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The genus Serratia has been studied for over a century and includes clinically-important and diverse environmental members. Despite this, there is a paucity of genomic information across the genus and a robust whole genome-based phylogenetic framework is lacking. Here, we have assembled and analysed a representative set of 664 genomes from across the genus, including 215 historic isolates originally used in defining the genus. Phylogenomic analysis of the genus reveals a clearly-defined population structure which displays deep divisions and aligns with ecological niche, as well as striking congruence between historical biochemical phenotyping data and contemporary genomics data. We show that Serratia is a diverse genus which displays striking plasticity and ability to adapt to its environment, including a highly-varied portfolio of plasmids, and provide evidence of different patterns of gene flow across the genus. This work provides an essential platform for understanding the emergence of clinical and other lineages of Serratia.

Since the 1980s, Shiga toxin-producing Escherichia coli (STEC), especially E. coli O157:H7, has been an important cause of food borne disease in industrial countries. In France, as there was no routine screening for STEC in clinical laboratories, enhanced surveillance of hemolytic uremic syndrome (HUS) in children less than 15 years of age was established in 1996 to monitor trends in the incidence of STEC infections.The surveillance system was based on a voluntary national network of pediatricians of 31 pediatric nephrology units in public hospitals.From 1996 to 2006, the mean annual incidence of HUS was 0.71 cases per 100,000 children less than 15 years of age and 1.87 cases per 100,000 children less than 5 years of age. STEC infections were confirmed in 66% of patients; STEC O157 was the most common serogroup identified in STEC-related HUS (83%). In this 11-year period, 96% of HUS cases were sporadic and only 2 outbreaks caused by STEC O157 and by a dual infection of STEC O26 and O80 were detected.An evaluation of the surveillance of pediatric HUS showed that it is a simple and useful system for monitoring trends in STEC infections in France. It provides the information needed to measure the impact of new and changing vehicles of STEC transmission, and evaluate the effectiveness of prevention measures.

Infections by Shigella species are an important cause of diarrhoeal disease worldwide. Of 4198 Shigella isolates received by the French National Reference Centre for Escherichia coli and Shigella, 180 from patients with diarrhoea and dysentery in 2000–2004 did not react with any available polyclonal rabbit antisera used to identify the established Shigella serogroups. This study describes the molecular and phenotypic characteristics of these isolates in seroagglutination tests, molecular serotyping (rfb-RFLP and fliC-RFLP), ribotyping, detection of invasivity and enterotoxins genes, and antibiotic sensitivity. All isolates gave biochemical reactions typical of Shigella boydii, were mannitol-positive and indole-negative. They all carried invasion-associated genes, enterotoxin 2 [ShET-2] and an IS630 sequence. They had a unique ribotype that was distinct from all other Shigella and E. coli patterns. Further characterization by rfb-RFLP clearly distinguished this serogroup from all other Shigella or E. coli O-groups. The fliC-RFLP pattern corresponded to P4, an F-pattern which is associated with 10 different serogroups of S. boydii. A new antiserum prepared against strain 00-977 agglutinated all 180 isolates and cross-agglutination and absorption studies with anti-00-977 serum and anti-CDC 99-4528 (reference for the newly described S. boydii serogroup 20) serum showed identical antigenic structure. Furthermore, strains 00-977 and CDC 99-4528 had the same molecular serotype, ribotype and virulence genes.

Summary Shigella dysenteriae type 1 (Sd1) represents a particular threat in developing countries because of the severity of the infection and its epidemic potential. Antimicrobial susceptibility testing and molecular subtyping by pulsed-field gel electrophoresis (PFGE) and plasmid profiling (PP) of Sd1 isolates collected during two dysentery outbreaks (2013 and 445 cases of bloody diarrhoea) in Central African Republic (CAR) during the period 2003–2004 were reported. Eleven Sd1 comparison strains (CS) acquired by travellers or residents of Africa (n = 10) or Asia (n = 1) between 1993 and 2003 were also analysed. The 19 Sd1 isolates recovered from CAR outbreaks were multidrug resistant, although susceptible to quinolones and fluoroquinolones. Molecular subtyping by PFGE was more discriminatory than PP. The PFGE using XbaI and NotI restriction enzymes indicated that the two outbreaks were due to two different clones and also revealed a genetic diversity among the CS recovered from outbreak or sporadic cases between 1993 and 2003. This study was the result of a fruitful collaboration between field physicians and microbiologists. The data collected will serve as the basis for establishing long-term monitoring of Sd1 in CAR.

Phylogenetic relationships within the genus Pseudomonas were examined by comparing partial (about 1000 nucleotides) rpoB gene sequences. A total of 186 strains belonging to 75 species of Pseudomonas sensu stricto and related species were studied. The phylogenetic resolution of the rpoB tree was approximately three times higher than that of the rrs tree. Ribogroups published earlier correlated well with rpoB sequence clusters. The rpoB sequence database generated by this study was used for identification. A total of 89 isolates (79.5%) were identified to a named species, while 16 isolates (14.3%) corresponded to unnamed species, and 7 isolates (6.2%) had uncertain affiliation. rpoB sequencing is now being used for routine identification of Pseudomonas isolates in our laboratory.

Shigella, the etiological agent of the bacillary dysentery, belongs to the extremely diverse species of Escherichia coli. In the evolutionary route of Shigella from commensal E. coli ancestors towards a pathogenic lifestyle, the critical events have been the acquisition of the pINV plasmid, through horizontal transfer and the inactivation of pre-existing genes. These so-called pathoadaptive mutations affect the expression of genes negatively interfering with the newly acquired functions necessary for the colonization of the host niche. Cadaverine, a small polyamine resulting from decarboxylation of lysine, has been shown to hamper the full expression of Shigella invasiveness mainly by altering the inflammatory response. Recent analysis of the evolution of the Shigella and enteroinvasive E. coli (EIEC) cad region indicates that silencing of the cad locus has been attained with several strategies. The increasing relevance of S. sonnei in both, developing and industrial countries, prompted us to analyze the molecular origin of the LDC- phenotype in these strains. The results obtained on several S. sonnei strains reveal that despite the difference in geographic origin and antibiotic resistance patterns, all the strains have undergone the same modifications. Multiple IS insertions into the cadBA operon have interrupted gene continuity without inducing deletions or inversions of the cadA and cadB genes which are remained entirely conserved. Moreover, by functional analysis we show that all the strains carry a defective cadC gene, thus strengthening the hypothesis that inactivation of the regulatory cadC gene might have been the first step towards a complete lack of the cad locus.

During the period of 9-27 March 2001, Pseudomonas putida strains were recovered from 10 neonates hospitalized in the neonatal intensive care unit of Farhat Hached Hospital, Sousse (Tunisia). Seven neonates developed bacteraemia, and three had an umbilical catheter-related infection (without bacteraemia). A total of 18 isolates were cultured from blood (N = 11) and catheters (N = 7). These isolates were identified as P. putida by routine biochemical methods (API 20 NE, bioMérieux, Lyon, France). Restriction endonuclease DNA profiles were determined by pulsed-field gel electrophoresis using two endonucleases XbaI and SpeI. They yielded the same patterns showing that the outbreak was caused by a single clone of P. putida. Although the antiseptic solutions used to clean the umbilicus were implicated circumstantially as probable sources, they were not sampled and so this could not be confirmed.

The nomenclature of Corynebacterium diphtheriae ribotypes is presented. A total of 86 ribotypes obtained after BstEII digestion were given a geographic name chosen to reflect the place where one of the strains was isolated or studied.

'Streptococcus bovis/Streptococcus equinus' is a large bacterial complex including different species frequently isolated from infections of humans (Streptococcus gallolyticus, Streptococcus infantarius) or animals (S. bovis, S. equinus, Streptococcus alactolyticus). The separation of S. bovis into three different biotypes has been partially correlated with genetic differentiation. In addition, recent advances in bacterial phylogeny have led to the inclusion of Streptococcus macedonicus and Streptococcus waius in this complex. The aim of this study was to improve physiological differentiation between species related to the complex and to clarify their respective phylogenetic positions. In this study, physiological, genetic and phylogenetic analyses of a set of 88 streptococcal strains were performed. The diversity of strains of S. bovis biotype II was analysed, and it was confirmed that they belong to different species, either S. equinus or S. infantarius. It was demonstrated that S. gallolyticus, S. bovis biotype II.2, S. macedonicus and S. waius form a single DNA cluster separated into three different subspecies. They are delineated by different biochemical traits, limited DNA-DNA relatedness and noticeable divergence in 16S rDNA sequences. According to the current definition of species, the names S. gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. pasteurianus subsp. nov. and S. gallolyticus subsp. macedonicus subsp. nov. are proposed for these three subspecies.

One hundred two Corynebacterium diphtheriae strains (93 of the gravis biotype and nine of the mitis biotype) isolated from clinical cases during the Belarus diphtheria epidemic were characterized by biotyping, toxigenicity testing by the Elek test and an indirect hemagglutination assay, phage typing, and ribotyping. The gravis biotype strains were characterized as high and medium toxin producers, and strains of biotype mitis were characterized as low and medium toxin producers. Most strains (82 of 102) were distributed among five phage types. Seventy-two strains (64 of the gravis biotype and 8 of the mitis biotype) belonged to phage type VI ls5,34add. Hybridization of genomic DNA digested with Bst EII and Pvu II revealed five ribotype patterns, namely, D1, D4, D6, D7, and D13. The majority of gravis biotype strains belonged to ribotypes D1 (49 of 93) and D4 (33 of 93) and included one clonal group of C . diphtheriae . This clone predominated in all regions in Belarus. There was a statistical association between ribotypes and phage types but not between ribotypes and levels of toxin production.

Streptococci are major pathogens for animals and human beings. Phenotypic studies and DNA-DNA hybridization assays have demonstrated that the genus Streptococcus may be divided in three major groups of species: pyogenic, oral, and group D streptococci (5, 6, 17, 20, 21, 27). Identification of streptococcal species is currently based on observation of the cultural and morphological characteristics, determination of the biochemical pattern (production of enzymes and production of acid from various carbohydrate sources) (4, 5, 6, 9, 11, 17, 20), and observation of the antigenic structure according to the classification of Rebecca Lancefield (22). Molecular tools as ribotyping (3, 8, 25, 26), oligonucleotide probing (2), PCR-based protocols (13, 23), and DNA sequence analysis (1, 19, 24) are now proposed to provide an accurate identification of streptococcal isolates. Among these, restriction fragment length polymorphism analysis was proposed as a general method for bacterial identification and typing (15). Amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) was recently reported to be a rapid and efficient method of identification of bacterial isolates to the species level in mollicutes (7) and in some genera such as Acinetobacter or Mycobacterium (30, 31). The initial protocol was substantially improved by amplification of a larger fragment of the rrn operon (16S rRNA gene, 16S-23S intergenic spacer, and 23S rRNA gene) followed by the digestion with one to three endonucleases selected according to the bacterial genus studied (12, 18). The latest modification of this method uses a unique combination of three restriction endonucleases (HhaI, MboII, and Sau3A) irrespective of the bacterial species studied. This modified protocol was successfully used for identification of the genus and the species of strains belonging to the family Enterobacteriaceae (G. Giammanco, P. Nogueira, O. Bouallegue, F. Grimont, and P. A. D. Grimont, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. R20, p. 482, 1998). In the present study, we investigate the interest of the modified rrn-ARDRA protocol for identification of streptococci. One hundred and forty-eight strains were amplified and analyzed with HhaI, MboII, and Sau3A endonucleases. A database suitable for identification of streptococcal species was constructed and used to identify some clinical isolates.

From 1984 to 1999, we collected 31 isolates of the rare serovar Salmonella bongori 48:z 35 :− in southern Italy. Twenty-four of the isolates were from cases of acute enteritis in humans. Pulsed-field gel electrophoresis analysis showed that all but one of our isolates were at least 80% similar. Our findings suggest that genetically related S. bongori 48:z 35 :− strains are endemically circulating in southern Italy.

A selection of 167 Corynebacterium diphtheriae strains isolated in Romania, the Russian Federation and the Republic of Moldova were analysed by biotyping, phage typing, the toxin production test and by molecular techniques such as ribotyping, pulsed field gel electrophoresis and random amplified polymorphic DNA, in order to establish the epidemiological relatedness, genetic divergence and strain circulation within and between the bordering countries. Using a set of five digoxigenin-labeled oligonucleotides and BstEII digestion, 34 ribotypes were identified. The strains isolated in the epidemic areas (Russia and Moldova) were very closely related but different from those isolated in Romania. C1 and C5 were the main ribotypes identified in these areas. Neither ribotype was found in Romania, where the main circulating types were C3 and C7. Field inversion gel electrophoresis was more discriminative than ribotyping and revealed 54 macrorestriction profiles after SfiI restriction. Both methods showed a significant homogeneity of the strains from epidemic areas and a large diversity among the Romanian strains. Random amplification was useful as an identification method for the epidemic strains, but not for the Romanian ones which displayed a large number of amplification profiles. The phenotypic methods associated with molecular typing techniques enabled distinguishing between strains, detecting the epidemic clone, and sustaining the absence of transmission across borders.

From September 1997 to November 1998, the French National Center for Salmonella and Shigella received 22 Shigella isolates recovered from 22 different patients suffering from dysentery. None of these isolates reacted with any of the antisera used to identify established Shigella serotypes, but all of them agglutinated in the presence of antisera to a previously described potentially new Shigella dysenteriae serotype (represented by strain 96–204) primarily isolated from stool cultures of imported diarrheal cases in Japan. All French isolates, as well as strain 96–204, showed biochemical reactions typical of S. dysenteriae and gave positive results in a PCR assay for detection of the plasmid ipaH gene coding for invasiveness. No Shiga toxin gene was detected by PCR. These isolates were indistinguishable by molecular analysis of ribosomal DNA (ribotyping) and seemed to be related to S. dysenteriae serotypes 3 and 12. However, further characterization by restriction of the amplified O-antigen gene cluster clearly distinguished this new serotype from all other Shigella or Escherichia coli serotypes.

The presence of cryptic fliC alleles in the genomes of 120 strains representative of the four Shigella species was investigated. One fragment was obtained by PCR amplification of fliC , with a size varying from 1.2 to 3.2 kbp, depending on the species or serotype. After digestion with endonuclease Hha I, the number of fragments in patterns varied from three to nine, with sizes of between 115 and 1,020 bp. Patterns sharing most of their bands were grouped to constitute an F type. A total of 17 different F types were obtained from all strains included in this study. A unique pattern was observed for each the following serotypes: Shigella dysenteriae 1, 2, 8, and 10 and S. boydii 7, 13, 15, 16, and 17. On the contrary, S. dysenteriae serotype 13 and S. sonnei biotype e were each subdivided into two different F types. S. flexneri serotypes 3a and X could be distinguished from the cluster containing S. flexneri serotypes 1 to 5 and Y. S. flexneri serotype 6 clustered with S. boydii serotypes 1, 2, 3, 4, 6, 8, 10, 11, 14, and 18 and S. dysenteriae serotypes 4, 5, 6, 7, 9, 11, and 12. Two other clusters were outlined: one comprising S. dysenteriae serotypes 3, 12, 13 (strain CDC598-77), 14, and 15 and the other one joining S. boydii serotypes 5 and 9. None of the 17 fliC patterns was found in the fliC Hha I pattern database previously described for Escherichia coli . Overall, this work supports the hypothesis that Shigella evolved from different ancestral strains of E. coli . Moreover, the method outlined here is a promising tool for the identification of some clinically important Shigella strains as well as for confirmation of atypical isolates as Shigella spp.

The precise serotyping of clinical Escherichia coli isolates is a crucial step for diagnostic and epidemiological purposes. Epidemiological knowledge associated with serotyping is so important that no alternative method may be considered if it does not correlate with serotyping. Unfortunately, E. coli are difficult to serotype. Genes specifically involved in O-antigen synthesis are clustered in E. coli, Shigella and Salmonella. Published oligonucleotide sequences complementary to JUMPstart and the gnd gene (the conserved flanking sequences upstream and downstream of O-antigen gene clusters, respectively) were used to amplify the O-antigen gene cluster of representative strains of 148 E. coli O-serogroups. A unique amplified fragment was observed for each serogroup (size ranging from 1.7 to 20 kbp). Clearly identifiable and reproducible O-patterns were obtained for the great majority of O-serogroups after MboII digestion of amplified products. The number of bands composing each pattern varied from five to 25. A database was built with the patterns obtained. A total of 147 O-patterns were obtained. Thirteen O-serogroups were subdivided into different O-patterns. However, each of 13 other O-patterns was shared by two or more O-serogroups. 0-serogroups of clinical isolates were deduced accurately from O-patterns in all cases, even for some rough or nonagglutinating isolates. The restriction method (rfb-RFLP) may prove to be better than serotyping since 100% of strains are typable, which is not the case with serotyping.

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.

A case-control study was conducted to establish the source of a community outbreak of typhoid fever in Utelle, France, a village located in the Alpes-Maritimes district of the French Riviera. Thirteen confirmed cases of typhoid fever and 41 confirmed community controls were included in the study. Cases and controls did not differ regarding ingestion of water. Multivariate logistic regression analysis identified consumption of pork meats during a village festival as the only statistically significant risk factor for typhoid fever after adjusting for age and sex (odds ratio, 76.0; 95% confidence interval, 3.5-1660). Assessment of food-handling procedures at the inn where the food had been prepared showed that the refrigeration and cooking facilities were inadequate to maintain a proper sanitary environment during the preparation of a meal for 350 people. Although the exact cause of the epidemic could not be confirmed, food contaminated by a chronic typhi carrier is the most plausible hypothesis.

During 1994, 10 isolates of extended-spectrum β-lactamase-producing Salmonella typhimurium were recovered from children transferred to our hospital from two different centers. Two additional isolates were recovered from two nurses from one of these centers. The aim of this study was to determine if there is any relationship between these isolates. The characterization was done by phenotypic and genotypic methods: biotyping, phage typing, antibiotic susceptibility pattern determination, plasmid analysis, ribotyping (by the four endonucleases Eco RI, Sma I, Bgl II, and Pvu II), pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with Xba I, and randomly amplified polymorphic DNA (RAPD) pattern determination (with the three primers 217 d2, B1, and A3). The same biotype, the same serotype, and an identical antibiotype were found. All isolates were resistant to oxyimino-β-lactams, gentamicin, tobramycin, and sulfamethoxazole-trimethoprim. All isolates showed an indistinguishable pattern by ribotyping and very similar patterns by PFGE and RAPD. The overall results indicated the spread of a closely related strain of S. typhimurium in children and nurses.

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.

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.

Epidemiologic relationships were investigated in 187 ampicillin-resistant Salmonella typhimurium strains (86 human, 101 animal) from >2000 strains isolated in 1994. Of 23 resistance patterns, the most frequent (ampicillin [Am], chloramphenicol [Cm], tetracycline [Tc], streptomycin and spectinomycin [Sm], and sulfonamides [Su]) was found in 69.5% of human and 64.8% of animal isolates. Four beta-lactamase genes were identified, blaTEM (24%), blaPSE-1 (78%), and blaSHV and oxa-2 (each

A total of 48 pathovars of Pseudomonas syringae and eight related species were studied by DNA-DNA hybridization (S1 nuclease method) and ribotyping. The existence of nine discrete genomospecies was indicated. Genomospecies 1 corresponded to P. syringae sensu stricto and included P. syringae pathovars syringae, aptata, lapsa, papulans, pisi, atrofaciens, aceris, panici, dysoxyli and japonica. Genomospecies 2 included P. syringae pathovars phaseolicola, ulmi, mori, lachrymans, sesami, tabaci, morsprunorum, glycinea, ciccaronei, eriobotryae, mellea, aesculi, hibisci, myricae, photiniae and dendropanacis and nomenspecies Pseudomonas savastanoi, Pseudomonas ficuserectae, Pseudomonas meliae and Pseudomonas amygdali, which are thus synonymous. P. amygdali is the earliest valid name for this genomospecies. Genomospecies 3 included P. syringae pathovars tomato, persicae, antirrhini, maculicola, viburni, berberidis, apii, delphinii, passiflorae, philadelphi, ribicola and primulae. We recommend strain CFBP 2212 of P. syringae pv. tomato to serve as the type strain. Genomospecies 4 included ‘Pseudomonas coronafaciens’ and P. syringae pathovars porri, garcae, striafaciens, atropurpurea, oryzae and zizaniae and corresponds to ‘P. coronafaciens’. Genomospecies 5 included P. syringae pv. tremae and corresponds to Pseudomonas tremae sp. nov. Genomospecies 6 included Pseudomonas viridiflava and the presently misidentified pathotype strains of P. syringae pv. ribicola and P. syringae pv. primulae and thus corresponds to P. viridiflava. Genomospecies 7 included P. syringae pv. tagetis and P. syringae pv. helianthi. We recommend strain CFBP 1694 of P. syringae pv. tagetis to serve as a reference strain. Genomospecies 8 included P. syringae pv. theae and Pseudomonas avellanae and thus corresponds to P. avellanae. Genomospecies 9 included P. syringae pv. cannabina and corresponds to Pseudomonas cannabina sp. nov. Ribotyping (Smal and Hincll endonucleases) could separate seven of the nine genomospecies. The unnamed genomospecies 3 and 7 will be named when phenotypic data are available for identification. Two species are described, P. tremae sp. nov. and P. cannabina sp. nov. Other species will be named when phenotypic data are available for identification.

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.

A total of 173 Vibrio cholerae O1 isolates from the recent cholera epidemic in Colombia was analysed by the polymerase chain reaction (PCR) for the genes encoding the A subunit of cholera toxin (ctx A) and the zonula occludens toxin (zot), and by ribotyping. All isolates were positive for ctx A and zot, which was confirmed by hybridisation. Ribotyping with restriction endonuclease Bg/I digestion of total DNA revealed three ribotypes: B5a comprising 165 (96.4%) isolates, and two new designated ribotypes B20 and B21a in six (3.5%) isolates and two (1.1%) isolates, respectively. These findings have significant public health implications.

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

Summary In the framework of a European project entitled “High Resolution Automated Microbial Identification”, the ability of rRNA gene restriction pattern determination (ribotyping) to uncover the taxonomic diversity of a collection of bacteria was tested on 226 strains of Pseudomonas sensu lato. Improved ribotyping methodology included automated DNA extraction and purification, low voltage electrophoresis, vacuum transfer of restricted, DNA fragments to nylon membranes, hybridization with a chemically labeled probe in a hybridization incubator, computer capture of restriction patterns with automatic migration measurements, fragment size interpolation, and comparison of patterns using programs of the Taxotron package (Institut Pasteur, Paris, France). Two endonucleases were used (SmaI and HincII). The strains, belonging to more than 40 nomenspecies, displayed 169 unique rRNA gene restriction patterns with SmaI, and 159 unique rRNA gene restriction patterns with HincII. A combined analysis of both restriction data yielded 79 ribogroups or isolated ribotypes. Most (92.7%) ribogroups were homogeneous with respects to nomenspecies. Some nomenspecies were split in several ribogroups (e.g. P. putida, P. fluorescens, P. marginalis, P. pseudoalcaligenes). Nomenspecies represented by single strains gave isolated ribotypes. Biovars of P. fluorescens and P. putida formed distinct ribogroups. However, P. marginalis pathovars were not separated by ribotyping. When the ribotyping data (ribogroups) were compared to published phenotypic data (phenons) obtained with Biotype-100 strips (BioMerieux, La Balme-les-Grottes, France) and Biolog GN Microplates (Biolog, Hayward, CA), an exclusive phenon-ribogroup correspondence was observed for 13 Biotype-100 phenonslribogroups and for 14 Biolog phenonslribogroups. Other Biotype-100 or Biolog phenons showed partial correspondence with ribogroups or were split into several ribogroups. Most unclustered strains in phenotypic analyses were also unclustered in the ribotyping study. DNA-DNA hybridization was used to verify some species delineation. Ribogroup R38 which contained most of P. fluorescens biovar I strains was found to constitute a DNA hybridization group distinct from P fluorescens strains in other ribogroups or biovars. The type strain of P fluorescens, which was unclustered by ribotyping, was found isolated by DNA relatedness. Ribogroup R1 which contained most of P. putida biovar A strains was found to constitute a DNA relatedness group distinct from biovar B strains. A total of 30 isolates from polluted environment were included in the study and 9 of them were identified to six known taxons. Thirteen other isolates constituted five ribogroups not represented by a reference strain and seven isolates gave isolated ribotypes. This work showed the identification otential of ribotyping and the need to extend the ribotype database. It is now clear that the ribotype carries taxonomic information in addition to typing information.

Molecular biology yielded universal typing methods. However, different methods have different applications. Multilocus enzyme electrophoresis allows precise population genetics studies. Random amplification is useful for quickly comparing a few strains. Pulse field gel electrophoresis, which is more discriminating than ribotyping, is indicated for the study of epidemics which do not last more than a few months. Ribotyping, which gives most stable types, is indicated for tracing clones world-wide and for many years. Automatization of key experimental steps and computer-aided interpretation are essential for an objective comparison of many strains. Databases of molecular typing patterns are in completion.

Objective: To assess the magnitude of a nationwide outbreak of infection with Salmonella enterica serotype paratyphi B and identify the vehicle and source of infection. Design: A case finding study of S paratyphi B infection between 15 August and 30 November 1993; a pair matched case-control study; an environmental investigation at a processing plant that produced a raw goats9 milk cheese incriminated in the outbreak; phage typing and genotyping of food and human S paratyphi B isolates. Setting: France, 15 August to 30 November 1993. Subjects: 273 patients with S paratyphi B infection; 59 pairs of cases and controls matched for age, sex, and city of residence. Main outcome measures: Numbers of cases and incidence rates by region of residence and age; matched odds ratios for dairy food preferences. Results: Among the 273 cases there was one death; 203 (78%) strains belonged to phage type 1 var 3. The incidence of infection was greatest in the region where goats9 milk cheese is commonly produced. Comparison of cases and controls showed a 12-fold greater risk of illness (95% confidence interval 1.6 to 92.3) from eating brand A unpasteurised goats9 milk cheese. S paratyphi B isolates of phage type 1 var 3 were recovered from cheese A, goats9 milk at the plant processing cheese A, and goats9 milk supplied to the plant by a single farm. Genotypic IS 200 typing of food and human 1 var 3 phage type isolates showed a common IS 200 pattern. Conclusion: This outbreak emphasises the potential health hazards of widely distributed unpasteurised milk products in France and the need for their close bacterial monitoring. Key messages Key messages Though the cheese was probably contaminated for more than two months, the outbreak continued undetected for a further two months The source of the infection was goats9 milk from one of the 40 farms that supplied the cheese processing plant Internal microbiological monitoring at the plant was not sensitive enough to detect the salmonella contamination initially Any batch of unpasteurised cheese or milk product should be closely monitored for Salmonella and should not be distributed until known to be clear

Summary The majority (91%) of 260 isolates initially identified as Vibrio anguillarum , that were obtained from a wide range of hosts, habitats and geographical locations, were recovered in a single cluster based on the ribotype and were pathogenic to Atlantic salmon. A significant proportion of isolates (78% of the total) were allocated to 15 serogroups (O1–O10 and five previously undescribed groups referred to as VaNT1, VaNT2, VaNT4, NaNT5 and VaNT7). A minority of isolates (6%) reacted with more than one antiserum or were self-agglutinating, and the remainder did not react with any of the antisera tested. Good correlation was noted between serogroups and lipopolysaccharide profiles, particularly with respect to isolates belonging to serogroups O1, 02 and 04ߝ010. Plasmids were recognized in some serogroups. especially O1, which contained the 67 kb plasmid associated with virulence. However, the 19 profiles based on outer membrane protein patterns did not correspond to the results obtained with the other typing methods. Generally, the isolates were heterogeneous in their biochemical characteristics; 117 profiles were obtained with the API 20E system, and 9 and 32 clusters recognised from the results of BIOLOG fingerprinting and Biotype-100 biotyping methods, respectively. Three dominant dusters were defined from fatty acid methyl esters profiles.

Phylogenetic analysis of partial rpoB gene sequences of type and clinical strains belonging to different 16S rRNA gene-fingerprinting ribogroups within 11 species of enterobacteria of the genera Proteus, Morganella and Providencia was performed and allowed the definition of rpoB clades, supported by high bootstrap values and confirmed by ≥2.5 % nucleotide divergence. None of the resulting clades included strains belonging to different species and the majority of the species were confirmed as discrete and homogeneous. However, more than one distinct rpoB clade could be defined among strains belonging to the species Proteus vulgaris (two clades), Providencia alcalifaciens (two clades) and Providencia rettgeri (three clades), suggesting that some strains represent novel species according to the genotypes outlined by rpoB gene sequence analysis. Percentage differences between the rpoB gene sequence of the type strain of Proteus myxofaciens and other members of the same genus (17.3–18.9 %) were similar to those calculated amongst strains of the genus Providencia (16.4–18.7 %), suggesting a genetic distance at the genus-level between Proteus myxofaciens and the rest of the Proteus–Providencia group. Proteus myxofaciens therefore represents a member of a new genus, for which the name Cosenzaea gen. nov., is proposed.

The intestinal colonization by Enterobacter cloacae strains with a derepressed cephalosporinase was studied in a paediatric ward between February 1990 and January 1991. Environmental sampling was performed simultaneously. Fifty-two isolates were recovered from 200 neonates (stool, blood) and 14 strains were isolated from the neonatal environment. An epidemiological study based on the typing of 36 Enterobacter cloacae isolates was carried out using antibiotyping, biotyping and ribotyping methods. The isolates selected were from 21 neonates (24 isolates), the neonatal ward environment (8 isolates) and from other wards (4 isolates). Thirty-two isolates had the same antibiotic resistance pattern, corresponding to a derepressed cephalosporinase and resistance to the following aminoglycosides: kanamycin, gentamicin, tobramycin and netilmicin. No predominant biotyping pattern could be established. Ribotyping done with two endonucleases (EcoRI and BamHI) showed 28 Enterobacter cloacae isolates to have a single pattern. Ribotyping was the most discriminating method used in this study, permitting identification of cross-contamination with Enterobacter cloacae in the paediatric ward.

In 1987, an outbreak of pneumonia and meningitis caused by an unknown bacterium occurred in a spa therapy centre. Nine isolates of this pathogen constituted a tight DNA hybridization group. rRNA-DNA hybridization and 16S rRNA sequencing showed that the studied bacteria represented a new branch in superfamily II (= gamma subclass) of the Proteobacteria, close to the genus Oceanospirillum. The new bacterium was highly polymorphic and, in young cultures, had curved Gram-negative cells, motile by polar single flagella. The new bacterium differed from the genus Oceanospirillum by its lacking the NaCl requirement and by reducing nitrate into nitrite, producing indole from tryptophan and producing acid from carbohydrates. The name Balneatrix alpica gen. nov., sp. nov. is proposed for the studied organism. The type strain is strain 4-87 (= CIP 103589).

A comparative study of six 01 serotype Vibrio anguillarum strains from different areas and fishes was realized with three methods, of which two for the first time in fish pathology. Three plasmid profiles were observed but all strains had the same rRNA gene restriction pattern.

An analysis of 23 Listeria ivanovii strains in which we used multilocus enzyme electrophoresis at 18 enzyme loci showed that this bacterial species could be divided into two main genomic groups. The results of DNA-DNA hybridizations and rRNA gene restriction patterns confirmed this finding. The DNA homology data suggested that the two genomic groups represent two subspecies, L. ivanovii subsp. ivanovii and L. ivanovii subsp. londoniensis subsp. nov. The two subspecies can be distinguished biochemically on the basis of the ability to ferment ribose and N-acetyl-β-d-mannosamine. The type strain of L. ivanovii subsp. londoniensis is strain CLIP 12229 (=CIP 103466).

In a prospective matched case-control study of sporadic pediatric hemolytic uremic syndrome related to Shiga-toxin producing Escherichia coli infection in France, eating undercooked ground beef, contact with a person with diarrhea, and drinking well water during the summer period were identified as risk factors. Prevention efforts in France should focus on reducing not only food-borne but also person-to-person transmission.

The rRNA gene restriction patterns of two species of nutritionally variant streptococci, Streptococcus defectivus and Streptococcus adjacens, were determined, and the results were compared with the electrophoretic migration profiles of penicillin-binding proteins. Reference strains belonging to various streptococcal species were used as controls. Our results correlated with the results of DNA-DNA hybridization experiments and confirmed the delineation of these two species. Moreover, they demonstrated that intraspecies variations occur and suggested that there are two subspecies of S. defectivus.

Background The reemergence of epidemic diphtheria in Belarus in 1990s has provided us with important information on the biology of the disease and the diversity of the causative agent Corynebacterium diphtheriae. Molecular investigations were conducted with the aim to analyze the genetic variability of C diphtheriae during the post-epidemic period. Methods The biotype and toxigenicity status of 3513 C. diphtheriae strains isolated from all areas in Belarus during a declining period of diphtheria morbidity (1996–2005) was undertaken. Of these, 384 strains were isolated from diphtheria cases, 1968 from tonsillitis patients, 426 from contacts and 735 from healthy carriers. Four hundred and thirty two selected strains were ribotyped. Results The C diphtheriae gravis biotype, which was prevalent during 1996–2000, was "replaced" by the mitis biotype during 2001–2005. The distribution of toxigenic C. diphtheriae strains also decreased from 47.1% (1996) to 5.8% (2005). Changes in the distribution of the epidemic ribotypes Sankt-Peterburg and Rossija were also observed. During 2001–2005 the proportion of the Sankt-Peterburg ribotype decreased from 24.3% to 2.3%, in contrast to the Rossija ribotype, that increased from 25.1% to 49.1%. The circulation of other toxigenic ribotypes (Otchakov, Lyon, Bangladesh), which were prevalent during the period of high diphtheria incidence, also decreased. But at the same time, the proportion of non-toxigenic strains with the Cluj and Rossija ribotypes dramatically increased and accounted for 49.3% and 30.1%, respectively. Conclusion The decrease in morbidity correlated with the dramatic decrease in the isolation of the gravis biotype and Sankt Peterburg ribotype, and the prevalence of the Rossija ribotype along with other rare ribotypes associated with non-toxigenic strains (Cluj and Rossija, in particular).