[show abstract][hide abstract] ABSTRACT: Pathogens of the genus Klebsiella have been classified into distinct capsular (K) types for nearly one century. K-typing of Klebsiella still has important applications in epidemiology and clinical microbiology, but the serological method has strong practical limitations. Our objective was to evaluate sequencing of wzi, a gene conserved in all capsular types coding for an outer membrane protein involved in capsule attachment to the cell surface, as a simple and rapid method to predict the K-type. Sequencing of a 447-nucleotides region of wzi distinguished K-type reference strains with only nine exceptions. A reference wzi sequence database was created by inclusion of multiple strains representing K-types associated with high virulence and multidrug resistance. A collection of 119 prospective clinical isolates of K. pneumoniae was then analyzed in parallel by wzi sequencing and classical K-typing. Whereas K-typing achieved 81% typeability and 94.4% discrimination, these figures were 98.1%, and 98.3% for wzi sequencing. Prediction of K-type once knowing the wzi allele was 94%. wzi sequencing is as a rapid and simple method to determine the K-type of most K. pneumoniae clinical isolates.
Journal of clinical microbiology 10/2013; · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Populations of the foodborne pathogen Listeria monocytogenes are genetically structured into a small number of major clonal groups, some of which have been implicated in multiple outbreaks. The goal of this study was to develop and evaluate an optimized multilocus variable number of tandem repeat (VNTR) analysis (MLVA) subtyping scheme for strain discrimination and clonal group identification. We evaluated 18 VNTR loci and combined the 11 best ones into two multiplexed PCR assays (MLVA-11). A collection of 255 isolates representing the diversity of clonal groups within phylogenetic lineages 1 and 2, including representatives of epidemic clones, were analyzed by MLVA-11, multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). MLVA-11 was less discriminatory than PFGE, except for some clones, and was unable to distinguish some epidemiologically unrelated isolates. Yet it distinguished all major MLST clones and therefore constitutes a rapid method to identify epidemiologically relevant clonal groups. Given its high reproducibility and high-throughput, MLVA represents a very attractive first-line screening method to alleviate PFGE workload in outbreak investigations and listeriosis surveillance.
Journal of Clinical Microbiology 04/2013; · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: SUMMARY Despite infection control measures, an important increase in the extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae incidence density occurred in our hospital from 2006 onwards. This study, focusing on the 2005-2007 period, was performed in an attempt to explain this increase. ESBLs were characterized, isolates were typed by ERIC2-PCR, and sequence type (ST) of clustered isolates was determined. Temporal-spatial relationships of patients were analysed to assess possible cross-contamination. Of the 74 ESBL-producing isolates, 30 (40%) were detected at admission, 53 (71·5%) produced CTX-M enzymes, 40 displayed unique ERIC2-PCR profiles and 34 were assigned into six clusters: ST16 (n = 21), ST101, ST48, ST35, ST13, and ST436. Relationships were identified in 22 of the 34 patients harbouring clustered isolates. This study highlights the complex epidemiology of ESBL-producing K. pneumoniae in the mid-2000s with potential cross-contamination for only 30% of the 74 patients in our hospital, and the emergence of clones that are currently spreading worldwide.
Epidemiology and Infection 10/2012; · 2.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.
PLoS ONE 01/2012; 7(5):e36995. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma.
[show abstract][hide abstract] ABSTRACT: Population diversity, susceptibility to antibiotics including carbapenems of 277 Acinetobacter baumannii strains collected in 17 Italian hospitals over a 6-months' period was assessed. Semi-automated rep-PCR was used for screening strains for genotypic relatedness. AFLP analysis and MLST were used as definitive methods for strain, species and/or clone identification. Among the 277 strains, 49 rep-PCR types were distinguished with four types (1-4) predominant, indicating both intra- and interhospital spread. AFLP analysis allowed to distinguish 51 types and largely confirmed rep-typing results. Isolates with predominant rep-types 1 and 2 (in 3 and 9 hospitals) were allocated to EU clones I and II, respectively. Rep-type 3 (8 hospitals) belonged to a new clone ("Italian clone"). Rep-type 4 was found in 2 neighbouring hospitals. Two isolates from 2 locations belonged to EU clone III. Twenty-five isolates were identified by AFLP-analysis to A. pittii, emphasizing misidentification by phenotypic methods. MLST confirmed clone identification by AFLP; demonstrating also that the "Italian clone" was ST78, recently detected in different Mediterranean countries. Multidrug resistance, defined as resistance to 9 out of the 11 drugs tested, was common in 10 out of 17 hospitals. The high prevalence of carbapenem resistance was associated with OXA-58 found in 9 out of the 10 hospitals. A high percentage of noted very major errors in susceptibility testing, especially for amikacin and meropenem, was probably due to heteroresistant strains. The occurrence of carbapenem and multidrug resistance in A. baumannii was mainly confined to a limited number of clonal lineages of A. baumannii.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 04/2011; 11(6):1319-26. · 3.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: Acinetobacter genomic species (gen. sp.) 3 and gen. sp. 13TU are increasingly recognized as clinically important taxa within the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex. To define the taxonomic position of these genomic species, we investigated 80 strains representing the known diversity of the ACB complex. All strains were characterized by AFLP analysis, amplified rDNA restriction analysis and nutritional or physiological testing, while selected strains were studied by 16S rRNA and rpoB gene sequence analysis, multilocus sequence analysis and whole-genome comparison. Results supported the genomic distinctness and monophyly of the individual species of the ACB complex. Despite the high phenotypic similarity among these species, some degree of differentiation between them could be made on the basis of growth at different temperatures and of assimilation of malonate, l-tartrate levulinate or citraconate. Considering the medical relevance of gen. sp. 3 and gen. sp. 13TU, we propose the formal names Acinetobacter pittii sp. nov. and Acinetobacter nosocomialis sp. nov. for these taxa, respectively. The type strain of A. pittii sp. nov. is LMG 1035(T) (=CIP 70.29(T)) and that of A. nosocomialis sp. nov. is LMG 10619(T) (=CCM 7791(T)).
Research in Microbiology 02/2011; 162(4):393-404. · 2.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: The genus Bifidobacterium comprises several species that are important contributors to the gut microbiome, with some strains having beneficial health effects. Understanding the evolutionary emergence of advantageous biological properties requires knowledge of the genetic diversity and clonal structure of species. We sequenced seven housekeeping genes in 119 Bifidobacterium strains of Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum. Phylogenetic analysis of concatenated sequences delineated sequence clusters that correspond to previously named taxa, and suggested that B. longum subsp. infantis is a nascent lineage emerging from within B. longum subsp. longum. Clear traces of recombination among distant bifidobacterial species indicate leaky species borders and warn against the practice of single gene-based identification. Multilocus sequence typing achieved precise strain genotyping, with discrimination indices above 99% in B. bifidum, B. breve and B. longum, providing a powerful tool for strain traceability, colonization dynamics and ecological studies. Frequent homologous recombination accelerates clonal diversification and may facilitate the transfer of biological properties among bifidobacterial strains.
Research in Microbiology 03/2010; 161(2):82-90. · 2.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Outbreaks of hospital infections caused by multidrug resistant Acinetobacter baumannii strains are of increasing concern worldwide. Although it has been reported that particular outbreak strains are geographically widespread, little is known about the diversity and phylogenetic relatedness of A. baumannii clonal groups. Sequencing of internal portions of seven housekeeping genes (total 2,976 nt) was performed in 154 A. baumannii strains covering the breadth of known diversity and including representatives of previously recognized international clones, and in 19 representatives of other Acinetobacter species. Restricted amounts of diversity and a star-like phylogeny reveal that A. baumannii is a genetically compact species that suffered a severe bottleneck in the recent past, possibly linked to a restricted ecological niche. A. baumannii is neatly demarcated from its closest relative (genomic species 13TU) and other Acinetobacter species. Multilocus sequence typing analysis demonstrated that the previously recognized international clones I to III correspond to three clonal complexes, each made of a central, predominant genotype and few single locus variants, a hallmark of recent clonal expansion. Whereas antimicrobial resistance was almost universal among isolates of these and a novel international clone (ST15), isolates of the other genotypes were mostly susceptible. This dichotomy indicates that antimicrobial resistance is a major selective advantage that drives the ongoing rapid clonal expansion of these highly problematic agents of nosocomial infections.
PLoS ONE 01/2010; 5(4):e10034. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe the benefit of using reconstructed ancestral sequences (RAS) on resequencing microarrays for rapid pathogen identification, with Enterobacteriaceae rpoB sequences as a model. Our results demonstrate a sharp improvement of call rate and accuracy when using RASs as compared to extant sequences. This improvement was attributed to the lower sequence divergence of RASs, which also expanded the sequence space covered by the microarray. Extension of this novel microarray design strategy to viruses, antimicrobial resistance elements or toxins is straightforward.
PLoS ONE 01/2010; 5(12):e15243. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: During a period of 6 years and 5 months (January 1999 to May 2005), 103 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates, each from an individual patient or site, were collected at Mongi Slim University Hospital Centre, Tunis, Tunisia. The objectives of our work were the characterization of the bla genes encoding ESBLs, the investigation of clonal diversity of strains, and identification of the transmission modes of the resistance genes. We carried out detection by PCR and sequencing of the bla(SHV), bla(CTX-M) and bla(TEM) genes, transferability studies, plasmid replicon typing, and analysis by multilocus sequence typing (MLST) on selected isolates. Forty-seven isolates were found to be producers of CTX-M-type ESBLs, of which 43 were CTX-M-15, two CTX-M-14 and two CTX-M-27. Fifty-eight isolates were producers of SHV-12, and three were producers of SHV-2a. More than one ESBL was detected in seven isolates, as five produced both CTX-M-15 and SHV-12, and two produced both CTX-M-27 and SHV-12. By a PCR-based replicon typing method, the plasmids carrying the bla(SHV-2a) or bla(CTX-M-15) genes were assigned to IncFII or, more rarely, to IncL/M types. Of 12 plasmids carrying the bla(SHV-12) gene, only one could be typed: it was positive for the HI2 replicon. The MLST results showed large genetic background diversity in the SHV-12-producing isolates and dissemination of specific clones of the CTX-M-15-producing isolates within the same ward and among wards, and suggested endemicity with horizontal dissemination of the bla(CTX-M-15) and the bla(SHV-12) genes.
Clinical Microbiology and Infection 09/2009; 16(2):157-64. · 4.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen (causing urinary, respiratory and blood infections) and the agent of specific human infections including Friedländer's pneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA). The identification and precise definition of virulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical to understand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K. pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content, biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clones that differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1, clone CC23(K1) and clone CC82(K1), were strongly associated with PLA and respiratory infection, respectively. Second, only one of the two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena and rhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association of virulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of the capsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae and CC82(K1) indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23(K1) remains metabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence of important virulent clones associated with specific infections and provide an evolutionary framework for research into the links between clones, virulence and other genomic features in K. pneumoniae.
PLoS ONE 02/2009; 4(3):e4982. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pantoea agglomerans and other Pantoea species cause infections in humans and are also pathogenic to plants, but the diversity of Pantoea strains and their possible association with hosts and disease remain poorly known, and identification of Pantoea species is difficult. We characterized 36 Pantoea strains, including 28 strains of diverse origins initially identified as P. agglomerans, by multilocus gene sequencing based on six protein-coding genes, by biochemical tests, and by antimicrobial susceptibility testing. Phylogenetic analysis and comparison with other species of Enterobacteriaceae revealed that the genus Pantoea is highly diverse. Most strains initially identified as P. agglomerans by use of API 20E strips belonged to a compact sequence cluster together with the type strain, but other strains belonged to diverse phylogenetic branches corresponding to other species of Pantoea or Enterobacteriaceae and to probable novel species. Biochemical characteristics such as fosfomycin resistance and utilization of d-tartrate could differentiate P. agglomerans from other Pantoea species. All 20 strains of P. agglomerans could be distinguished by multilocus sequence typing, revealing the very high discrimination power of this method for strain typing and population structure in this species, which is subdivided into two phylogenetic groups. PCR detection of the repA gene, associated with pathogenicity in plants, was positive in all clinical strains of P. agglomerans, suggesting that clinical and plant-associated strains do not form distinct populations. We provide a multilocus gene sequencing method that is a powerful tool for Pantoea species delineation and identification and for strain tracking.
Journal of clinical microbiology 01/2009; 47(2):300-10. · 4.16 Impact Factor