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The current study describes a novel species with the strain name ERMR1:05

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.

Wave upon wave of diseaseThe cholera pathogen,Vibrio cholerae, is considered to be ubiquitous in water systems, making the design of eradication measures apparently fruitless. Nevertheless, local and globalVibriopopulations remain distinct. Now, Weillet al.and Dommanet al.show that a surprising diversity between continents has been established. Latin America and Africa bear different variants of cholera toxin with different transmission dynamics and ecological niches. The data are not consistent with the establishment of long-term reservoirs of pandemic cholera or with a relationship to climate events.Science, this issue p.785, p.789

International audience; Four bacterial strains isolated from peat bog soil or swampy meadow in Baden-Württemberg (Germany) and found to have rrs sequences close to that of Rouxiella chamberiensis were compared to this species by using multi-locus sequence analysis and phenotypic tests. The four strains constituted two discrete groups (referred to as the Baden and the Silva groups) belonging to the genus Rouxiella. These groups differed in their ability to grow at 37 °C, reduce nitrate into nitrite, and to produce acid from several carbohydrates. Two novel species are, therefore, proposed: Rouxiella badensis sp. nov. for the Baden group (type strain, 323T=CIP 111153T=DSM 100043T) and Rouxiella silvae for the Silva group (type strain, 213T=CIP 111154T=DSM 103735T). The definition of the genus Rouxiellahas also been emended in order to take these two novel species into account.

Wave upon wave of disease The cholera pathogen, Vibrio cholerae , is considered to be ubiquitous in water systems, making the design of eradication measures apparently fruitless. Nevertheless, local and global Vibrio populations remain distinct. Now, Weill et al. and Domman et al. show that a surprising diversity between continents has been established. Latin America and Africa bear different variants of cholera toxin with different transmission dynamics and ecological niches. The data are not consistent with the establishment of long-term reservoirs of pandemic cholera or with a relationship to climate events. Science , this issue p. 785 , p. 789

Latin America has experienced two of the largest cholera epidemics in modern history; one in 1991 and the other in 2010. However, confusion still surrounds the relationships between globally circulating pandemic

This supplement (no. 48) of the White-Kauffmann-Le Minor scheme reports on the characterization of 63 new Salmonella serovars and 25 new variants of previously described Salmonella serovars recognized by the WHO Collaborating Centre for Reference and Research on Salmonella between 2008 and 2010. Forty-four new serovars were assigned to Salmonella enterica subspecies enterica, 12 to subspecies salamae, two to subspecies arizonae, two to subspecies diarizonae and three to subspecies houtenae. All these new serovars or new variants are described with their multilocus sequence type.

Phylogenetic analysis of strains from Burkholderia, Ralstonia, Cupriavidus, Comamonas, Delftia, Acidovorax, Brevundimonas, Herbaspirillum huttiense and "Pseudomonas butanovora" was performed based on the protein-coding genes rpoB and gyrB and on the 16S rRNA-coding gene rrs. Overall, the phylogenies deduced from the three genes were concordant among themselves and with current taxonomy. However, a few differences among individual gene phylogenies were noted. For example, the separation of Cupriavidus from Ralstonia was not supported in the rpoB tree, as Ralstonia was nested within Cupriavidus. Similarly, the separation of Delftia from Comamonas was not supported in the gyrB tree. Based on rrs and rpoB, the genus Burkholderia contained four groups: (i) the B. cepacia complex, (ii) the B. pseudomallei-B. thailandensis group, (iii) a 6-species group including B. caledonica and B. glathei and (iv) the B. plantarii-B. glumae-B. gladioli group. However, B. caribensis and B. glathei stood as a fifth group based on gyrB. It appears that a phylogeny cannot be reliably based on a single gene. Using rpoB and gyrB, better separation of closely related species was obtained compared to rrs, indicating the potential of these two genes for identification and species definition. Nevertheless, intraspecific sequence diversity will need to be determined to fully establish the value of these genes for strain identification.

Salmonella enterica serotype Typhi clinical isolates ( n = 91) resistant to nalidixic acid (Nal r ) were collected from sporadic cases and minor outbreaks throughout Vietnam between 1996 and 2004. These isolates were typed and compared by four methods: Vi phage typing, PstI ribotyping, XbaI and SpeI pulsed-field gel electrophoresis (PFGE), and single-nucleotide polymorphism (SNP) analysis. The results indicated that 65% of the isolates were not typeable by Vi phage typing. In contrast, the ribotyping and, with more accuracy, the SNP analysis methods indicated that all Nal r isolates belonged to a single clone (ribotype 3a, haplotype H58) that was found previously and that largely consisted of plasmid-encoded multidrug-resistant serotype Typhi isolates. PFGE demonstrated the occurrence of microevolution within this clone. We identified two major combined PFGE profiles: X1-S1 and X3-S6. X3-S6 predominated between 1996 and 2002 but was replaced by X1-S1 after 2002. Nevertheless, PFGE, with a Simpson's index of 0.78, was not considered an optimal discriminatory method for investigating typhoid fever outbreaks in Vietnam. The rate of quinolone resistance increased and the rate of multidrug resistance decreased during the study period. From 2002 to 2004, 80.6% of the isolates from South Vietnam were resistant only to Nal. The mechanism of Nal resistance in most of the isolates (94%) was a mutation in the quinolone resistance-determining chromosomal region of gyrA that led to the amino acid substitution Ser83Phe. No plasmid-located qnrA , qnrB , or qnrS was detected.

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.

In the last three decades, high rates of resistance to common first-line antimicrobial agents have been reported in Salmonella enterica serotype Typhi (Typhi), the causative organism of typhoid fever (TF), in many regions of the world, especially in South East Asia. Analysis of Typhi strains isolated from outbreaks and sporadic cases of TF in Son La province, northwest Vietnam, in 2002 revealed that 94.5% (85/90) of the isolates were fully susceptible to amoxicillin, chloramphenicol, cotrimoxazole, tetracycline, and nalidixic acid. There was a clear decline in the occurrence of multi-drug resistant (MDR) Typhi isolates collected in this province in 2002 (4.4%) compared with the period 1995-1999 in the same province (30.8-100%). By using molecular (IS200 profiling, PstI-ribotyping, XbaI-pulsed-field gel electrophoresis, and haplotyping) and phage-typing methods, we showed that the Typhi isolates from Son La province in 2002 were genetically related; however, they were unrelated to the previous MDR clones established in Vietnam.

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.

Antibiotic treatment is not required in cases of Salmonella enterica gastroenteritis but is essential in cases of enteric fever or invasive salmonellosis or in immunocompromised patients. Although fluoroquinolones and extended-spectrum cephalosporins are the drugs of choice to treat invasive Salmonella , resistance to these antibiotics is increasing worldwide. During the period 2000 to 2003, 90 Salmonella enterica serovar Virchow poultry and poultry product isolates and 11 serovar Virchow human isolates were found to produce an extended-spectrum β-lactamase, CTX-M-2, concomitantly with a TEM-1 β-lactamase. The bla CTX-M-2 gene was located on a large conjugative plasmid (>100 kb). Pulsed-field gel electrophoresis indicated a clonal relationship between the poultry and human isolates. All these isolates displayed additional resistance to trimethoprim-sulfamethoxazole and tetracycline as well as a reduced susceptibility to ciprofloxacin (MICs of between 0.5 and 1 μg/ml). CTX-M-2-producing Salmonella with a reduced susceptibility to fluoroquinolones constitutes a major concern, since such strains could disseminate on a large scale and jeopardize classical antibiotic therapy in immunocompromised patients.

Two bla OKP subgroups were found, diverging by 4.2%. Subgroups bla OKP-A (10 enzyme variants, pIs from 7.1 to 8.3) and bla OKP-B (11 variants, pI 7.1) showed similar antibiotic susceptibilities. Sequencing of rpoB , gyrA , and mdh demonstrated a concordant subdivision of Klebsiella pneumoniae phylogenetic group KpII into two subgroups, KpII-A and KpII-B.

The diversity and evolution of the class A OXY β-lactamase from Klebsiella oxytoca were investigated and compared to housekeeping gene diversity. The entire bla OXY coding region was sequenced in 18 clinical isolates representative of the four K. oxytoca β-lactamase gene groups bla OXY-1 to bla OXY-4 and of two new groups identified here, bla OXY-5 (with four isolates with pI 7.2 and one with pI 7.7) and bla OXY-6 (with four isolates with pI 7.75 and three with pI 8.1). Genes bla OXY-5 and bla OXY-6 showed 99.8% within-group nucleotide similarity but differed from each other by 4.2% and from bla OXY-1 , their closest relative, by 2.5% and 2.9%, respectively. Antimicrobial susceptibility to β-lactams was similar among OXY groups. Nucleotide sequence diversity of the 16S rRNA (1,454 bp), rpoB (940 bp), gyrA (383 bp), and gapDH (573 bp) genes was in agreement with the β-lactamase gene phylogeny. Strains with bla OXY-1 , bla OXY-2 , bla OXY-3 , bla OXY-4 , and bla OXY-6 genes formed five phylogenetic groups, named KoI, KoII, KoIII, KoIV, and KoVI, respectively. Isolates harboring bla OXY-5 appeared to represent an emerging lineage within KoI. We estimated that the bla OXY gene has been evolving within K. oxytoca for approximately 100 million years, using as calibration the 140-million-year estimation of the Escherichia coli - Salmonella enterica split. These results show that the bla OXY gene has diversified along K. oxytoca phylogenetic lines over long periods of time without concomitant evolution of the antimicrobial resistance phenotype.

Until recently, morphotyping, a method evaluating fringe and surface characteristics of streak colonies grown on malt agar, has been recommended as a simple and unexpensive typing method for Candida albicans isolates. The discriminatory power and reproducibility of Hunter's modified scheme of Phongpaichit's morphotyping has been evaluated on 28 C. albicans isolates recovered from the oral cavity of asymptomatic human immunodeficiency virus-positive subjects, and compared to two molecular typing methods: randomly amplified polymorphic DNA (RAPD) fingerprinting, and contour clamped homogeneous electric field (CHEF) electrophoretic karyotyping. Morphological features of streak colonies allowed to distinguish 11 different morphotypes while RAPD fingerprinting yielded 25 different patterns and CHEF electrophoresis recognized 9 karyotypes. The discriminatory power calculated with the formula of Hunter and Gaston was 0.780 for morphotyping, 0.984 for RAPD fingerprinting, and 0.630 for karyotyping. Reproducibility was tested using 43 serial isolates from 15 subjects (2 to 6 isolates per subject) and by repeating the test after one year storage of the isolates. While genetic methods generally recognized a single type for all serial isolates from each of the subjects studied, morphotyping detected strain variations in five subjects in the absence of genetic confirmation. Poor reproducibility was demonstrated repeating morphotyping after one year storage of the isolates since differences in at least one character were detected in 92.9% of the strains.

A multilocus sequence typing (MLST) scheme was developed for Klebsiella pneumoniae . Sequences of seven housekeeping genes were obtained for 67 K. pneumoniae strains, including 19 ceftazidime- and ciprofloxacin-resistant isolates. Forty distinct allelic profiles were identified. MLST data were validated against ribotyping and showed high (96%) discriminatory power. The MLST approach provides unambiguous data useful for the epidemiology of K. pneumoniae isolates.

This study was conducted to investigate the occurrence of multiple-antibiotic resistance among 261 clinical isolates of Salmonella enterica serotype Paratyphi B strains collected between 2000 and 2003 through the network of the French National Reference Center for Salmonella . The 47 multidrug-resistant (MDR) isolates identified (18%), were characterized on the basis of the presence of several resistance genes ( bla TEM , bla PSE-1 , bla CTX-M , floR , aadA2 , qacEΔ1 , and sul1 ), the presence of Salmonella genomic island 1 (SGI1) by PCR mapping and hybridization, and the clonality of these isolates by several molecular (ribotyping, IS 200 profiling, and pulsed-field gel electrophoresis [PFGE]) and phage typing methods. The results of PCR and Southern blot experiments indicated that 39 (83%) of the 47 S. enterica serotype Paratyphi B biotype Java MDR isolates possessed the SGI1 cluster (MDR/SGI1). Among these 39 MDR/SGI1 isolates, only 3 contained variations in SGI1, SGI1-B ( n = 1) and SGI1-C ( n = 2). The 39 MDR/SGI1 isolates showed the same specific PstI-IS 200 profile 1, which contained seven copies from 2.6 to 18 kb. Two PstI ribotypes were found in MDR/SGI1 isolates, RP1 ( n = 38) and RP6 ( n = 1). Ribotype RP1 was also found in two susceptible strains. Analysis by PFGE using XbaI revealed that all the MDR/SGI1 isolates were grouped in two related clusters, with a similarity percentage of 82%. Isolation of MDR/SGI1 isolates in France was observed mainly between the second quarter of 2001 and the end of 2002. The source of the contamination has not been identified to date. A single isolate possessing the extended-spectrum β-lactamase bla CTX-M-15 gene was also identified during the study.

Three clinical isolates of Salmonella enterica recovered between 2000 and 2003 in France and Senegal were found to produce extended-spectrum b-lactamase CTX-M-15. The two isolates from Senegal were recovered from stool of a hospitalized patient with gastroenteritis in 2000 and from an urine specimen of an out-patient with urinary tract infection in 2001. These S. enterica isolates belonged to serotype Kentucky and were clonally related as determined by pulsed-field gel electrophoresis and automated ribotyping. The third isolate of serotype Typhimurium was recovered from a patient hospitalized in France for an acute gastroenteritis acquired in Lebanon. The blaCTX-M-15 gene was located on two different transferable plasmids, one of which also carried blaTEM-1, blaOXA-30, aminoglycoside-, tetracycline-, and sulfamethoxazole–trimethoprim resistance genes. ISEcp1 element was found to be located upstream of blaCTX-M-15 in the same position as reported previously in CTX-M-15-producing Escherichia coli from India and Turkey. This is the first report of blaCTX-M-15 in the genus Salmonella. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Screening of aerobic culturable hydrocarbon (HC)-degrading microorganisms isolated from petroleum-polluted soils and cyanobacterial mats from Indonesia resulted in the collection of 33 distinct species. Eight bacteria, 21 fungi and 4 yeasts were identified to the specific level by molecular and phenotypic techniques. Bacterial strains belonged to the genera Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia and Mycobacterium. Four species are new and not yet described. Fungi belonged to Aspergillus, Penicillium, Fusarium, Amorphoteca, Neosartorya, Paecilomyces, Talaromyces and Graphium. Yeasts were Candida, Yarrowia and Pichia. All strains were cultivated axenically in synthetic liquid media with crude oil as sole carbon and energy source. After incubation, the detailed chemical composition of the residual oil was studied by gravimetric and gas-chromatographic techniques. Thirteen parameters for assessing the biodegradation potential were defined and computed for each strain. Maximum degradation was observed on the saturated HCs (n- and isoalkanes, isoprenoids), whereas aromatic HC degradation was lower and was related to the structural composition of the molecules. A principal components analysis (PCA) permitted grouping and classifying the strains as a function of their degradative capacities. It was shown that the most active strains produced polar metabolites which accumulated in the resins and asphaltene fractions. These fractions are highly resistant to microbial metabolism. No taxonomic trend could be defined between microbial phyla in terms of HC biodegradation activity.

From December 2002 to June 2003, 14 cultures of Salmonella enterica serotype Babelsberg and 6 cultures of serotype Enteritidis, isolated in France from internationally adopted children, were identified at the French National Reference Center for Salmonella . All serotype Babelsberg isolates were related, as determined by pulsed-field gel electrophoresis, and all serotype Enteritidis strains displayed the same phage type. All serotype Enteritidis and seven serotype Babelsberg isolates produced an SHV-12-like extended-spectrum β-lactamase as determined by sequencing of PCR products and by isoelectrofocusing. Some serotype Enteritidis isolates exhibited additional antimicrobial resistance (aminoglycosides, tetracycline, chloramphenicol, sulfonamides, and trimethoprim). Our investigation indicated that these Salmonella isolates were certainly acquired in the same orphanage in Bamako, Mali, before the children were adopted by French families. An inappropriate use of ceftriaxone was probably the cause of the emergence of such strains. There is an urgent need to determine the origin of the contamination and to introduce adequate antibiotic protocols into this orphanage to prevent further transmission and dissemination. Screening for infections and follow-up, adapted to the origin of the internationally adopted children, should be recommended.

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.

Many molecular typing methods are difficult to interpret because their repeatability (within-laboratory variance) and reproducibility (between-laboratory variance) have not been thoroughly studied. In the present work, ribotyping of coryneform bacteria was the basis of a study involving within-gel and between-gel repeatability and between-laboratory reproducibility (two laboratories involved). The effect of different technical protocols, different algorithms, and different software for fragment size determination was studied. Analysis of variance (ANOVA) showed, within a laboratory, that there was no significant added variance between gels. However, between-laboratory variance was significantly higher than within-laboratory variance. This may be due to the use of different protocols. An experimental function was calculated to transform the data and make them compatible (i.e., erase the between-laboratory variance). The use of different interpolation algorithms (spline, Schaffer and Sederoff) was a significant source of variation in one laboratory only. The use of either Taxotron (Institut Pasteur) or GelCompar (Applied Maths) was not a significant source of added variation when the same algorithm (spline) was used. However, the use of Bio-Gene (Vilber Lourmat) dramatically increased the error (within laboratory, within gel) in one laboratory, while decreasing the error in the other laboratory; this might be due to automatic normalization attempts. These results were taken into account for building a database and performing automatic pattern identification using Taxotron. Conversion of the data considerably improved the identification of patterns irrespective of the laboratory in which the data were obtained.

'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.

Culturable cells and non-culturable cells of fecal coliforms, obtained by irradiation at 312 nm were submitted to the combined stress conditions of salinity and starvation. After 14 days, β-galactosidase activity of UV-irradiated cells was at least twice the value of non-irradiated cells. UV-irradiated cells thus contribute more than non-irradiated cells to the enzyme assay after incubation in saline water. This finding is essential for the interpretation of quantitative investigations into the environment using enzymatic methods.

On the basis of phenotypic and DNA relatedness data, three novel species of the genus Bosea are proposed, Bosea massiliensis (63287T =CIP 106336T =CCUG 43117T), Bosea vestrisii (34635T =CIP 106340T =CCUG 43114T) and Bosea eneae (34614T =CIP 106338T =CCUG 43111T). The original description of the genus Bosea included thiosulphate oxidation as a phenotypic feature, when the sole and type species of the genus, Bosea thiooxidans, was proposed. The three novel species described herein were not able to oxidize thiosulphate; thus, it is proposed that this characteristic be removed from the description of the genus and considered as specific for B. thiooxidans. The novel species of the genus Bosea proposed here form a well-separated cluster in the Bradyrhizobium group of the alpha-2 subclass of the Proteobacteria, on the basis of 16S rDNA gene sequence analysis. However, 16S rDNA gene sequence analysis was not sufficient to delineate the species; hence, DNA-DNA relatedness and phenotypic data were also required. All of the novel species described in this study are fastidious bacteria isolated from a hospital water supply, using co-cultivation with amoebae. This group of bacteria are hypothesized to be a potential cause of nosocomial infections. For treatment of infections caused by these novel bacteria, doxycycline appears to be the sole antibacterial compound with a consistently low MIC value.

Kluyvera ascorbata produces a β-lactamase that results in an atypical susceptibility pattern, including low-level resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate. Ten nucleotide sequences of the corresponding gene, bla KLUA , were obtained and were found to have minor variations (96 to 100%). Otherwise, bla KLUA was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type β-lactamases. Finally, mobilization of bla KLUA on a plasmid was found to be mediated probably by a genetic mobile element like IS Ecp1 .

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.

Advances in molecular taxonomy of bacteria have generated many tools allowing identification of any bacteria, whether culturable or not. In addition to taxonomic identification, growing knowledge on pathogenicity mechanisms allows the detection of bacteria with given virulence genes. The problem is in asking the proper questions so that the most appropriate tools can be chosen. Some rapid identification tools (gene amplification, in situ hybridisation) require a starting hypothesis. Other tools (rrs gene amplification and sequencing) can be used without prior hypothesis, but take longer. To face the bioterrorist threat, particular attention should be given to laboratory and strain dispatch (within- and between-laboratory) organisation.

International audience; An ad hoc committee for the re-evaluation of the species definition in bacteriology met in Gent, Belgium, in February 2002. The committee made various recommendations regarding the species definition in the light of developments in methodologies available to systematists.

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.

Six isolates recovered from coffee seeds giving off a potato-like flavour were studied. Gene sequencing (rrs and rpoB) showed they belong to the genus Pantoea . By DNA–DNA hybridization, the isolates constituted a genomic species with less than 17 % relatedness to 96 strains representing enterobacterial species. Multilocus sequence analysis (gyrB, rpoB, atpD and infB genes) showed the isolates to represent a discrete species of the genus Pantoea . Nutritional versatility of the novel species was poor. The novel species is proposed as Pantoea coffeiphila sp.nov. and its type strain is Ca04T ( = CIP 110718T = DSM 28482T).

Strains previously classified as members ofKlebsiella pneumoniaephylogroups KpI, KpII-A, KpII-B and KpIII were characterized by 16S rRNA (rrs) gene sequencing, multilocus sequence analysis based onrpoB,fusA,gapA,gyrAandleuSgenes, average nucleotide identity and biochemical characteristics. Phylogenetic analysis demonstrated that KpI and KpIII corresponded toK. pneumoniaeandKlebsiella variicola, respectively, whereas KpII-A and KpII-B formed two well-demarcated sequence clusters distinct from other members of the genusKlebsiella. Average nucleotide identity between KpII-A and KpII-B was 96.4 %, whereas values lower than 94 % were obtained for both groups when compared withK. pneumoniaeandK. variicola. Biochemical properties differentiated KpII-A, KpII-B,K. pneumoniaeandK. variicola, with acid production from adonitol andl-sorbose and ability to use 3-phenylproprionate, 5-keto-d-gluconate and tricarballylic acid as sole carbon sources being particularly useful. Based on their genetic and phenotypic characteristics, we propose the namesKlebsiella quasipneumoniaesubsp.quasipneumoniaesubsp. nov. andK. quasipneumoniaesubsp.similipneumoniaesubsp. nov. for strains of KpII-A and KpII-B, respectively. The type strain ofK. quasipneumoniaesp. nov. and ofK. quasipneumoniaesubsp.quasipneumoniaesubsp. nov. is 01A030T( = SB11T = CIP 110771T = DSM 28211T). The type strain ofK. quasipneumoniaesubsp.similipneumoniaesubsp. nov. is 07A044T( = SB30T = CIP 110770T = DSM 28212T). Both strains were isolated from human blood cultures. This work also showed thatKlebsiella singaporensisis a junior heterotypic synonym ofK. variicola.

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.

Nonculturable segmented filamentous bacteria (SFB) have been described in the gut of rats, mice and chickens, and 16S rRNA sequences for these organisms are available. These organisms, peripherically related to Clostridium phylogenetic group I, have been provisionally named 'Candidatus Arthromitus'. This work reports the observation of similar bacteria in the intestinal content of the distal intestine, preferentially, in the adult rainbow trout (Oncorhynchus mykiss) that exhibited episodic acute diarrhea, usually during the summer. Abdominal distension, intestinal fluid-mucus content and epithelium detachment were observed in trout. The demonstration that the observed microorganisms are bacteria and belong in the 'Candidatus Arthromitus' group was achieved by in situ hybridization with, respectively, a eubacterial probe and an oligonucleotide probe designed to react specifically with SFB 16S rRNA (encoded by the rrs gene) sequences. The sequenced rrs gene was compared with published sequences and found to be closely related to (although distinct from) other SFB sequences. Implication of these bacteria in trout diarrheic illness remains hypothetical.

The diversity of symbiotic dinoflagellates from reef-building corals collected in the lagoon of Tahiti (South Pacific ocean) was investigated by using a molecular approach. Populations of symbionts (strains or species) of 7 coral species (Fungia scutaria, F. paumotensis Stutchbury, Pavona cactus Forskal, Leptastrea transversa Kluzinger, Pocillopora verrucosa Ellis and Solender, Montastrea curta Dana, and Acropora formosa Dana) were delimited by phylogenetic analysis of small subunit rDNA sequences. Coral P. verrucosa harbored 2 populations of symbiont SSU rDNA sequences that may correspond to two different Symbiodinium species. Corals F. scutaria and M. curta also seemed to contain two different Symbiodinium species. SSU rDNA dinoflagellate sequences from P. cactus, L. transversa, F. scutaria, F. paumotensis, and P. verrucosa were in the same phylogenetic cluster and showed low variability. For these distantly related coral species, dinoflagellate strains from the same species, rDNA paralogues from the same strain, or closely related Symbiodinium species could not be distinguished because monophyletic subgroups were not observed. SSU rDNA dinoflagellate sequences from A. formosa and M. curta were clearly different from the other Symbiodinium sequences and may represent specific species. This molecular approach highlighted a greater diversity of symbiotic dinoflagellates from corals in South Pacific (Symbiodinium groups A, B, and C) than that observed in the rest of the Pacific ocean (Symbiodinium group C). The diversity of symbiotic associations in a restricted area of the lagoon of Tahiti may reflect the complexity of interactions between species of Symbiodinium and corals.

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.

There are several occasions when enumeration of Escherichia coli cells is needed. These include examination of urine specimens and water or food samples. Present methods rely on growth in more or less selective media (colony-forming units on agar or the most probable number method using liquid media). Unfortunately, no really selective medium with 100% efficiency of plating is available for E. coli. A 24-mer oligonucleotide probe (Colinsitu), complementary to a piece of 16S ribosomal ribonucleic acid, has been tested for specifically visualizing E. coli cells by in situ hybridization and epifluorescence microscopy. The fluorescent dye-labeled probe was able to stain cells of E. coli, Shigella spp. and E. fergusonii. Shigella spp. are known to belong to the E. coli genomospecies and E. fergusonii is the nomenspecies closest to E. coli by DNA–DNA hybridization. The probe did not stain any strain of 169 other genomospecies of the family Enterobacteriaceae or of a few other species frequently encountered in the environment. Revivification without cell division allowed the visualization of E. coli cells in contaminated water. In situ hybridization using the Colinsitu probe is a potential tool for the confirmation of (atypical) E. coli in reference centers and the rapid (3–6 h) detection and enumeration of E. coli in urine specimens, contaminated water and food. More work is needed to include in situ hybridization in laboratory routine.

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.

To determine risk factors for the occurrence of sporadic Salmonella typhimurium infections among children in France, we conducted a matched case-control study. Cases were identified between 15 June and 30 September 1996. We interviewed 101 pairs of case patients and control subjects, matched for age and place of residence. The risk of illness was greater for children who ate undercooked ground beef than for those who did not (odds ratio [OR], 5.0; 95% confidence interval [CI], 1.9-13.1). Case patients were more likely than control subjects to have taken antibiotics during the month before onset of disease (OR, 2.2; 95% CI, 1.0-4.9). Case patients

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.

The influence of growth temperature on the ability to ferment d -sorbitol was investigated in Escherichia coli O157:H7. It was found that O157:H7 strains have a temperature-sensitive sorbitol phenotype. d -Sorbitol transport and sorbitol-6-phosphate dehydrogenase activities were expressed in sorbitol-fermenting cells grown at 30°C but only at a low level at 40°C. Sorbitol-positive variants able to transport d -sorbitol were easily selected at 30°C from culture of Sor − E. coli O157:H7 strains.