Ribosomal RNA Gene Restriction Patterns Research Articles

Fingerprinting of Flavobacterium psychrophilum isolates by ribotyping and plasmid profiling.

Flavobacterium psychrophilum is the agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. Ribosomal RNA gene restriction patterns (ribotypes) and plasmid profiles were determined on a collection of 85 strains isolated from different countries and fish species. Several ribotypes were obtained by using the restriction endonucleases Hinc II and Pvu II. Computer analysis of the ribotypes revealed that some of them were clearly associated with the fish species from which the strains were isolated, whereas no correlation with the geographical origin was found. Most of the strains harboured at least one plasmid and several different plasmid profiles were observed, even among strains sharing the same ribotype. These methods, used alone or in combination with other typing techniques, can be considered powerful tools for the epidemiological tracing of F. psychrophilum infections.

Fingerprinting of Salmonella enterica subsp. enterica serovar Enteritidis by ribotyping.

OBJECTIVE: To carry out an epidemiologic evaluation of Salmonella enterica subsp. enterica serovar Enteritidis outbreaks in households and small communities by means of rRNA gene restriction pattern analysis (ribotyping). METHODS: One hundred Enteritidis isolates dating from 1989 to 1994 which could be allocated epidemiologically to different sources or to small community outbreaks were investigated with ribotyping, a fingerprinting method in which bacterial DNA is hybridized with the biotin-labeled plasmid pKK 3535 containing a ribosomal RNA operon of Escherichia coli to determine the ribosomal RNA gene restriction patterns. RESULTS: Four different ribotyping patterns were found with the restriction endonuclease Smal and nine with Sphl. Ribotypes of isolates which could be allocated epidemiologically to a common source usually corresponded. Almost 60% of the Enteritidis infections had the ribotyping pattern Sphl-A. In contrast, this pattern was not found in any of the five Enteritidis strains isolated in 1989. The suspicion that Enteritidis phage type 4 infections are caused by consumption of insufficiently heated eggs is supported by the fact that the ribotyping pattern Sph1-A was found in isolates from eggs and from human specimens. CONCLUSIONS: As patterns Sphl-A and Smal-J appeared in 58% and 75% of the isolates, respectively, ribotyping cannot be used for the differentiation between various outbreaks with these two patterns. In cases where the Enteritidis strains showed less frequent patterns, ribotyping seems to be a practical tool for the identification of infection chains. In addition newly appearing ribotyping patterns can give information about the epidemiologic development of Enteritidis infection.

Outbreak of typhoid fever in a non-endemic area: comparison of three molecular typing methods

An outbreak of typhoid fever in Zurich, Switzerland, which involved seven customers and three employees of a city mall restaurant, was investigated by comparing three molecular typing methods: pulsed-field gel electrophoresis (PFGE), ribosomal RNA gene restriction patterns (ribotyping), and random amplified polymorphic DNA (RAPD). Both PFGE and ribotyping identified two molecular patterns among the outbreak-related isolates which differed in one band: these isolates were considered clonally related and differed clearly from other unrelated S. typhi strains. RAPD could not distinguish among outbreak isolates and control strains.

Recognition of mutans streptococci by monoclonal antibodies.

The mutans group of streptococci were previously identified either biochemically or serologically using antisera(3). Recently they have been examined serologically with monoclonal antibodies(mAbs) (2,4), analyses by SDS-PAGE patterns, DNA hybridisation and gene restriction enzyme patterns. In this report, serological types and ribosomal RNA gene restriction patterns(ribotyping) of the mutans group were conducted to demonstrate that S. mutans show more heterogeneity than reported previously.KeywordsClinical IsolateDental CariesStreptococcus MutansMutans GroupPolysaccharide AntigenThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Differentiation of Rhodococcus species by ribotyping.

The ribosomal RNA gene restriction patterns (ribotyping) was investigated with respect to the characterization of Rhodococcus species and R. rhodochrous strains. Chromosomal DNA was prepared, digested with BamH I, blotted, and hybridized with acetylaminofluorene-labelled 16 + 23S rRNA from E. coli. The type strains of seven Rhodococcus species studied gave different hybridization patterns in each case. All strains tested were clearly distinguishable by ribotyping. Patterns contained two to eight bands between 1.4 kb and 11.8 kb and demonstrated the high genetic divergence of genus Rhodococcus. Investigation of nine R. rhodochrous strains resulted in patterns with seven or eight bands. One fragment, 4.4 kb in size, was common to all R. rhodochrous strains and appears to be characteristic of R. rhodochrous. Ribotyping was evaluated as a tool for distinguishing between Rhodococcus species.

Characterization of Bacillus thuringiensis and related bacteria by ribosomal RNA gene restriction fragment length polymorphisms

Ribosomal RNA gene restriction patterns have been determined for 43 strains of Bacillus thuringiensis representing 10 serovars and eight reference strains of B. anthracis, B. cereus and B. mycoides. Strains within a B. thuringiensis serovar produced highly related or identical ribotype patterns: in particular, 12 strains of serovar israelensis, five strains of serovar kurstaki, two strains of serovar galleriae and three strains of serovar aizawa produced ribotype patterns consistent with serotype designations. Moreover, variety tenebrionis (serotype 8a8b), a coleopteran pathogen, could be distinguished from the more common lepidopteran pathogens of this serotype (serovar morrisoni) by ribotyping. The correlation of ribotype patterns with serotype suggests a clonal population structure for B. thuringiensis.

Ribotyping shows intrafamilial similarity in Actinobacillus actinomycetemcomitans isolates.

This study reports ribosomal RNA gene restriction patterns of 54 Actinobacillus actinomycetemcomitans isolates obtained from 9 families (12 children and 11 parents). The isolates represented serotypes a, b, c and d. The chromosomal DNA extracted from A. actinomycetemcomitans isolates was digested with the restriction endonucleases EcoRI, BamHI, HindIII, ClaI and Bg/I. The DNA fragments were hybridized to the rrnB ribosomal RNA operon of the Escherichia coli chromosome. In 5 families, isolates belonging to the same family (mother and/or father and the children) had identical hybridization patterns when analyzed with all 5 enzymes. In 3 families, each family member harbored only one ribotype of A. actinomycetemcomitans, but at least one member harbored isolates that were of a different ribotype than the other members of the family. In one family, the mother harbored 2 ribotypes (and serotypes), one common with the daughter and one different. In conclusion, the study confirms the previous results that A. actinomycetemcomitans is transmitted intrafamilially.

Molecular typing of thermotolerant species of Campylobacter with ribosomal RNA gene patterns

Ribosomal RNA gene restriction patterns (ribopatterns) of 72 strains representing Campylobacter jejuni (subspecies jejuni and doylei), C. coli, C. upsaliensis and C. lari including the urealytic (UPTC) biovar were determined using four common restriction endonucleases ( HaeIII, HindIII, PstI and PvuII). The relative effectiveness of these enzymes for general molecular typing of the thermotolerant campylobacters was assessed. Ribotypes ( HaeIII) were defined on the basis of computer-assisted numerical analysis. For C. jejuni, C. coli and C. lari, the HaeIII ribopatterns provided a high level of typability and discrimination, with patterns that were reproducible and easy to code for numerical analysis. There was evidence of diversity within three of the HaeIII types, and PstI ribopatterns proved the most reliable for detecting such differences. C. upsaliensis also could be ribotyped with HaeIII, but HindIII, PvuII and PstI were less satisfactory for this species. For such campylobacters, the HindIII ribopatterns generally were complex and difficult to compare, and the PvuII profiles provided the least discrimination. We conclude that the choice of restriction endonuclease is of critical importance when applying ribotyping to different species of Campylobacter. HaeIII ribopatterns were the most effective means of typing strains of different thermotolerant species of campylobacters and, when combined with PstI ribopatterns, offered a highly discriminatory basis for molecular typing.

Biotype and molecular fingerprints of metronidazole-resistant strains of Helicobacter pylori from antral gastric mucosa.

Biotypes, ribosomal RNA gene restriction patterns (ribopatterns), whole-cell protein patterns and plasmid profiles of paired Helicobacter pylori isolates from 17 patients were examined. Each pair comprised a pre- and a post-treatment isolate; nine of the 17 post-treatment isolates were obtained after treatment with tripotassium dicitrate bismuthate (De-Nol) and metronidazole. All strains of H. pylori had identical biotypes, but exhibited diversity between pairs in their molecular fingerprints. Each of the 17 strain pairs had unique ribopatterns; the pre- and post-treatment isolates in most pairs (16 of 17) were similar or identical, irrespective of metronidazole susceptibility. DNA subtype variants were detected in three patient sets. Although nine post-treatment isolates had acquired resistance to metronidazole, most (six of nine) resembled the pre-treatment isolates in their ribopattern, protein and plasmid profiles. No significant correlation was observed between metronidazole resistance and plasmid content in these H. pylori isolates. Emergence of post-treatment metronidazole-resistant isolates of H. pylori was associated only rarely with colonisation by a novel strain or acquisition of a plasmid and, in most patients, probably resulted from spontaneous emergence of resistance in the original infecting strain.

Use of ribosomal RNA gene restriction patterns to investigate two outbreaks of Campylobacter enteritis in Melbourne, Australia.

The analysis of ribosomal RNA (rRNA) gene patterns (ribotyping) has been used to differentiate strains within bacterial species. We used this method to investigate two outbreaks of campylobacter enteritis that occurred recently in Melbourne, Australia. The first outbreak involved seven patients although isolates from only five patients were available for typing. The second outbreak consisted of three patients infected with human immunodeficiency virus (HIV) on the same ward of a hospital. Analysis of the rRNA gene patterns revealed identical patterns for the isolates from five patients in the first outbreak, suggesting that these isolates were from the same source. However, ribotyping of the four isolates from the second outbreak showed three distinct ribotypes indicative of contact with unrelated sources. This study demonstrated that ribotyping is a useful, reliable and convenient typing scheme for epidemiological purposes.

Genetic variation among Malaysian isolates of Salmonella typhi as detected by ribosomal RNA gene restriction patterns.

Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.

Characterization ofNeisseria meningitidisisolates by ribosomal RNA gene restriction patterns and restriction endonuclease digestion of chromosomal DNA

The use of ribosomal RNA (rRNA) gene restriction patterns to study the molecular epidemiology of Neisseria meningitidis was investigated. Ninety-four isolates of Neisseria meningitidis were characterized by their rRNA gene restriction patterns with 16 + 23 S rRNA from Escherichia coli as a probe. Thirteen rRNA gene restriction patterns were recognized; each of these patterns represented between 1 and 30 isolates. Isolated with the outbreak-associated phenotype B15P1.16 (sulphonamide resistant) all gave a single rRNA gene restriction pattern but this pattern also contained isolates with other phenotypes. Further discrimination between isolates was achieved by comparison of banding patterns resulting from restriction endonuclease digestion of chromosomal DNA with Bgl II. This gave a banding pattern consisting of about ten bands which was simple to interpret. Using this technique 94 isolates were classified in 54 patterns containing between 1 and 14 isolates. Restriction endonuclease analysis with Bgl II characterized outbreak-associated isolates with the phenotype B15P1.16 and enabled strains not typable by conventional methods to be identified as probable outbreak-associated isolates. The techniques should prove useful for epidemiological studies.

Typing of methicillin resistant and susceptibleStaphylococcus aureus strains by ribosomal RNA gene restriction patterns using a biotinylated probe

Nine methicillin-sensitive (MSSA) and 37 methicillin-resistant (MRSA) Staphylococcus aureus isolates of various phage types and resistotypes from seven countries were investigated. Chromosomal DNA was restricted with HindIII or EcoRI, Southern blotted and hybridised with a cDNA probe to 16S+23S rRNA derived from MRSA NCTC 10442. Resulting rDNA profiles could be differentiated on the basis of 22 patterns which were unaffected by changes in plasmid, transposon, enterotoxin A or phage content. Percentage similarity values were calculated using the Dice coefficient and UPGA clustering. Australian and epidemic (EMRSA-1) isolates from the UK showed a high degree of similarity, but the pattern was not unique and was also found in MSSA and other MRSA, e.g. NCTC 10442. An MSSA of phage group II was the most distinct isolate. The method shows potential as an additional tool in a complex typing system, types non-phage typable strains and may provide clues to the clonal evolution of MRSA and MSSA.

RRNA gene restriction patterns and plasmid analysis as a tool for typing Salmonella enteritidis

We have evaluated the usefulness of ribosomal RNA gene restriction patterns and of plasmid analysis for the typing of Salmonella serotype enteritidis isolates. Strains were isolated from single patients excreting the organism for up to about 4 months, as well as from clusters of patients who had probably been exposed to the same source of infection. The sensitivity of ribosomal DNA (rDNA) analysis was dependent on the restriction enzyme used; of 14 different enzymes, only SmaI and SphI showed different rDNA patterns. Differences were detected between clusters, but all strains belonging to a given cluster showed identical rDNA patterns. In one single strain (isolated at day 66), we observed an SphI pattern that was not identical to that of the original isolate from the same patient. Strains were also typed by plasmid analysis. All strains harboured a plasmid of approximately 55 kb with identical restriction patterns. Smaller plasmids of 4.5 and 4.3 kb were isolated from all the strains belonging to one and two clusters, respectively. We have demonstrated that the analysis of rRNA genes is a sensitive and useful method for epidemiological typing of Salmonella serotype enteritidis, which can be complemented by analysis of plasmids if such structures are present.

Recognition ofFusobacterium nucleatumsubgroups Fn-1, Fn-2 and Fn-3 by ribosomal RNA gene restriction patterns

DNA from representative strains of Fusobacterium nucleatum subgroups Fn-1, Fn-2 and Fn-3 was digested with restriction enzymes EcoRI and TaqI and the electrophoretically separated fragmetns hybridized with a 32P-16S rRNA gene probe from E. coli. The rRNA gene restriction patterns from DNA digested with either enzyme allowed the clustering of strains into the three subgroups. However, TaqI digested DNA yielded a wider distribution of taxonomically useful bands (ca 0.65 ± 14.3 kbp) and the pattern produced was characteristic of each subgroup. The present method is a simple and reliable means of identifying the three subgroups of F. nucleatum and provides a useful method for further studies of the heterogeneity of F. nucleatum.

Recognition of Fusobacterium nucleatum subgroups Fn-1, Fn-2 and Fn-3 by ribosomal RNA gene restriction patterns.

DNA from representative strains of Fusobacterium nucleatum subgroups Fn-1, Fn-2 and Fn-3 was digested with restriction enzymes EcoRI and TaqI and the electrophoretically separated fragments hybridized with a 32P-16S rRNA gene probe from E. coli. The rRNA gene restriction patterns from DNA digested with either enzyme allowed the clustering of strains into the three subgroups. However, TaqI digested DNA yielded a wider distribution of taxonomically useful bands (ca 0.65 +/- 14.3 kbp) and the pattern produced was characteristic of each subgroup. The present method is a simple and reliable means of identifying the three subgroups of F. nucleatum and provides a useful method for further studies of the heterogeneity of F. nucleatum.

Ribosomal RNA Gene Restriction Patterns Provide Increased Sensitivity for Typing Salmonella typhi Strains

To date, epidemiologic associations among strains of Salmonella typhi are based exclusively on phage typing, which may be of limited value if a common phage type is involved. Analysis of ribosomal RNA gene restriction patterns allows separation of most independently isolated strains of identical phage types. The sensitivity of the method is dependent on the restriction enzymes used to digest chromosomal DNA. It was highest for PstI, which separated 16 of 20 strains that belonged to 8 phage types including 3 untypable strains. Three strains differed in their phage types but had identical ribosomal RNA gene restriction patterns. Also, two pairs of strains indistinguishable by phage typing exhibited identical patterns; however, two of these strains were expected to be identical because they were isolated from two patients who were likely exposed to the same source. Ribosomal RNA gene restriction patterns appear to be stable. Thus, the method may complement phage typing and aid in further differentiation of strains.

Differentiation of Staphylococcal Species and Strains by Ribosomal RNA Gene Restriction Patterns

Staphylococcal DNA was digested with endonucleases and probed with labelled ribosomal RNA (rRNA) from Escherichia coli. Reproducible restriction patterns containing between seven and 22 bands were obtained for seven different species of staphylococci. These profiles were species-specific with different strains of a particular species sharing an identical or similar restriction pattern. The results reported here indicate that rRNA gene restriction pattern analyses have an application in the taxonomy of staphylococci.

Characterization of Staphylococcus Species by Ribosomal RNA Gene Restriction Patterns

The rRNA gene restriction pattern sof 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindIII and EcoRI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilis inserted in a plasmid vector, pBR322. Fourty-four distinct HindIII patterns and 44 distinct EcoRI patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed with some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcus xylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g.S.aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.