Dissemination of blaVIM in Greece at the peak of the epidemic of 2005–2006: clonal expansion of Klebsiella pneumoniae clonal complex 147

VIM-producing Klebsiella pneumoniae (<n = 21) isolated from ten Greek hospitals during 2003–2007 were characterized with multilocus sequence typing (MLST), semi-automated repetitive sequence-based PCR (rep-PCR) (Diversilab), plasmid replicon typing, serotyping and screening for multiple resistance determinants. The isolates were selected to represent different strain clusters (defined by >80% similarity) according to pulsed-field gel electrophoresis. MLST identified three major clonal complexes (CCs); CC147 (n = 8), CC18 (n = 5) and CC14 (n = 3). Plasmid replicon typing showed that IncA/C and/or IncFII_K replicons were detected among isolates in each of the major CCs. Good concordance was observed between semi-automated-rep PCR genotyping and MLST.     

Genetic diversity,the virulence gene profile and antimicrobial resistance of clinical mastitis-associated Escherichia coli

Escherichia coli is a common cause of bovine mastitis, particularly around parturition and early lactation when the host is immunosuppressed. Isolates (n = 37) recovered from cases of clinical mastitis in Ireland were characterised with respect to genotypic diversity, phylogenetic group, virulence gene profile and antimicrobial susceptibility. The isolates were genotypically diverse, belonging to 19 different sequence types. However, the majority (86%) belonged to phylogenetic groups A or B1, groups commonly associated with commensal E. coli. The isolates encoded few virulence genes with iss (increased serum survival, 41%), lpfA (long polar fimbriae, 19%) and astA (enteroaggregative heat-stable toxin, 14%) among the most common virulence genes detected. The only virulence gene to differ in frequency between the phylogenetic groups was lpfA, found exclusively in B1. Resistance to at least one antimicrobial was detected in 16% of isolates. Three isolates were multidrug-resistant, with one resistant to seven antibiotics. There was no relationship between antimicrobial resistance and phylogenetic group. These results indicate that many cases of clinical E. coli mastitis in Ireland may be caused by opportunistic commensal organisms lacking specific virulence genes. However, the organisms represent a reservoir of antimicrobial resistance determinants with the potential to disseminate determinants to other organisms.     

Molecular epidemiology and resistance profiles of Clostridium difficile in a tertiary care hospital in Spain

Epidemiological surveillance of Clostridium difficile infection has gained importance in recent years as a result of the rapid spread of epidemic strains, including hypervirulent strains and strains with reduced susceptibility to antimicrobials. The molecular epidemiology and antimicrobial susceptibility of C. difficile in the reference hospital of the Balearic Islands (Spain) is reported in this study. One hundred isolates of toxigenic C. difficile from different patients were selected using rapid dual EIA screening test. All isolates were characterized through toxin profile, PCR ribotyping and, in addition, multi-locus sequence typing (MLST) was performed on fifty selected strains. MICs to metronidazole, vancomycin, erythromycin and moxifloxacin were also determined. A total of 43 different ribotypes were distinguished, with higher prevalence of ribotype 014 (34%). Twenty one per cent of the isolates expressed binary toxin and it is noteworthy that 62% of these were identified as the hypervirulent ribotype 078, the second most prevalent ribotype found in our hospital (13%). A total of 20 different sequence types (STs) were found, including a new described allele and ST. MLST data showed a clear concordance between some ribotypes and STs, mainly represented by ribotype 014/ST-2, ribotype 078/ST-11 and ribotype 001/ST-3. Phylogenetic analysis also revealed that most of the isolates were genetically related, forming a large clonal complex. Finally, ribotypes 078 (ST-11) and 001 (ST-3) were associated with higher resistance to erythromycin and to erythromycin and moxifloxacin, respectively. All these data suggest that the combination of ribotyping and MLST is a good tool for the surveillance of the changing epidemiology of C. difficile. A wide dissemination of clones has been observed in our setting, ribotype 014 (ST-2) being the most prevalent followed by the hypervirulent ribotype 078 (ST-11) and ribotype 001 (ST-3), their spread in our setting probably influenced by their higher resistance.     

Molecular epidemiology of extended-spectrum β-lactamase-producing Klebsiella pneumoniae strains in a university hospital in Tunis,Tunisia, 1999–2005

During a period of 6 years and 5 months (January 1999 to May 2005), 103 extended-spectrum β-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 H12 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.     

Characterisation of Neisseria meningitidis C:2b:P1.2,5 isolates in Poland

This study aimed to characterise Neisseria meningitidis C:2b:P1.2,5 isolates from Poland, which have now become predominant among serogroup C isolates in this country. Overall, 44 isolates (25 invasive and 19 from contact carriers) were typed by whole-cell ELISA and pulsed-field gel electrophoresis. Additionally, the invasive isolates were analysed by multilocus sequence typing, which revealed that they all belonged to the ST-8 complex/cluster A4. The emergence of this clone in other countries has resulted in mass immunisation campaigns and has been associated with a higher level of decreased susceptibility to penicillin; however the present study detected only one isolate that was penicillin-non-susceptible.     

Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia

A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004–2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes – emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies.     

Genetic characterization of Vibrio vulnificus strains isolated from oyster samples in Mexico

Vibrio vulnificus strains were isolated from oysters that were collected at the main seafood market in Mexico City. Strains were characterized with regard to vvhA, vcg genotype, PFGE, multilocus sequence typing (MLST), and rtxA1. Analyses included a comparison with rtxA1 reference sequences. Environmental (vcgE) and clinical (vcgC) genotypes were isolated at nearly equal percentages. PFGE had high heterogeneity, but the strains clustered by vcgE or vcgC genotype. Select housekeeping genes for MLST and primers that were designed for rtxA1 domains divided the strains into two clusters according to the E or C genotype. Reference rtxA1 sequences and those from this study were also clustered according to genotype. These results confirm that this genetic dimorphism is not limited to vcg genotyping, as other studies have reported. Some environmental C genotype strains had high similarity to reference strains, which have been reported to be virulent, indicating a potential risk for oyster consumers in Mexico City.     

Phylogeny and Disease Association of Shiga Toxin�Cproducing Escherichia coli O91

The diversity and relatedness of 100 Shiga toxin–producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence types (ST) and 4 distinct clonal groups. ST442 was significantly associated with hemolytic uremic syndrome.     

Molecular characterization of novel sequence type of carbapenem-resistant New Delhi metallo-β-lactamase-1-producing Klebsiella pneumoniae in the neonatal intensive care unit of an Indian hospital

Emergence of multi-drug resistance, especially carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major threat to public health. The aim of this study was to characterize CRKP isolates from infants admitted to the Neonatal Intensive Care Unit (NICU) to find the clonal outbreak of New Delhi metallo-β-lactamase (NDM) producers. In this study 17 CRKP isolates were analysed. Antimicrobial susceptibility of the isolates was determined by the disc diffusion and micro-dilution method. Carba-NP test and double-disk synergy test (DDST) were performed for the detection of carbapenemase and metallo-β-lactamase-producing K. pneumoniae. Antibiotic-resistant markers were detected by polymerase chain reaction (PCR) followed by sequencing. Clonal relatedness of the isolates was checked by multi-locus sequence typing. Conjugation experiments were performed to determine the transferability of the plasmids. All 17 CKRP isolates were found to carry bla_NDM (13 bla_NDM-1, 1 bla_NDM-4 and 3 bla_NDM-5), seven isolates carried bla_OXA-48, 13 isolates had bla_CTX-M-15, seven isolates carried bla_CMY-1 and five isolates were found to carry bla_SHV-1 on conjugative plasmids of different types (IncFIA, IncFIB, IncFIIAs, IncFIC, IncA/C, IncF, IncK, IncX, IncW and IncY). Of six different sequence types (STs) identified, ST3344 was detected as a novel ST in two K. pneumoniae isolates. Genetic environment analysis revealed ISAba125 and bleomycin genes flanking to all bla_NDM variants. This is the first report of novel ST3344 in two NDM-1-producing K. pneumoniae isolates from neonates admitted to the NICU of a North Indian Hospital. This study is provides understanding of the genetic features of this newly emerged strain type.     

Neisseria meningitidis sequence type and risk for death, Iceland

Invasive meningococcal infections are hyperendemic in Iceland, a relatively isolated country in the mid-Atlantic. We performed a nationwide study on all viable meningococcal strains (N = 362) from 1977 to 2004. We analyzed the association of patient's age and sex, meningococcal serogroups, and sequence types (STs) with outcomes. Overall, 59 different STs were identified, 19 of which were unique to Iceland. The most common STs were 32 (24.6%), 11 (19.9%), and 10 (10.2%). The unique ST-3492 ranked fourth (7.7%). The most common serogroups were B (56.4%), C (39.8%), and A (2.2%). Age (p < 0.001) and infection with a unique ST (p = 0.011) were independently associated with increased death rates, whereas isolation of meningococci from cerebrospinal fluid only was associated with lower death rates (p = 0.046). This study shows evolutionary trends of meningococcal isolates in a relatively isolated community and highlights an association between unique STs and poor outcome.