Rahnella sikkimica sp. nov., a novel cold-tolerant bacterium isolated from the glacier of Sikkim Himalaya with plant growth-promoting properties.

The current study describes a novel species with the strain name ERMR1:05T isolated from the forefield soil of East Rathong Glacier in West Sikkim Himalaya (India). The isolate was facultatively anaerobic, gram-stain negative, non-spore-forming, rod-shaped, and oxidase negative. Whole-genome-based bacterial core gene phylogenetic analysis placed the strain in the genus Rahnella, well separated from Rouxiella spp. The digital DNA-DNA hybridisation and average nucleotide identity values between strain ERMR1:05T and other members of genus Rahnella were below the proposed thresholds for the species delineation. Based on these results, a new species, Rahnella sikkimica sp. nov., is proposed with strain ERMR1:05T (CIP 111636T, MTCC 12598T) as the type strain. The bacterium showed upregulation of cold-stress genes in cold conditions. Additionally, the genome analysis of the bacterium showed the presence of plant growth-promotion factors suggesting its role in crop improvement in cold hilly regions.

Rouxiella badensis sp. nov. and Rouxiella silvae sp. nov. isolated from peat bog soil and emendation description of the genus Rouxiella.

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.

Pantoea coffeiphila sp. nov., cause of the 'potato taste' of Arabica coffee from the African Great Lakes region.

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 Ca04(T) ( =CIP 110718(T) =DSM 28482(T)).

Description of Klebsiella quasipneumoniae sp. nov., isolated from human infections, with two subspecies, Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a junior heterotypic synonym of Klebsiella variicola.

Strains previously classified as members of Klebsiella pneumoniae phylogroups KpI, KpII-A, KpII-B and KpIII were characterized by 16S rRNA (rrs) gene sequencing, multilocus sequence analysis based on rpoB, fusA, gapA, gyrA and leuS genes, average nucleotide identity and biochemical characteristics. Phylogenetic analysis demonstrated that KpI and KpIII corresponded to K. pneumoniae and Klebsiella variicola, respectively, whereas KpII-A and KpII-B formed two well-demarcated sequence clusters distinct from other members of the genus Klebsiella. Average nucleotide identity between KpII-A and KpII-B was 96.4 %, whereas values lower than 94 % were obtained for both groups when compared with K. pneumoniae and K. variicola. Biochemical properties differentiated KpII-A, KpII-B, K. pneumoniae and K. variicola, with acid production from adonitol and l-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 names Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and K. quasipneumoniae subsp. similipneumoniae subsp. nov. for strains of KpII-A and KpII-B, respectively. The type strain of K. quasipneumoniae sp. nov. and of K. quasipneumoniae subsp. quasipneumoniae subsp. nov. is 01A030(T) ( = SB11(T) = CIP 110771(T) = DSM 28211(T)). The type strain of K. quasipneumoniae subsp. similipneumoniae subsp. nov. is 07A044(T) ( = SB30(T) = CIP 110770(T) = DSM 28212(T)). Both strains were isolated from human blood cultures. This work also showed that Klebsiella singaporensis is a junior heterotypic synonym of K. variicola.

Serological cross-reaction between O-antigens of Shigella dysenteriae type 4 and an environmental Escherichia albertii isolate.

An environmental freshwater bacterial isolate, DM104, appearing as Shigella-like colonies on selective agar plates was found to show strong and specific serological cross-reactivity with Shigella dysenteriae type 4. Biochemical identification according to the analytical profile index, molecular serotyping by restriction of the amplified O-antigen gene cluster (rfb-RFLP), together with phylogenetic analysis of the 16S rRNA gene and multi-locus sequence analysis, identified the isolate as Escherichia albertii. rfb-RFLP of DM104, revealed a profile different from that of S. dysenteriae type 4. However, western blot analysis of extracted lipopolysaccharides demonstrated strong cross-reactivity with S. dysenteriae type 4 using specific monovalent antisera and a lipopolysaccharide gel banding profile similar to that of S. dysenteriae type 4. The observed O-antigen cross-reaction between an E. albertii isolate and S. dysenteriae extends our knowledge of the extent of O-antigen cross-reaction within the Escherichia/Shigella group of organisms, and offers the possibility of using DM104 and similar cross-reacting strains as shigellosis vaccine candidates.

The genus Serratia revisited by genomics.

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 highlight the genomic, phenotypic and plasmid diversity of Serratia, and provide evidence of different patterns of gene flow across the genus. Our work provides a framework for understanding the emergence of clinical and other lineages of Serratia.

Phylogenetic analysis of the genera Proteus, Morganella and Providencia by comparison of rpoB gene sequences of type and clinical strains suggests the reclassification of Proteus myxofaciens in a new genus, Cosenzaea gen. nov., as Cosenzaea myxofaciens comb. nov.

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.

Cholera in Ethiopia in the 1990 s: epidemiologic patterns, clonal analysis, and antimicrobial resistance.

In 1993, after 6 years of absence, cholera re-emerged in the Horn of Africa. Following its introduction to Djibouti, the disease spread to the central and southern areas of Ethiopia reaching Somalia in 1994. Cholera outbreaks persisted in Ethiopia with a recrudescence of cases in 1998. Twenty-two Vibrio cholerae O1 strains, selected to represent the 1998 history of cholera in Ethiopia, were characterized by random amplified polymorphic DNA patterns, BglI ribotyping and antimicrobial susceptibility. All isolates showed a unique amplified DNA pattern and a prevalent ribotype B8a. All strains were multidrug-resistant and harboured an IncC plasmid which conferred resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and trimethoprim. These findings indicate that a group of closely related V. cholerae O1 strains was responsible for the cholera epidemic in Ethiopia in 1998.

Clonal relationship among Vibrio cholerae O1 El Tor strains isolated in Somalia.

One hundred and three Vibrio cholerae O1 strains, selected to represent the cholera outbreaks which occurred in Somalia in 1998-1999, were characterized by random amplified polymorphic DNA patterns, ribotyping, and antimicrobial susceptibility. All strains showed a unique amplified DNA pattern and 2 closely related ribotypes (B5a and B8a), among which B5a was the more frequently identified. Ninety-one strains were resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim, conferred, except for spectinomycin, by a conjugative plasmid IncC. These findings indicated that the group of strains active in Somalia in the late 1990s had a clonal origin.

Ceftriaxone-resistant salmonella enterica serotype Newport, France.

The multidrug-resistant (MDR) Salmonella enterica serotype Newport strain that produces CMY-2 beta-lactamase (Newport MDR-AmpC) was the source of sporadic cases and outbreaks in humans in France during 2000-2005. Because this strain was not detected in food animals, it was most likely introduced into France through imported food products.

Comparative phylogenies of Burkholderia, Ralstonia, Comamonas, Brevundimonas and related organisms derived from rpoB, gyrB and rrs gene sequences.

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.

Prevalence and characterization of extended-spectrum cephalosporin-resistant nontyphoidal Salmonella isolates in adults in Saint Petersburg, Russia (2002-2005).

We studied the prevalence of resistance to extended-spectrum cephalosporins (ESC) among 1,078 Salmonella enterica isolates collected from adults admitted to Botkin Hospital, St. Petersburg, Russia, for gastroenteritis between 2002 and 2005. Only two ESC-resistant isolates were detected, giving a low percentage of strains resistant to ESC (0.2%). One multidrug-resistant (MDR) isolate of the Virchow serotype produced a CTXM-3 extended-spectrum beta-lactamase (ESBL). The bla(CTX-M-3) gene was located downstream from an ISEcp1 element, on an 80-kb conjugative plasmid. The Virchow isolate possessed a class 1 integron with a 2.2-kb gene cassette (dhfrXII-orfF-aadA2). The second ESC-resistant isolate belonged to serotype Newport, was also MDR and produced a CMY-2 cephamycinase. This CMY-2-producing isolate (also called Newport MDR-AmpC) possessed a class 1 integron with a 1-kb gene cassette including a new variant of the aadA gene, aadA24. A large plasmid (>125 kb) was involved in transfer of the bla(CMY-2) gene. The ESC-resistant S. enterica isolates detected in this study were different from those (S. enterica serotype Typhimurium DT193 producing CTXM-4 or CTX-M-5 ESBLs) involved in several nosocomial outbreaks between 1994 and 2003 in Russia. This is the first description of both CTX-M-3 ESBL-producing S. enterica and Newport MDR-AmpC in Russia.

Clonal reconquest of antibiotic-susceptible Salmonella enterica serotype Typhi in Son La Province, Vietnam.

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.

Identification of a group of shigella-like isolates as Shigella boydii 20.

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.

Integrated view of Vibrio cholerae in the Americas.

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 Vibrio cholerae clones and local bacterial populations. We used whole-genome sequencing to characterize cholera across the Americas over a 40-year time span. We found that both epidemics were the result of intercontinental introductions of seventh pandemic El Tor V. cholerae and that at least seven lineages local to the Americas are associated with disease that differs epidemiologically from epidemic cholera. Our results consolidate historical accounts of pandemic cholera with data to show the importance of local lineages, presenting an integrated view of cholera that is important to the design of future disease control strategies.

Genomic history of the seventh pandemic of cholera in Africa.

The seventh cholera pandemic has heavily affected Africa, although the origin and continental spread of the disease remain undefined. We used genomic data from 1070 Vibrio cholerae O1 isolates, across 45 African countries and over a 49-year period, to show that past epidemics were attributable to a single expanded lineage. This lineage was introduced at least 11 times since 1970, into two main regions, West Africa and East/Southern Africa, causing epidemics that lasted up to 28 years. The last five introductions into Africa, all from Asia, involved multidrug-resistant sublineages that replaced antibiotic-susceptible sublineages after 2000. This phylogenetic framework describes the periodicity of lineage introduction and the stable routes of cholera spread, which should inform the rational design of control measures for cholera in Africa.

Interstitial pulmonary inflammation due to Microbacterium sp. after heart transplantation.

A coryneform bacterium was isolated from the bronchoalveolar aspirate of a patient with interstitial pulmonary inflammation. Commercial systems identified the isolate as Corynebacterium sp. or Aureobacterium sp./Corynebacterium aquaticum, but 16S rRNA gene analysis unequivocally attributed it to the genus Microbacterium. This represents the first documented case of Microbacterium pulmonary infection.

Molecular epidemiology of multidrug-resistant Shigella dysenteriae type 1 causing dysentery outbreaks in Central African Republic, 2003-2004.

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.

Molecular epidemiology of C. diphtheriae strains during different phases of the diphtheria epidemic in Belarus.

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).

Repeatability and reproducibility of ribotyping and its computer interpretation.

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.