Umaer Naseer

Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing plasmid-mediated KPC carbapenemase in Norway and Sweden

BACKGROUND The class A carbapenemase KPC has disseminated rapidly worldwide, challenging the treatment of Gram-negative infections. This report describes the first KPC-producing Klebsiella pneumoniae isolates identified in Norway (n=6) and the second isolate from Sweden. METHODS Antimicrobial susceptibility profiles were determined using Etest. PCR and sequencing were used to determine the bla(KPC) variant, the surrounding genetic structure and the presence of AmpC and extended-spectrum beta-lactamase genes. PFGE and multilocus sequence typing (MLST) were used for epidemiological comparisons. Localization of bla(KPC) was investigated by S1 nuclease digestion, followed by PFGE and Southern blot hybridization. RESULTS All isolates expressed a multidrug-resistant phenotype with some variability in the carbapenem susceptibility profile. The Norwegian isolates carried bla(KPC-2), while the Swedish isolate carried bla(KPC-3). All isolates carried TEM-1, but were negative for bla(CTX-M) and bla(AmpC) genes. SHV-11 and SHV-12 were detected in the Norwegian isolates, while the Swedish isolate carried only SHV-11. Isolates from four patients were associated with import from Greece (n=3) and Israel. The other isolates were probably associated with local transmissions. PFGE and MLST showed that the isolates were clonally related, with three isolates displaying ST258, a single locus variant of ST11 previously associated with the clonal spread of CTX-M-15-producing K. pneumoniae in Hungary. In all isolates, bla(KPC) was located on plasmids as part of isoform a of Tn4401. CONCLUSIONS The emergence of KPC-producing isolates of K. pneumoniae in Norway and Sweden is associated with multiple import events and probable local transmission of a successful multiresistant ST258 clone, closely related to the CTX-M-15-producing ST11 clone previously described in Hungary.

The CTX-M Conundrum: Dissemination of Plasmids and Escherichia coli Clones

The ongoing global spread and increased prevalence of CTX-M-type extended-spectrum β-lactamases in Enterobacteriaceae is of great concern. The successful distribution of CTX-M enzymes mainly involves Escherichia coli causing systemic as well as urinary tract infections in patients worldwide. CTX-M expression is often associated with coresistance that critically reduces treatments options. The mobilization of bla(CTX-M) genes from their original chromosomal position in various Kluyvera species has been facilitated by mobile genetic elements such as ISEcp1 or ISCR1. Molecular epidemiological studies have revealed a thriving linkage of bla(CTX-M) genes to conjugative plasmids and successful bacterial clones. Multireplicon FII plasmids are shown to carry the most widely distributed bla(CTX-M-15) across continents, paving the way for bla(CTX-M-15) into different genetic lineages of E. coli. Dissemination of virulent clones ST131-O25:H4-B2 and ST405-O102:H6-D is now being described worldwide. Importantly, CTX-M-producing strains are uncovering their ability of long-term gastrointestinal colonization often associated with travel to high-prevalent areas. Thus, we are witnessing a global epidemic of bla(CTX-M)-encoding E. coli strains and plasmids, which require serious attention and efficient infection control measures.

Molecular characterization of CTX-M-15-producing clinical isolates of Escherichia coli reveals the spread of multidrug-resistant ST131 (O25:H4) and ST964 (O102:H6) strains in Norway.

Nationwide, CTX‐M‐producing clinical Escherichia coli isolates from the Norwegian ESBL study in 2003 (n=45) were characterized on strain and plasmid levels. BlaCTX‐M allele typing, characterization of the genetic environment, phylogenetic groups, pulsed field gel electrophoresis (PFGE), serotyping and multilocus sequence typing were performed. Plasmid analysis included S1‐nuclease‐PFGE, polymerase chain reaction‐based replicon typing, plasmid transfer and multidrug resistance profiling. BlaCTX‐M‐15 (n=23; 51%) and blaCTX‐M‐14 (n=11; 24%) were the major alleles of which 18 (78%) and 6 (55%), respectively, were linked to ISEcp1. Thirty‐two isolates were of phylogenetic groups B2 and D. Isolates were of 29 different XbaI‐PFGE‐types including six regional clusters. Twenty‐three different O:H serotypes were found, dominated by O25:H4 (n=9, 20%) and O102:H6 (n=9, 20%). Nineteen different STs were identified, where ST131 (n=9, 20%) and ST964 (n=7, 16%) were dominant. BlaCTX‐M was found on ≥100 kb plasmids (39/45) of 10 different replicons dominated by IncFII (n=39, 87%), FIB (n=20, 44%) and FIA (n=19, 42%). Thirty‐nine isolates (87%) displayed co‐resistance to other classes of antibiotics. A transferable CTX‐M phenotype was observed in 9/14 isolates. This study reveals that the majority of CTX‐M‐15‐expressing strains in Norway are part of the global spread of multidrug‐resistant ST131 and ST‐complex 405, associated with ISEcp1 on transferrable IncFII plasmids.

Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway

Clinical isolates of Escherichia coli with reduced susceptibility to oxyimino-cephalosporins and not susceptible to clavulanic acid synergy (n = 402), collected from Norwegian diagnostic laboratories in 2003-2007, were examined for the presence of plasmid-mediated AmpC beta-lactamases (PABLs). Antimicrobial susceptibility testing was performed for beta-lactam and non-beta-lactam antibiotics using Etest and Vitek2, respectively. The AmpC phenotype was confirmed using the boronic acid test. PABL-producing isolates were detected using ampC multiplex-PCR and examined by bla(AmpC) sequencing, characterization of the bla(AmpC) genetic environment, phylogenetic grouping, XbaI- pulsed-field gel electrophoresis (PFGE), multi-locus sequence typed (MLST), plasmid profiling and PCR-based replicon typing. For the PABL-positive isolates (n = 38), carrying bla(CMY-2) (n = 35), bla(CMY-7) (n = 1) and bla(DHA-1) (n = 2), from out- (n = 23) and in-patients (n = 15), moderate-high MICs of beta-lactams, except cefepime and carbapenems, were determined. All isolates were resistant to trimethoprim-sulphamethoxazole. Multidrug resistance was detected in 58% of the isolates. The genes bla(CMY-2) and bla(CMY-7) were linked to ISEcp1 upstream in 32 cases and in one case, respectively, and bla(DHA-1) was linked to qacEDelta1sul1 upstream and downstream in one case. Twenty isolates were of phylogenetic groups B2 or D. Thirty-three XbaI-PFGE types, including three clusters, were observed. Twenty-five sequence types (ST) were identified, of which ST complexes (STC) 38 (n = 7), STC 448 (n = 5) and ST131 (n = 4) were dominant. Plasmid profiling revealed 1-4 plasmids (50-250 kb) per isolate and 11 different replicons in 37/38 isolates; bla(CMY-2) was carried on transferable multiple-replicon plasmids, predominantly of Inc groups I1 (n = 12), FII (n = 10) and A/C (n = 7). Chromosomal integration was observed for bla(CMY-2) in ten strains. CMY-2 is the dominant PABL type in Norway and is associated with ISEcp1 and transferable, multiple-replicon IncI1, IncA/C, or IncFII plasmids in nationwide strains of STC 448, STC 38 and ST131.

A long-term low-frequency hospital outbreak of KPC-producing Klebsiella pneumoniae involving Intergenus plasmid diffusion and a persisting environmental reservoir.

Background To study the molecular characteristics of a long-term, low frequency outbreak of bla KPC-2 in a low prevalence setting involving the hospital environment. Methodology/Principal Findings KPC-producing bacteria were screened by selective chromogenic agar and Real-Time PCR. The presence of antibiotic resistance genes was ascribed by PCRs and subsequent sequencing, and the KPC-producing isolates were phylogenetically typed using PFGE and multi-locus sequence typing. Bla KPC-2-plasmids were identified and analysed by S1-nuclease-PFGE hybridization and PCR based replicon typing. A ∼97 kb IncFII plasmid was seen to carry bla KPC-2 in all of the clinical isolates, in one of the isolates recovered from screened patients (1/136), and in the Klebsiella pneumoniae and Enterobacter asburiae isolates recovered from the environment (sinks) in one intensive care unit. The K. pneumoniae strain ST258 was identified in 6 out of 7 patients. An intergenus spread to E. asburiae and an interspecies spread to two different K. pneumoniae clones (ST27 and ST461) of the bla KPC-2 plasmid was discovered. K. pneumoniae ST258 and genetically related E. asburiae strains were found in isolates of both human and environmental origins. Conclusions/Significance We document a clonal transmission of the K. pneumoniae ST258 strain, and an intergenus plasmid diffusion of the IncFII plasmid carrying bla KPC-2 in this outbreak. A major reservoir in the patient population could not be unveiled. However, the identification of a persisting environmental reservoir of strains with molecular determinants linked to human isolates, suggests a possible role of the environment in the maintenance of this long-term outbreak.

Nosocomial outbreak of CTX-M-15-producing E. coli in Norway.

Seven E. coli isolates expressing resistance to 3rd generation cephalosporins were recovered from blood (n=2), kidney and lung tissue (n=1), and urinary tract (n=4) samples from seven patients hospitalised or recently discharged from the Divisions of Geriatrics and Pulmonary Medicine, Central Hospital of Rogaland, between July and September 2004. All isolates expressed a typical ESBL‐cefotaximase profile (cefotaxime MIC>ceftazidime MIC) with clavulanic acid synergy. A blaCTX‐M‐15 genotype was confirmed in six strains that were coresistant to gentamicin, nitrofurantoin, trimethoprim‐sulfamethoxazole and ciprofloxacin. A blaCTX‐M‐3 genotype was detected in the last strain. XbaI‐PFGE patterns of the six blaCTX‐M‐15 isolates revealed a clonal relationship. BlaCTX‐M‐15 strains were also positive for the ISEcp1‐like insertion sequences that have been shown to be involved in the mobilization of blaCTX‐M. Further analyses revealed two blaCTX‐M‐15‐positive E. coli urinary isolates clonally related to the outbreak strain from two different patients at the same divisions in January and February 2004. These patients were later re‐hospitalised and one had E. coli with an ESBL‐cefotaximase profile in sputum and nasopharyngeal specimen during the outbreak period. Clinical evaluation suggests that the CTX‐M‐producing E. coli strains contributed to death in three patients due to delayed efficient antimicrobial therapy. The outbreak emphasises the epidemic potential of multiple‐antibiotic‐resistant CTX‐M‐15‐producing E. coli also in a country with low antibiotic usage and low prevalence of antimicrobial resistance.

Long-term faecal carriage in infants and intra-household transmission of CTX-M-15-producing Klebsiella pneumoniae following a nosocomial outbreak

OBJECTIVES To investigate the duration of faecal carriage of CTX-M-15-producing Klebsiella pneumoniae in infants colonized during a nosocomial neonatal intensive care unit (NICU) outbreak after discharge from hospital, possible risk factors for long-term colonization and transmission to household contacts (HCs). METHODS Fifty-one infants colonized with two unrelated clones of CTX-M-15 K. pneumoniae [sequence type (ST) 17 and ST485] during an NICU outbreak and 60 HCs provided faecal and rectal samples, respectively, every 1-3 months after hospital discharge. Extended-spectrum β-lactamase (ESBL)-producing strains of K. pneumoniae were identified on Chrom ID ESBL agar and examined by antimicrobial susceptibility testing. blaCTX-M-15 was detected by PCR and DNA sequencing. Clonal relationship was examined by PFGE. RESULTS The median carriage time in infants after discharge was 12.5 months (IQR 9.5-17.5). Stable antimicrobial susceptibility patterns in PFGE-related strains confirmed the intestinal persistence of both outbreak strains. Risk factors for prolonged faecal carriage in infants were delivery by caesarean section [hazard ratio (HR) 2.4, 95% CI 1.1-5.5, P = 0.029] and treatment with antibiotics during hospitalization (HR 4.5, 95% CI 1.6-12.6, P = 0.004). Transmission of CTX-M-15 K. pneumoniae was observed in 9/28 (32%) households. Median carriage length in parents was 2.5 months (IQR 1.0-5.0) (P < 0.001 compared with infants). CONCLUSIONS Infants may be long-term faecal carriers of ESBL-producing K. pneumoniae after colonization during hospitalization in the neonatal period. Delivery by caesarean section and antibiotic treatment during hospitalization are possible risk factors for prolonged carriage. Faecal ESBL carriage in infants represents a reservoir for intra-household spread of ESBL-producing K. pneumoniae.

Sequence types and plasmid carriage of uropathogenic Escherichia coli devoid of phenotypically detectable resistance

OBJECTIVES Plasmids play a major role in the dissemination of antibiotic resistance, and several studies have shown the association between specific resistance mechanisms and certain plasmid types and/or Escherichia coli lineages. This study describes the distribution of plasmids, replicon types, sequence types (STs) and ST complexes (STCs) of E. coli devoid of phenotypic resistance to 24 antibiotics. METHODS Eighty E. coli isolates from urinary tract infections from four European countries were selected because of their lack of phenotypically detectable antibiotic resistance. The isolates were characterized to the phylogenetic group level using PCR and to ST by multilocus sequence typing. Plasmid carriage was assessed using S1 nuclease PFGE profiling and PCR-based replicon typing. RESULTS Plasmids were detected in only 38/80 (47%) of the isolates; one (n = 18), two (n = 14), three (n = 5) and four (n = 1) plasmids. Six different replicon types were identified, the most common being a combination of IncFII and IncFIB. Most isolates belonged to phylogenetic group B2 and STC73 (n = 20), STC95 (n = 7) and ST420 (n = 6). A high proportion of STC73 isolates (75%) was devoid of plasmids. No association could be found between specific STs and replicon type. CONCLUSIONS A large proportion of E. coli strains phenotypically devoid of antibiotic resistance were plasmid naive. Those isolates that harboured plasmids displayed replicon types similar to those of resistant isolates, but the distributions of STs and STCs were different. This may indicate chromosomally encoded mechanisms important for the stabilization of plasmids harbouring antibiotic resistance.

Identification of Enterobacteriaceae isolates with OXA-48 and coproduction of OXA-181 and NDM-1 in Norway

References 1 Bonomo RA. New Delhi metallo-b-lactamase and multidrug resistance: a global SOS? Clin Infect Dis 2011; 52: 485–7. 2 Yong D, Toleman MA, Giske CG et al. Characterization of a new metallo-b-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009; 53: 5046–54.

Multi-Locus Variable Number of Tandem Repeat Analysis for Rapid and Accurate Typing of Virulent Multidrug Resistant Escherichia coli Clones

One hundred E. coli isolates from Norway (n = 37), Sweden (n = 24), UK (n = 20) and Spain (n = 19), producing CTX-M-type - (n = 84), or SHV-12 (n = 4) extended spectrum β-lactamases, or the plasmid mediated AmpC, CMY-2 (n = 12), were typed using multi-locus sequence typing (MLST) and multi-locus variable number of tandem repeat analysis (MLVA). Isolates clustered into 33 Sequence Types (STs) and 14 Sequence Type Complexes (STCs), and 58 MLVA-Types (MTs) and 25 different MLVA-Type Complexes (MTCs). A strong agreement between the MLST profile and MLVA typing results was observed, in which all ST131-isolates (n = 39) and most of the STC-648 (n = 10), STC-38 (n = 9), STC-10 (n = 9), STC-405 (n = 8) and STC-23 (n = 6) isolates were clustered distinctly into MTC-29, -36, -20, -14, -10 and -39, respectively. MLVA is a rapid and accurate tool for genotyping isolates of globally disseminated virulent multidrug resistant E. coli lineages, including ST131.