O148 Shiga toxin-producing Escherichia coli outbreak: microbiological investigation as a useful complement to epidemiological investigation.

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O148 infection occurred among wedding attendees in France in June 2002. A retrospective cohort study was performed and ten cases were identified, including two adults with haemolytic uraemic syndrome (HUS). The analytical study revealed that > 80% of affected individuals had eaten lightly roasted mutton and poultry pâté, but only the consumption of pâté tended to be associated with illness (relative risk 3.4; 95% CI 0.8-14.4). Left-overs (cooked mutton and raw offal) and processed foods (pâté) from the same batches as served at the party were sampled. Human, food and environmental samples were examined for the Shiga toxin (stx) gene and virulence traits by PCR. Stx-positive samples were cultured for STEC. HUS cases were tested for serum antibodies against 26 major STEC serogroups. An STEC O26 strain (stx1, eae, ehxA) was isolated from one case with diarrhoea, and an STEC O148 strain (stx2c) from one case of HUS. Serum antibodies against O26 were not detected in either of these patients; antibodies against O148 were not tested. Three STEC strains were isolated from the mutton and the offal (stx2c, O148), and two from the pâté (stx2c, O-X and O-Y). The isolates from the mutton were indistinguishable from the human stx2c isolate, whereas the pâté isolates differed. Although four different STEC strains were identified in patients and foods, the results of molecular subtyping, in conjunction with analysis of food consumption patterns, strongly suggested that this outbreak was caused by mutton contaminated with STEC O148.

Clonal diversity of Vibrio cholerae O1 evidenced by rRNA gene restriction patterns.

The rRNA gene restriction patterns of 89 Vibrio cholerae O1 isolates from different geographic origins were studied. The probe was Escherichia coli 16 + 23S rRNA labelled with "ECL Gene detection system". A total of 17 rRNA gene restriction patterns were observed after BglI cleavage. Four patterns (B1 to B4) were only given by biotype cholerae (14 strains studied). Thirteen patterns (B5 to B17) were only given by biotype El Tor (75 strains studied). There was no correlation between serotypes and rRNA gene restriction patterns. This study provides arguments that (1) strains of biotypes cholerae and El Tor are different clones, (2) a cholera pandemic is not a single world-wide epidemic (due to a single clone) but rather a simultaneous occurrence of several epidemics (several clones involved), and (3) epidemic waves of biotype El Tor could be due to the emergence of new clones.

Nucleotide sequences of 16S rRNA from ten Serratia species.

Comparison of 16S ribosomal ribonucleic acid (rRNA) sequences has emerged as a powerful tool for bacterial phylogeny. However, earlier studies often only included one or a few species per genus, and it is not sure whether the rRNA sequences could discriminate closely related species. The genus Serratia is composed of ten species, some being up to 60% related by DNA hybridization. The reverse transcriptase/primer extension method was used to determine 1,492 to 1,509 nucleotides in each of ten Serratia 16S rRNA sequences. All rRNA sequences determined were unique. The phylogenetic tree obtained with the neighbour-joining method showed a cluster of Serratia species distinct from both Escherichia coli and Proteus vulgaris. S. fonticola--whose position in the genus Serratia is questioned--was clearly included in the Serratia branch and grouped within the psychrophilic Serratia species. Variable regions in the Serratia rRNA molecules were identified and could serve as the basis for a specific probe design.

Comparison of 14 PCR systems for the detection and subtyping of stx genes in Shiga-toxin-producing Escherichia coli.

The specificity of 14 polymerase chain reaction (PCR) systems designed for the detection and subtyping of stx genes was tested on a set of Escherichia coli strains with known sequences of stx genes. Systems designed for the detection of genes of the stx1 type did not detect any variant genes of the stx2 type and conversely, no stx2 type-specific systems detected stx1 variant genes. Among five stx2 type-specific systems, none detected the stx2ev gene, and two detected the stx2e gene. Among systems designed for screening genes of the both stx1 and stx2 types with a single primer pair, only one system (the Lin system) was able to detect stx genes in all studied strains. Shiga-toxin-producing E. coli frequently carry more than one stx variant gene. Coamplification of stx genes present in the same strain was demonstrated by restriction of PCR products with endonucleases generating fragments of variant-specific size. The amplification product obtained by the Lin system restricted by Hincll yielded fragments of different size for stx1, stx2, stx2c, stx2e and stx2ev. Thus it was possible to identify different genes carried in a single strain with a simple two-step PCR/endonuclease restriction protocol.

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 MluI, ClaI or HindIII restriction endonucleases and hybridization of fragments with acetylaminofluorene-labelled 16 + 23S rRNA. A wide diversity of ribotypes was observed with endonucleases MluI (104 patterns), ClaI (90 patterns) and HindIII (98 patterns). When MluI 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 MluI and ClaI ribotypes were built. Automatic identification was obtained after setting the percent fragment size variation tolerance (error) at 5%. MluI ribotyping is recommended as a primary epidemiological marker. Strains with similar MluI ribotype should then be submitted to ClaI ribotyping. Ribotyping with HindIII 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.

Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene.

A total of 182 strains of Escherichia coli (133 reference strains, 22 clinical strains, nine nonmotile strains and 18 strains derived from K-12) were characterized by HhaI restriction of the amplified flagellin gene (fliC). The amplified fliC product was a single band between 0.9 and 2.6 kbp. With the collection of reference strains which represented 48 flagellar types (H-types), a total of 62 patterns (F-types) were observed after HhaI restriction. A single F-type was associated with each of 39 H-types and more than one F-type was associated with the other nine H-types. Antigenically related H-types 12 and 45 gave a single F-type. The determination of HhaI-fliC F-types could allow deduction of all H-types and subdivision of some of these. Application of this identification system to 22 E. coli clinical isolates yielded nine F-patterns and the deduced H-types were confirmed by serotyping in all cases. Nine nonmotile strains were studied and their F-types were also identified. The proposed determination of fliC restriction patterns should be helpful for epidemiological studies.

Identification of diary Propionibacterium species by rRNA gene restriction patterns.

A total of 78 strains of dairy propionibacteria, 4 reference strains of Propionibacterium and 8 related bacteria were characterized by ribosomal ribonucleic acid (rRNA) gene restriction patterns (ribotyping). The patterns were obtained after cleavage of total DNA with either BamHI or ClaI restriction endonucleases and hybridization of fragments with acetylaminofluorene-labelled 16 + 23S rRNA from Escherichia coli. The four different species of dairy propionibacteria, P. freudenreichii, P. jensenii, P. thoenii and P. acidipropionici, gave different restriction patterns with species-specific fragments. Moreover, ribotyping allowed the differentiation of P. freudenreichii subsp. freudenreichii from P. freudenreichii subsp. shermanii. The patterns of dairy propionibacteria were different from those of closely related bacteria and other bacteria used in the dairy industry.

Universal ribotyping method using a chemically labelled oligonucleotide probe mixture.

Some of the present problems in ribotyping are associated with a lack of uniform reactivity of probes when bacterial DNAs are of phylogenetically diverse origins. To overcome these problems, a set of five oligonucleotides (referred to as OligoMix5) was selected to react with conserved sequences located near both extremities of rrs (16S rRNA gene) and near both extremities and the middle of rrl (23S rRNA gene). DNA samples from 13 bacterial species selected to represent various phylogenetic branches within the Eubacteria were cleaved by a restriction endonuclease and electrophoresed in 0.8% agarose, and the fragments were vacuum-transferred to nylon membranes and hybridized with digoxigenin-labelled OligoMix5, plasmid DNA from pKK3535 (cloned rrn operon from Escherichia coli) or pBA2 (cloned rrs from Bacillus subtilis), or acetylamino-fluorene-labelled E. coli 16 + 23S rRNA. The results showed OligoMix5 to visualize patterns in DNA from phylogenetically diverse bacteria with comparable intensity. Banding patterns (not band intensity) obtained with OligoMix5 were identical with those obtained with 16 + 23S rRNA or plasmid pKK3535 for each strain studied and represented complete ribotypes. For DNA from Gram-positive bacteria, complete ribotypes were observed after prolonged enzymatic detection of bands when probes were either E. coli 16 + 23S rRNA or pKK3535. Patterns given by plasmid pBA2 were subsets of the complete ribotypes for 9/13 strains. Each oligonucleotide of the OligoMix5 set was used as a probe to determine its contribution to the complete ribotype. The five oligonucleotide probes, used individually, visualized one to four patterns per DNA sample. Use of DNA from Xenorhabdus sp. CIP 105189 cleaved by EcoRI is suggested to control the quality of the oligonucleotide probes composing OligoMix5. Probe OligoMix5 was found to be an essential tool for ribotyping phylogenetically diverse eubacteria.

Identification of Shigella serotypes by restriction of amplified O-antigen gene cluster.

Due to the scarcity of distinctive biochemical reactions for differentiation of Shigella-Escherichia coli, antigenic analysis has long been used for identification and typing of Shigella isolates. Nevertheless, several intra- and interspecific cross-reactions have been reported to disturb serotyping assays. Shigella serotyping is also occasionally affected by the transition from the smooth (S) form to the rough (R) form. Thus, there is a need for the development of novel robust and discriminating methods for Shigella identification and typing. Characteristically, all genes specifically involved in O-antigen synthesis are clustered in E. coli, Shigella, and Salmonella. Published oligonucleotide sequences complementary to JUMPstart and gene gnd, the conserved flanking sequences upstream and downstream of O-antigen gene clusters, were used to amplify the O-antigen gene cluster of representative strains of each Shigella serotype. A unique, amplified fragment was generally observed for each serotype (size ranging from 6 kbp to 17 kbp). Clearly identifiable and reproducible patterns were obtained for each serotype after MboII digestion of the products, except for S. boydii 12 which showed two distinct patterns, and S. flexneri serotypes 1 to 5 and X and Y which showed a single pattern. A database was built with the Taxotron package allowing automated identification of clinical Shigella isolates to all known serotypes.

rRNA gene restriction patterns of Legionella species: a molecular identification system.

A total of 28 species of Legionella could be differentiated by rRNA gene restriction patterns generated after cleavage of total DNA with either EcoRV or HindIII restriction endonucleases, and hybridization of fragments with 32P-labelled Escherichia coli 16 + 23S rRNA. Different species gave different fragment patterns. When several isolates of a species were tested, the patterns obtained were often identical. However, more than one pattern was often observed when more than one serotype was considered. The method should be useful for the identification of all species of Legionella including those exhibiting immunological cross-reactions.

Molecular typing of Salmonella enterica subsp. enterica serovar Wien by rRNA gene restriction patterns.

Analysis of digested DNA from 40 Salmonella enterica subsp. enterica serovar Wien isolates revealed five different rRNA gene restriction patterns for HindIII (H1 to H5) and five for PstI (P1 to P5). The isolates had been selected on the basis of the year of isolation, the geographic origin and the antibiotic resistance pattern and were distinguished as follows: a) 13 pre-epidemic isolates from different French towns in 1958-69 and from Senegal in 1968; b) 7 epidemic isolates from Algiers in 1969; c) 20 post-epidemic isolates from different French and Italian towns in 1970-90. Three different rRNA patterns (H1P1, H3P3 and H4P4) were observed among the pre-epidemic isolates. Conversely, the epidemic isolates, which were characterized by the previously described large antibiotic resistance patterns and by the presence of 1.3 and 80-109 MDa plasmids, belonged to the same H1P1 ribotype. All but two post-epidemic isolates were of the H1P1 ribotype. The determination of rRNA gene restriction patterns together with the plasmid content proved to be useful for a better characterization of the serovar Wien endemic and epidemic isolates.

Replicon typing of 71 multiresistant Serratia marcescens strains.

Replicon typing is the identification of plasmids by hybridization with specific DNA probes which contain the genes involved in plasmid maintenance. This new method has been used to classify plasmids into replicon (rep) groups which can often be correlated with incompatibility (Inc) groups. We studied 71 multiresistant Serratia marcescens strains with 19 rep probes constructed from reference plasmid replicons belonging to known Inc groups. These probes are known to react with enteric bacterial plasmids. However, they did not represent the totality of the thirty known Inc groups. For 52% of the studied strains, plasmids were identified and classified into groups FIB, FIC, FIIA, HI2, L/M, N, B/O, P, W, Y and Com9. Most (79%) of the plasmid preparations hybridized with a single rep probe, and 21% hybridized with two different probes. Electrophoretic analysis of DNA suggested that double hybridization could result from the presence of either two different Inc plasmids in the same strain (e.g. S37) or one single plasmid with a multireplicon (e.g. S113).

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.

Ribotyping of Streptococcus agalactiae strains isolated from vaginas of asymptomatic women.

One hundred and eleven strains of Streptococcus agalactiae isolated from vaginas of asymptomatic women were characterized by determination of restriction length polymorphism profiles of rDNA regions (rDNA RFLP patterns) and serotyping. Thirty-five different PstI rDNA RFLP patterns were identified. Half of the strains fell into only seven of these 35 groups. No correlation between serotypes and rDNA RFLP patterns was found. These results indicate that (i) the genetic diversity of the S. agalactiae species is relatively limited and (ii) determination of rDNA RFLP patterns can be used as a typing system only in conjunction with serotyping.

DNA fingerprinting of Chlamydia trachomatis by use of ribosomal RNA, oligonucleotide and randomly cloned DNA probes.

DNA fingerprinting of 15 reference strains and 24 clinical isolates of Chlamydia trachomatis, 2 strains of C. psittaci and one strain of C. pneumoniae was studied by use of universal 16 + 23S RNA from Escherichia coli, 16S rDNA-directed oligonucleotide and randomly cloned chlamydial DNA probes. The rRNA-gene restriction patterns (ribotypes) enabled the differentiation of chlamydial species. Following DNA cleavage by restriction endonuclease PvuII, lymphogranuloma venereum and trachoma biovars of C. trachomatis could be differentiated. An oligonucleotide, designed to hybridize the C. trachomatis 16S rDNA, also allowed for both species-specific identification and biovar typing of C. trachomatis human strains. Molecular typing system using 3 lambda clones containing C. trachomatis serotype E random DNA inserts, combined to ribotyping, revealed 12 groups of variable banding patterns within C. trachomatis, and could provide an alternative epidemiological tool.

Classification of Brucella strains isolated from marine mammals using DNA-DNA hybridization and ribotyping.

DNA-DNA hybridization showed that the Brucella strains recently isolated from marine mammals belong to the monospecific genus Brucella (more than 77% DNA relatedness). Ribotyping (HindIII rDNA restriction patterns) showed that they may represent a separate subgroup (marine type) specifically associated with marine mammals.

rRNA gene restriction patterns of Leptospira: a molecular typing system.

A total of 67 serovar reference strains and 7 isolates belonging to the genus Leptospira were characterized by ribosomal ribonucleic acid (rRNA) gene restriction patterns. Fifty patterns were observed. Strains belonging to different genomic species always gave different patterns. However, genomic species were subdivided into several patterns. Forty-three serovars gave a specific pattern. Some serovars could not be separated by rRNA gene restriction patterns: strains of serovars icterohaemorrhagiae, copenhageni, lai, pyrogenes and jalna gave pattern 1; serovars birkini, mankarso and wolffi gave pattern 4; serovars canicola, gem, hebdomadis, pomona and hardjo (strain hardjoprajitno) gave pattern 12; serovars valbuzzi and zanoni gave pattern 14; serovars jonsis, malaya and sumneri gave pattern 16; serovars arborea, ballum, castellonis and kenya gave pattern 35; and serovars borincana and shermani gave pattern 43. These data provide the bases for a molecular typing system for the genus Leptospira.

Molecular typing of Chlamydia trachomatis by random amplification of polymorphic DNA.

The random amplification of polymorphic DNA (RAPD) was used for epidemiological typing of Chlamydia trachomatis strains. DNA samples from 39 C. trachomatis, 1 C. pneumoniae and 2 C. psittaci strains were screened by the use of 4 single 10-mer primers. Different and reproducible banding profiles were observed on agarose gel electrophoresis. No common profiles were recorded for strains from different Chlamydia species. All C. trachomatis strains of trachoma biovar were distinguished from lymphogranuloma venereum biovar. Moreover, serotypes A to C were separated from serotypes D to K, and some groups of strains sharing the same serotype D to K were further subdivided by RAPD. Conversely, strains of different serotypes could produce identical patterns of amplification, indicating that RAPD did not reflect serotyping. The patterns of amplified products were compared to the restriction fragment length polymorphism of the omp1 gene after amplification and to DNA fingerprinting by use of ribosomal RNA or randomly cloned DNA probes. RAPD seemed to be an alternative molecular typing procedure for epidemiological study and strain identification in urogenital infections due to serotypes D to K.

Molecular characterization of Vibrio cholerae O1 strains isolated in Romania.

A collection of 89 Vibrio cholerae O1 strains, isolated in Romania between 1977 and 1994, and 6 strains from the Republic of Moldavia, was characterized by ribotyping, toxin gene restriction pattern (toxinogenotype) and distribution of cholera toxin gene (ctx), accessory toxin gene (ace) and zonula occludens toxin gene (zot). After Bg/I endonuclease restriction of chromosomal DNA, a total of 18 ribotypes and 21 toxinogenotypes were distinguished. Deletions in the core region of the toxin gene cassette were found in 20% of strains; however, with the exception of one strain, all the isolates contained the ctx gene. Used in association, the three methods of molecular typing provided an accurate characterization of V. cholerae O1 isolates.

Palindromic unit highly repetitive DNA sequences exhibit species specificity within Enterobacteriaceae.

Palindromic units (PU, or REP for repetitive extragenic palindrome) constitute a family of DNA sequences of 40 nucleotides which is highly repeated in the genome of Escherichia coli. We analysed the presence of PU sequences in 99 different bacterial genomes by cross-hybridization. When PU sequences were used as a probe, only DNA from Enterobacteriaceae closely related to E. coli exhibited an appreciable hybridization signal: Shigella sonnei, Shigella boydii, Salmonella enteritica serotype Typhimurium, Citrobacter freundii and Levinea malonatica. Furthermore, these bacteria could be divided into two groups which corresponded to a slight difference in their PU sequence: the E. coli group includes S. sonnei and S. boydii; the S. enteritica serotype Typhimurium group includes C. freundii and L. malonatica.