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.

Undercooked ground beef and person-to-person transmission as major risk factors for sporadic hemolytic uremic syndrome related to Shiga-toxin producing Escherchia coli infections in children in France.

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.

Surveillance of hemolytic uremic syndrome in children less than 15 years of age, a system to monitor O157 and non-O157 Shiga toxin-producing Escherichia coli infections in France, 1996-2006.

BACKGROUND: 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. METHODS: The surveillance system was based on a voluntary national network of pediatricians of 31 pediatric nephrology units in public hospitals. RESULTS: 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. CONCLUSIONS: 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.

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.

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).

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.

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.

Value of morphotyping for the characterization of Candida albicans clinical isolates.

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.

Value of morphotyping for the characterization of Candida albicans clinical isolates

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 percent of the strains.

Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates.

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.

Molecular evolution of the lysine decarboxylase-defective phenotype in Shigella sonnei.

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.

Investigation of the clonal dissemination of Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases in a neonatal ward, Sousse, Tunisia.

This study was designed to investigate the spread of extended-spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-Kp) strains in Sousse hospital, during 7-month period by using phenotypic and genotypic markers. A total of 57 clinical isolates of ESBL-Kp, 22 strains recovered from seriously infected neonates and 35 strains recovered from colonized neonates and hospitalized in the neonatal ward of Sousse hospital, Tunisia, was subjected to 99 carbon source utilization tests, ribotyping and pulse-field gel electrophoresis (PFGE) profiles of total genomic DNA. Biotyping, ribotyping and PFGE typing showed that four different clones circulated in the neonatal ward between January and July 1997 and suggested that the epidemic strain belonged to the same biotype, ribotype and PFGE pattern, and was represented by 18 isolates from infected neonates and 28 isolates from colonized neonates. Biotyping, ribotyping and PFGE typing appeared to be reliable methods for distinguishing K. pneumoniae strains. Biotyping, which has the advantage of simplicity and rapidity, may be used as a first screening method.

Characterization of Escherichia coli O157 : H7 isolates causing haemolytic uraemic syndrome in France.

Forty-seven non-epidemic Escherichia coli O157 : H7 isolates causing haemolytic uraemic syndrome in France were characterized. The isolates clustered into 36 clones using PFGE typing. All the isolates harboured eae and one or more copies of stx2 and belonged to phylogenetic group D. Nine per cent were resistant to amoxicillin.

Molecular typing of Agrobacterium species isolates from catheter-related bloodstream infections.

Agrobacterium isolates from intravenous catheters of three hospitalized patients were initially identified as A. tumefaciens, but inability to produce 3-ketolactose revealed that two of them were A. vitis. However, rDNA analysis correlated all of the isolates to A. tumefaciens. Pulsed-field gel electrophoresis analysis ascertained the nosocomial transmission of the infection.

New group D streptococcal species.

BACKGROUND & OBJECTIVES: The heterogeneity of group D streptococci led to the identification of various biotypes of Streptococcus equinus and Streptococcus bovis and to the description of new species. The objective of the present study was to improve the phenotypic delineation between species and to clarify their respective phylogenetic position. METHODS: Physiological and genomic analyses were carried out in 84 representative strains of the group D streptococci. Biotypes were determined with the API 20 strep and rapid ID 32 STREP systems of identification. Quantitative DNA-DNA hybridization under stringent conditions and values of the deltaT(m) allowed to delineate species and subspecies. The phylogenic position of the different genomic groups was determined by comparing the sequences of their 16S rDNA. RESULTS: Four DNA-clusters, including seven species or subspecies, were characterized. Differential associations of biochemical characters allowed their identification. S. equinus and the type strain of S. bovis belonged to a single species. S. gallolyticus, S. bovis biotype II.2, and S. macedonicus formed a single DNA-cluster including three different subspecies. These were designated as S. gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus, and S. gallolyticus subsp. macedonicus. The two other DNA-clusters corresponded to the two subspecies of S. infantarius, and to S. alactolyticus. INTERPRETATION & CONCLUSION: This study presented a new classification associated with an identification scheme of group D streptococci. The changes in this classification demonstrate the interest of a polyphagic approach of the bacterial identification.

Endemic, epidemic clone of Salmonella enterica serovar typhi harboring a single multidrug-resistant plasmid in Vietnam between 1995 and 2002.

Salmonella enterica serovar Typhi strains resistant to ampicillin, chloramphenicol, tetracyclines, streptomycin, and cotrimoxazole, isolated from sporadic cases and minor outbreaks in Vietnam between 1995 and 2002, were typed and compared. Plasmid fingerprinting, Vi bacteriophage typing, XbaI pulsed-field gel electrophoresis, and PstI ribotyping showed that endemic, epidemic multidrug-resistant typhoid fever was due, for at least 74.1% of the isolates, to one or two clones of serovar Typhi harboring a single resistance plasmid. PstI ribotyping was used as a basic technique to ensure that a serovar Typhi expansion was clonal.

International nomenclature for Corynebacterium diphtheriae ribotypes.

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.

Shigella dysenteriae serotype 1 in west Africa: intervention strategy for an outbreak in Sierra Leone.

In November 1999, a Médecins Sans Frontières team based in the southeastern part of Sierra Leone reported an increased number of cases of bloody diarrhoea. Shigella dysenteriae serotype 1 (Sd1) was isolated in the early cases. A total of 4218 cases of dysentery were reported in Kenema district from December, 1999, to March, 2000. The overall attack rate was 7.5%. The attack rate was higher among children younger than 5 years than in the rest of the population (11.2% vs 6.8%; relative risk=1.6; 95% CI 1.5-1.8). The case fatality was 3.1%, also higher for children younger than 5 years (6.1% vs 2.1%; relative risk=2.9; 95% CI 2.1-4.1]). Among 583 patients regarded at increased risk of death who were treated with ciprofloxacin in isolation centres, case fatality was 0.9%. A 5-day ciprofloxacin regimen, targeted to the most severe cases of bloody diarrhoea, was highly effective. This is the first time a large outbreak caused by Sd1 has been reported in west Africa.