[Show abstract][Hide abstract] ABSTRACT: Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected.
[Show abstract][Hide abstract] ABSTRACT: Hypervirulent Klebsiella pneumoniae isolates of capsular serotype K2 (hvKP-K2) that cause community-acquired invasive infections represent several unrelated clones, which all belong to phylogenetic group KpI. These clones were recognized using multilocus sequence typing (MLST) and genomic analyses, but no rapid method currently exists to differentiate them. In this work, a multiplex PCR assay was developed for identification of three hvKP-K2 groups: (i) sequence type (ST) 86; (ii) ST380 and ST679 (i.e. clonal group 380); and (iii) ST65 and ST375. A specific genetic marker, Kp50233, allowing to distinguish K. pneumoniae sensu stricto (corresponding to phylogroup KpI) from closely related species, was included in the assay. This PCR will be useful to better define the epidemiology and clinical features of emerging virulent K. pneumoniae clones.
[Show abstract][Hide abstract] ABSTRACT: Strains previously classified as members of Klebsiella pneumoniae phylogroups KpI, KpII-A, KpII-B and KpIII were characterized by rrs sequencing, multilocus sequence analysis based on genes rpoB, fusA, gapA, gyrA and leuS, average nucleotide identity and biochemical characteristics. Phylogenetic analysis demonstrated that KpI and KpIII corresponded to K. pneumoniae and K. variicola, respectively, whereas KpII-A and KpII-B formed two well-demarcated sequence clusters distinct from other Klebsiella taxa. 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 source being particularly useful. Based on their genetic and phenotypic characteristics, we propose the names K. quasipneumoniae subsp. quasipneumoniae and K. quasipneumoniae subsp. similipneumoniae for strains of KpII-A and KpII-B, respectively. The type strain of K. quasipneumoniae and of K. quasipneumoniae subsp. quasipneumoniae is 01A030T (SB11T, CIP 110771T, DSM 28211T). The type strain of K. quasipneumoniae subsp. similipneumoniae is 07A044T (SB30T, CIP 110770T, DSM 28212T). Both strains were isolated from human blood cultures. This work also showed that K. singaporensis is a junior heterotypic synonym of K. variicola.
International journal of systematic and evolutionary microbiology. 06/2014;
[Show abstract][Hide abstract] ABSTRACT: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are amongst the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044.
[Show abstract][Hide abstract] ABSTRACT: Background. Novel therapies are urgently needed to treat Carbapenem-resistant Klebsiella pneumoniae (CR-Kp)-mediated infection, which constitute a major health threat in the US. In order to assess if it is feasible to develop anti-capsular antibodies as a potential novel therapy it is crucial to first systematically characterize capsular polysaccharide (CPS) and virulence traits in these strains.Methods. Forty CR-Kp were genotyped by Pulsed Field Gel Electrophoresis, Multilocus Sequence typing (MLST) and molecular capsule typing (C-patterns and wzi sequencing). Their biofilm formation, serum resistance, macrophage-mediated killing, and virulence in Galleria mellonella were compared. MAb (1C9) was generated by co-immunization with two CPSs and cross-reactivity was investigated.Results. MLST assigned 80% of CR-Kp isolates to the ST258-clone. Molecular capsule typing identified new C-patterns; including C200/wzi-154, which was widely represented and associated with blaKPC-3-bearing strains. Heterogeneity was detected in biofilm formation and macrophage-mediated killing. Differences in serum resistance correlated with virulence in G. mellonella. ST258 strains carrying blaKPC-3 were less virulent than those with blaKPC-2. MAb 1C9 cross-reacted with 58% of CR-Kp CPSs.Conclusions. CR-Kp ST258 strains exhibit variability of virulence-associated traits. Differences were associated with the type of KPC gene and CPS. Identification of cross-reacting anti-CPS mAbs encourages their development as adjunctive therapy.
The Journal of Infectious Diseases 03/2014; · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Populations of the food-borne 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 I and II, including representatives of epidemic clones, were analyzed by MLVA-11, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). MLVA-11 had less discriminatory power 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 the PFGE workload in outbreak investigations and listeriosis surveillance.
[Show abstract][Hide abstract] ABSTRACT: The genome sequence and annotation of Campylobacter coli strain IPSID-1 are reported here. This bacterial isolate is the first to be cultured from a patient with immunoproliferative small intestinal disease (IPSID). The draft genome sequence is 1.683 Mb long, comprises 64 contigs, and has 31.26% G+C content.
[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. · 3.53 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