guizhou.

Blotting, Southern↗
PubMed1997

Comparative genomic analysis of isolates belonging to the six species of the genus Thermus using pulsed-field gel electrophoresis and ribotyping.

Fifty isolates belonging to the six validly described species of the genus Thermus (T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus, T. brockianus, and T. oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping. With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct. However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring. The estimated genomic size of strains of the Thermus spp. varied between approximately 1.8 and 2.5 Mbp. Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively. In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring.

Bacterial Typing Techniques↗
PubMed1995

Epidemiological evaluation by PCR ribotyping of sporadic and outbreak-associated strains of Salmonella enterica serotype Typhimurium.

Salmonella enterica serotype Typhimurium has a very large diffusion worldwide within human and non-human hosts. The simultaneous circulation in the same geographical areas of many bacterial clones requires the use of reliable, reproducible and highly discriminatory typing techniques for epidemiological studies. Molecular biological methods, such as plasmid profile analysis, restriction endonuclease digestion of plasmid and chromosomal DNA and hybridization-based procedures have proven to be useful tools for strain differentiation. More recently, detection of polymorphisms in the intergenic spacer regions of rRNA genes by polymerase chain reaction (PCR ribotyping) has been successfully applied to characterize bacterial strains. In this study, PCR ribotyping was performed on 45 epidemiologically related and unrelated strains of S. enterica serotype Typhimurium isolated in northern and southern Italy during 1992. Isolates were simultaneously characterized by traditional ribotyping. Results suggest that PCR ribotyping is a rapid, easy-to-perform and reproducible typing method able to determine relatedness among isolates of this serotype.

Bacterial Typing Techniques↗
PubMedDate not supplied

Ribotyping and DNA macrorestriction analysis of isolates of Burkholderia pseudomallei from cases of melioidosis in Malaysia.

Forty-nine isolates of Burkholderia pseudomallei from sporadic cases of melioidosis in Malaysia over the past 18 years were examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of total deoxyribonucleic acid (DNA). Twenty-four patients had septicaemic melioidosis with a mortality of 70%; mortality in the non-septicaemic disease was 16%. Five ribotype patterns were identified, 2 of which accounted for 90% of all isolates. PFGE revealed a number of different strains within these ribotypes, but some pairs of isolates from unrelated cases gave closely similar DNA profiles. These results are in agreement with Australian studies which showed a high prevalence of a few ribotypes of B. pseudomallei which are further divisible by genotyping, in areas where melioidosis is endemic.

Adult↗
PubMed1996

WHO-sponsored international collaborative study to evaluate methods for subtyping Listeria monocytogenes: restriction fragment length polymorphism (RFLP) analysis using ribotyping and Southern hybridization with two probes derived from L. monocytogenes chromosome.

Seven laboratories participated in a WHO-sponsored international collaborative study, to evaluate methods for subtyping Listeria monocytogenes, by performing restriction fragment length polymorph sm (RFLP) analysis-based subtyping of an international study set of 80 strains of L. monocytogenes that included 22 epidemiologically related groups. The RFLP analysis was done by Southern hybridization with one of two types of probes found in multiple copies on the chromosome of L. monocytogenes. Six laboratories performed ribotyping. These laboratories used EcoRI enzyme to restrict the L. monocytogenes DNA and ribosomal RNA or DNA as the probe for Southern hybridizations. The seventh laboratory used Ncil to restrict the DNA, and two probes, one randomly cloned and the other containing repeat sequences cloned from L. monocytogenes DNA. The overall discriminating power of ribotyping, as estimated by calculation of Simpson's index of diversity, ranged from 0.83 to 0.88 for the six laboratories. The discriminating power of the combination of two probes used by Laboratory 7 was 0.91. Ribotyping and the cloned probes used by Laboratory 7 discriminated poorly between serotype 4b strains. Neither method identified three atypical strains (identified by other subtyping methods) included in three apparently epidemiologically related groups. Ribotyping did not discriminate between strains of serotypes 4b and 4b(X) in one epidemiologically related group of strains; one cloned probe used by Laboratory 7 discriminated between these strains. Intra-laboratory reproducibilities for the seven laboratories ranged from 80.0 to 100%. as determined by their abilities to correctly identify 11 pairs of duplicate strains included in the study set. Inter-laboratory reproducibilities were generally very good considering that no attempt was made to standardize protocols used by the participants.

Bacterial Typing Techniques↗
PubMed1999

Comparison between automatic ribotyping and random amplified polymorphic DNA analysis of Bacillus cereus isolates from the dairy industry.

Discrimination by automatic ribotyping and random amplified polymorphic DNA PCR, RAPD, was compared for 40 different B. cereus dairy isolates, 4 different B. mycoides isolates and 6 culture collection strains. RAPD-PCR has previously shown to be useful for tracing contamination routes of B. cereus to milk. Automatic ribotyping using EcoRI and PvuII separated the B. cereus and B. mycoides isolates/strains into 36 different ribotypes. RAPD-typing with primers generated 40 different RAPD-profiles. However, 17 isolates clustered into eight groups, irrespective of the primer and restriction enzyme used, and in all but one case, the isolates with the same pattern were isolated from the same dairy. Automatic ribotyping proved to be a useful, standardized and quick method to discriminate between B. cereus strains, only slightly less discriminatory than RAPD-typing.

Animals↗
PubMed1996

The use of plasmid profile analysis and ribotyping for typing Acinetobacter baumannii isolates.

Plasmid profiles were used to analyse 39 Acinetobacter baumannii isolates from 36 patients at three hospitals. The isolates were prevously classified by biotyping and rDNA fingerprinting. Ribotyping was useful to establish the lineage of isolates and to confirm genospecies identification. Thirty-seven isolates (94.9%) contained plasmids. The variable number of plasmids with different molecular weights in each isolate enabled the identification of 13 profiles without the need for endonuclease digestion. Fifteen A. baumannii biotype 2 isolates of similar ribotype and antibiotype contained identical plasmids over a two-month outbreak at one hospital. Plasmid typing discriminated these isolates from sporadic A. baumannii isolates of close ribotype obtained from different hospitals. A few isolates of different lineage, however, showed similar plasmid profile. Our results suggest that plasmid typing is a practical method to assist infection control of nosocomial A baumannii. A combination of plasmid typing and ribotyping is suggested to confirm genospecies classification and to identify strains against reference band profiles.

Acinetobacter↗
PubMed1999

Evaluation of canine Bordetella bronchiseptica isolates using randomly amplified polymorphic DNA fingerprinting and ribotyping.

Bordetella bronchiseptica is a respiratory tract pathogen in a variety of species. Previous studies suggest little genetic variation among canine B. bronchiseptica isolates. The degree of genetic diversity in 26 canine B. bronchiseptica strains was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprinting and ribotyping. Strains evaluated include historic reference strains (N=3). vaccine strains (N=5) and clinical isolates (N=18). RAPD fingerprinting with the 10-nucleotide primer OPA-4 resulted in four distinct fingerprint patterns. RAPD fingerprinting consistently separated four previously characterized electromorphotype (EMT) 6 strains into two fingerprint types. Ribotyping, using the restriction endonuclease PvuI, resulted in six distinct ribotypes. With the exception of vaccine strains, considerable genetic diversity exists in the canine B. bronchiseptica isolates examined. These findings indicate the genetic variability within canine strains of B. bronchiseptica is greater than appreciated previously. Additionally, OPA-4 RAPD fingerprinting and PvuI ribotyping will be useful tools in epidemiologic studies of canine B. bronchiseptica isolates.

Animals↗
PubMed1998

Typing of nosocomial strains of Serratia marcescens: comparison of pulsed-field gel electrophoresis of macrorestriction fragments with biotyping, esterase typing and ribotyping.

Fifty nosocomial isolates of Serratia marcescens, collected in six Belgian hospitals between 1986 and 1990, were characterized by using pulsed-field gel electrophoresis (PFGE) with XbaI. The results were compared with those previously obtained by three other methods: biotyping, esterase electrophoresis typing and ribotyping with EcoRI and HindIII. Macrorestriction analysis (42 PFGE groups) and esterase typing (42 zymotypes) proved to be the most discriminating, followed by ribotyping (28 ribotypes) and biotyping (10 biochemical profiles). Biotyping would serve as a screen to identify isolates, due to its accessibility. Esterase typing provided a reliable tool to make subdivisions within biotypes because of congruence between biochemical groups and esterase patterns. Additional discrimination was still achieved by ribotyping and PFGE. It is concluded that the combined results of these four markers were useful for distinguishing all epidemic and sporadic isolates.

Bacterial Typing Techniques↗