rRNA Gene Restriction Patterns Research Articles

Characterisations of Probiotic <i>Lactobacillus</i> Strains by Amplified Ribosomal DNA Restriction Analysis (ARDRA)

Twelve probiotic Lactobacillus strains for poultry were characterised by amplified ribosomal DNA restriction analysis (ARDRA) using Sau3AI, TaqI, HaeIII and AluI restriction endonucleases. Species-specific and strain-specific restriction patterns were observed from the bacterial strains. Numerical analysis of composite analysis of ARDRA exhibited D value of 0.8456. Whereas, the caculated D values of ARDRA patterns generated by Sau3AI, TaqI, HaeIII and AluI were 0.8309, 0.8382,0.8088 and 0.8088, repectively. Composite analysis of ARDRA was the most discriminative method when compared to the individual analysis. ARDRA could distinguished L. reuteri C 10 and L. panis C 17 into single strains. The 16S rRNA gene restriction patterns were also able to group L. gallinarum I 16 and I 26 into single strains. Lactobacillus brevis I 12, I 23, I 25, I 211 and I 218 seem to be multiple clones of the same bacterial strain as are L. reuteri C 1 and C 16. ARDRA is a valuable fingerprinting method to discriminate probiotic Lactobacillus strains.

Lactobacillus sakei/ curvatus is the prevailing lactic acid bacterium group in spoiled maatjes herring

A total of 164 lactic acid bacteria (LAB) isolated from spoiled maatjes herring stored in air and under modified atmosphere at 4 or 10 °C were characterised and identified using an rRNA gene restriction pattern (ribotype) database. The isolates were initially grouped according to their HindIII restriction endonuclease profiles and further identified to species level using numerical analysis. Lactobacillus sakei, Lactobacillus curvatus and strains of the L. curvatus spp./ Lactobacillus fuchuensis group were the main species detected. Of all the isolates, six were identified as Lactococcus spp.

PandemicVibrio parahaemolyticusO3:K6 Spread, France

Pandemic<i>Vibrio parahaemolyticus</i>O3:K6 Spread, France

Development and use of fluorescent 16S rRNA-targeted probes for the specific detection of Methylophaga species by in situ hybridization in marine sediments

Methylotrophic bacteria are widespread in nature. They may play an important role in the cycling of carbon and in the metabolism of dimethylsulfide in a marine environment. Bacteria belonging to the genus Methylophaga are a unique group of aerobic, halophilic, non-methane-utilizing methylotrophs. Two 16S rRNA-targeted oligonucleotide probes were developed for the specific detection of Methylophaga species, marine methylobacteria, by fluorescence in situ hybridization. Probe MPH-730 was highly specific for all members of the genus Methylophaga while probe MPHm-994 targeted exclusively M. marina. The application of these probes were demonstrated by the detection of Methylophaga species in enrichment cultures from various marine sediments. All isolates recovered were visualized by using the genus specific probe MPH-730. The results were confirmed by 16S rDNA sequencing which demonstrated that all selected isolates belong to Methylophaga. Five isolates could be detected by the M. marina-specific probe MPHm-994 and were confirmed by rRNA gene restriction pattern (ribotyping). With the development of these specific probes, fluorescence in situ hybridization shows that the genus Methylophaga is widespread in marine samples.

Identification of lactic acid bacteria from spoiled, vacuum-packaged ‘gravad’ rainbow trout using ribotyping

A total of 296 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged ‘gravad’ rainbow trout stored at 3 and 8 °C were characterised and identified using a molecular approach. The isolates were initially grouped according to their HindIII restriction endonuclease profiles and further identified to species level using an rRNA gene restriction pattern (ribotype) identification database. Lactobacillus sakei, L. curvatus and Carnobacterium piscicola were the three main species detected. Only one isolate was identified as C. divergens. Most of the carnobacteria were found in the samples stored at 3 °C. The relative proportion of L. sakei was higher in the samples stored at 8 °C.

Characterization of Different Pathovars and Reisolates of Xanthomonas Campestris by Xanthan Synthesis and rRNA Gene Restriction Patterns

ABSTRACTThe characterization of exopolysaccharide production and rRNA gene restriction patterns of Xanhtomonas campestris were studied. The pathovars of glycines, phaseoli and vesicatoria major patterns correlated with pathogenicity on the host plant and this can be used to characterize strains. The synthesis of xanthan in the strains and the reisolates is different and can not be used for the identification of different pathovars.

Natural endophytic association between Rhizobium etli and maize ( Zea mays L.)

Maize ( Zea mays) and bean ( Phaseolus vulgaris) have been traditionally grown in association for thousands of years in Mesoamerica. From surface sterilized maize roots, we have isolated over 60 Rhizobium strains that correspond to Rhizobium etli bv. phaseoli (the main symbiont of bean) on the basis of 16S rRNA gene restriction patterns, metabolic enzyme electropherotypes, organization of nif genes, and the ability to nodulate beans. The colonization capacity of some of the isolates was tested with an unimproved maize cultivar and with 30 maize land races. Increases in plant dry weight upon R. etli inoculation were recorded with some of the land races, and these increases may be related to plant growth promotion effects. Additionally, from within maize grown in monoculture we have also recovered R. etli isolates recognizable by their 16S rRNA gene types, which lack nif genes and are incapable of nodulating bean. These strains are presumed to correspond to the earlier described non-symbiotic R. etli obtained from bean rhizosphere.

Identification of Strains of Alcaligenes and Agrobacterium by a Polyphasic Approach

The number of stable discriminant biochemical characters is limited in the genera Alcaligenes and Agrobacterium, whose species are consequently difficult to distinguish from one another by conventional tests. Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example, Alcaligenes xylosoxidans was transferred to the genus Achromobacter in 1998. Twenty-five strains of Achromobacter xylosoxidans, three strains of an Agrobacterium sp., five strains of an Alcaligenes sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined. These strains were characterized by conventional tests, including biochemical tests. The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with EcoRI or PstI restriction endonuclease. This polyphasic approach allowed the two subspecies of A. xylosoxidans to be clearly separated. Relationships between five strains and the Ralstonia paucula type strain were demonstrated. Likewise, three strains were found to be related to the Ochrobactrum anthropi type strain. We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying A. xylosoxidans subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing.

Helicobacter pylori intrafamilial infections: change in source of infection of a child from father to mother after eradication therapy.

Biopsy specimens of the antrum and corpus were obtained from four Helicobacter pylori-infected members of a family and from the same boy (son 1) in whom the infection reappeared after simultaneous successful eradication treatment of three family members, excluding the mother. A total of 18 to 60 H. pylori isolates were obtained from each specimen and subjected to rRNA gene restriction pattern analysis. The father's isolates and the initial isolates from son 1 showed the same HindIII type, which was divided into three HaeIII subtypes. Isolates from the mother and a brother (son 2) and posttreatment isolates from son 1 showed a distinct HindIII type (with one minor subtype), which was divided into six HaeIII subtypes. All subtypes of the initial isolates from son 1 were present in the father's isolates, and all subtypes of the posttreatment isolates from son 1 were present in the mother's isolates but not in son 2's. Electron microscopic analysis of the biopsy specimens demonstrated extremely high levels of H. pylori colonization in the father's gastric mucosa. H. pylori adherence with a ruffle formation was also demonstrated. The findings suggest that son 1 was infected initially with the H. pylori strain of the father and son 2 was infected with the H. pylori strain of the mother and that after eradication therapy son 1 was reinfected with the H. pylori strain of the mother, who did not undergo eradication therapy.

Computer identification of Shigella species by rRNA gene restriction patterns

We describe a MluI ribotyping scheme for Shigella which approaches correlation with serotyping. One hundred and seventeen reference strains and previously serotyped clinical isolates representing the 57 Shigella serotypes and biotypes were included in this study. A total of 51 distinct ribotypes were obtained and a database was built with them. The number of bands composing each ribotype varied from 9 to 15. The fragments ranged in size from 1.6 to 18.8 kbp. One hundred and eleven clinical isolates were successfully identified in a double blind study with standard biochemical/serologic methods, by automatic comparison of their ribotypes with our database using the software Taxotron.

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms.

To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate. 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.

Characterisation of lactic acid bacteria from spoiled, vacuum-packaged, cold-smoked rainbow trout using ribotyping

A total of 405 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged, salted, sodium nitrite- or potassium nitrate-treated, cold-smoked rainbow trout stored at 4°C or 8°C were characterised and identified using a molecular method. The isolates were initially classified according to their restriction endonuclease profiles using HindIII and EcoRI restriction endonucleases and further characterised by rRNA gene restriction patterns (ribotypes). Numerical analysis of these ribopatterns was performed together with 19 reference LAB strain patterns in order to identify the isolates to species level. The strains were divided with HindIII and EcoRI ribopatterns into ten and nine clusters at the similarity level of 65% and 50%, respectively. The Leuconostoc-clusters and the Lb. sakei/Lb. curvatus-clusters formed the two main groups. Only one isolate was identified as Lactobacillus plantarum and no Carnobacterium strains were discovered. For both enzymes, the 35 isolates possessing six individual ribotypes and forming five clusters could not be identified further with the reference strains used. The relative proportion of Leuconostoc mesenteroides subsp. mesenteroides was higher in all samples stored at 4°C. Most of the Leuconostoc citreum were found in the samples stored at 8°C, and particularly in the nitrite-treated samples.

Characterization of Methylophaga species by rRNA Gene Restriction Patterns (Ribotyping)

Summary A total of 24 strains of Methylophaga , marine methylotrophic bacteria, were characterized by rRNA gene restriction patterns (ribotypes), after cleavage of total DNA with Eco RI or Hin dIII restriction endonuclease. Manual ribotyping and the RiboPrinter® Microbial Characterization System were used. The two genomic DNA groups could be differentiated each by ribotyping. Our study showed that ribotyping could characterize Methylophaga species and identify unknown isolates. All results were confirmed by DNA/DNA hybridization. Methylophaga sulfidovorans species was confirmed to be a third species by ribotyping and DNA/DNA hybridization.

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.

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.

Analysis of Clinical and Environmental Strains of Nontoxigenic Vibrio cholerae for Susceptibility to CTXΦ: Molecular Basis for Origination of New Strains with Epidemic Potential

Toxigenic Vibrio cholerae strains are lysogens of CTXPhi, a filamentous phage which encodes cholera toxin. The receptor for CTXPhi for invading V. cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of V. cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTXPhi attachment sequence attRS, as well as for susceptibility of the strains to CTXPhi, to investigate the molecular basis for the emergence of new clones of toxigenic V. cholerae. DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island, toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS. An analysis of the strains for susceptibility to CTXPhi, using a genetically marked derivative of the phage CTX-KmPhi, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice. The phage genome integrated into the chromosome of infected V. cholerae O1 cells forming stable lysogens. Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V. cholerae. To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V. cholerae strains by CTXPhi. The results of this study further indicated that strains belonging to the O1 serogroup of V. cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXPhi, leading to the origination of potential new epidemic clones.

Computer identification of Escherichia coli rRNA gene restriction patterns

A total of 191 strains of Escherichia coli comprising 164 serovar reference strains and 28 clinical strains were characterized by rRNA gene restriction patterns (ribotypes) generated after cleavage of total DNA with Mlul, Clal or Hindlll restriction endonucleases and hybridization of fragments with acetylaminofluorene-labelled 16+23S rRNA. A wide diversity of ribotypes was observed with endonucleases Mlul (104 patterns), Clal (90 patterns) and Hindlll (98 patterns). When Mlul was used, 85% of patterns (11 to 15 fragments) shared five fragments 17.09, 3.94, 3.06, 2.23 and 1.76 kb in size. When these fragments were used as internal standards, the percent errors in fragment length determination was half of that obtained with an external standard. Two fragment size databases of Mlul and Clal ribotypes were built. Automatic identification was obtained after setting the percent fragment size variation tolerance (error) at 5%. Mlul ribotyping is recommended as a primary epidemiological marker. Strains with similar Mlul ribotype should then be submitted to Clal ribotyping. Ribotyping with Hindlll can only be the third choice, since the patterns were often uncertain due to the frequent occurrence of faint bands. Most of the studied serovars gave discrete patterns and these data provide the basis for a molecular typing system for E. coli which could possibly substitute for serotyping when the latter is not available.

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.

Molecular and phenotypic features of aeromonads isolated from snails (Helix aspersa) affected with a new summer disease.

Forty-four aeromonads were isolated during the summer of 1994 from snails (Helix aspersa) affected or not by a newly described disease. Snta 1 rRNA gene restriction patterns (ribotyping) and nine selected conventional phenotypic tests were performed to retrospectively determine relationships among these isolates regarding the disease. Results from ribotyping were found highly discriminant in strain differentiation and suggest the possible existence of a group of Aeromonas associated with the disease. Biochemical and molecular characterizations indicated that most of these disease-associated aeromonads could belong to the phenospecies A. hydrophila. Phenospecies of aeromonads from healthy snails were distinct from those isolated from diseased snails and could be identified as A. caviae or remained undetermined. To the authors' knowledge, this is the first report of a probable pathogenic aeromonad found in snails, extending the host-pathogen spectrum of Aeromonas.

Isolation and identification of Salmonella species from chelonians using combined selective media, serotyping and ribotyping.

Thirty-two faecal samples from various pet chelonians were bacteriologically examined using various combinations of selective media for Salmonella detection. Strains identified as Salmonella were clustered by rRNA gene restriction pattern (ribotyping) analysis prior to serological typing. The combination of selenite broth and Rambach or Salmonella-Shigella agars gave the maximum rate of Salmonella recovery. Of the 32 samples examined, 13 (40%), were positive for Salmonella. Five serovars were identified, Muenchen, Rubislaw, Newport, Ferruch and a non-motile O:4,5,12, with Salmonella Muenchen the most common. This result indicates that biochemical characterization combined with ribotyping prior to serotyping can contribute to Salmonella detection on a large scale.