Clonal dissemination of Klebsiella pneumoniae ST258 harbouring KPC-2 in Argentina

The present work describes the abrupt emergence of Klebsiella pneumoniae carbapenemase (KPC) and characterizes the first 79 KPC-producing enterobacteria from Argentina (isolated from 2006 to 2010). The emergence of bla_KPC-2 was characterized by two patterns of dispersion: the first was the sporadic occurrence in diverse enterobacteria from distant geographical regions, harbouring plasmids of different incompatibility groups and bla_KPC-2 in an unusual genetic environment flanked by ISKpn8-Δbla_TEM-1 and ISKpn6-like. bla_KPC-2 was associated with IncL/M transferable plasmids; the second was the abrupt clonal spread of K. pneumoniae ST258 harbouring bla_KPC-2 in Tn4401a.     

Characterization of carbapenem-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> from a healthcare region in Hong Kong

Carbapenem-resistant Enterobacteriaceae represents a major public health issue. This study investigated the clonality and resistance mechanisms of 92 carbapenem-resistant E. coli (n?=?21) and K. pneumoniae (n?=?71) isolates collected consecutively from clinical specimens and patients at high risk of carriage between 2010 and 2012 in a healthcare region in Hong Kong. Combined disk tests (CDTs) and the Carba NP test were used for phenotypic detection of carbapenemases. PCR assays were used to detect carbapenemase genes. All isolates were intermediate or resistant to at least one carbapenem. Nine (9.8 %) isolates were genotypic carbapenemase producers and included six K. pneumoniae (one ST1306/bla _IMP-4, one ST889/bla _IMP-4, two ST11/bla _KPC-2, one ST258/bla _KPC-2, one ST483/bla _NDM-1) and three E. coli (one ST131/bla _IMP-4, two ST744/ bla _NDM-1) isolates. All nine isolates carrying carbapenemase genes could be detected by the CDTs and the Carba NP test. PCR identified bla _CTX-M and bla _AmpC alone or in combination in 77.8 % (7/9) and 96.4 % (80/83) of the carbapenemase-producers and non-producers, respectively. Porin loss was detected in 22.2 % (2/9) and 59.0 % (49/83) of the carbapenemase-producers and non-producers, respectively. Overall, the E. coli clones were diverse (14 different STs), but 36.6 % (26/71) of the K. pneumoniae isolates belonged to ST11. In conclusion, the prevalence of carbapenemases among carbapenem-nonsusceptible E. coli and K. pneumoniae remained low in Hong Kong. Porin loss combined with AmpC and/or CTX-M type ESBL was the major mechanism of carbapenem resistance in the study population.     

Rapid Detection and Statistical Differentiation of KPC Gene Variants in Gram-Negative Pathogens by Use of High-Resolution Melting and ScreenClust Analyses

In the United States, the production of the Klebsiella pneumoniae carbapenemase (KPC) is an important mechanism of carbapenem resistance in Gram-negative pathogens. Infections with KPC-producing organisms are associated with increased morbidity and mortality; therefore, the rapid detection of KPC-producing pathogens is critical in patient care and infection control. We developed a real-time PCR assay complemented with traditional high-resolution melting (HRM) analysis, as well as statistically based genotyping, using the Rotor-Gene ScreenClust HRM software to both detect the presence of bla_KPC and differentiate between KPC-2-like and KPC-3-like alleles. A total of 166 clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with various β-lactamase susceptibility patterns were tested in the validation of this assay; 66 of these organisms were known to produce the KPC β-lactamase. The real-time PCR assay was able to detect the presence of bla_KPC in all 66 of these clinical isolates (100% sensitivity and specificity). HRM analysis demonstrated that 26 had KPC-2-like melting peak temperatures, while 40 had KPC-3-like melting peak temperatures. Sequencing of 21 amplified products confirmed the melting peak results, with 9 isolates carrying bla_KPC-2 and 12 isolates carrying bla_KPC-3. This PCR/HRM assay can identify KPC-producing Gram-negative pathogens in as little as 3 h after isolation of pure colonies and does not require post-PCR sample manipulation for HRM analysis, and ScreenClust analysis easily distinguishes bla_KPC-2-like and bla_KPC-3-like alleles. Therefore, this assay is a rapid method to identify the presence of bla_KPC enzymes in Gram-negative pathogens that can be easily integrated into busy clinical microbiology laboratories.     

Interspecies Transfer of blaIMP-4 in a Patient with Prolonged Colonization by IMP-4-Producing Enterobacteriaceae

A patient was colonized by IMP-4-producing Enterobacter cloacae and Escherichia coli strains for 7 months. IMP-4-producing E. cloacae strains were first and last isolated at day 33 and at 8 months after admission, respectively. IMP-4-producing E. coli strains were first and last isolated at days 88 and 181 after admission, respectively. The E. cloacae and E. coli isolates shared identical genetic features in terms of bla_IMP-4, bla_TEM-1, qnrB2, aacA4, HI2 plasmids, and ISCR1. This study shows the first prolonged colonization with in vivo interspecies transfer of bla_IMP-4.     

Coproduction of KPC-18 and VIM-1 Carbapenemases by Enterobacter cloacae: Implications for Newer β-Lactam–β-Lactamase Inhibitor Combinations

Enterobacter cloacae strain G6809 with reduced susceptibility to carbapenems was identified from a patient in a long-term acute care hospital in Kentucky. G6809 belonged to sequence type (ST) 88 and carried two carbapenemase genes, bla_KPC-18 and bla_VIM-1. Whole-genome sequencing localized bla_KPC-18 to the chromosome and bla_VIM-1 to a 58-kb plasmid. The strain was highly resistant to ceftazidime-avibactam. Insidious coproduction of metallo-β-lactamase with KPC-type carbapenemase has implications for the use of next-generation β-lactam–β-lactamase inhibitor combinations.     

A novel KPC-113 variant conferring carbapenem and ceftazidime-avibactam resistance in a multidrug-resistant Pseudomonas aeruginosa isolate

ObjectivesThis study aimed to characterize a novel KPC-113 variant from a clinical Pseudomonas aeruginosa isolate R20-14.MethodsGenomic DNA of R20-14 was subjected to Illumina and Oxford Nanopore sequencing. The horizontal transmission of plasmid was evaluated with conjugation experiments. Minimum inhibitory concentrations of bacterial strains were obtained using broth microdilution methods. KPC-113 detectability of different carbapenemase detection methods was tested. The kinetic parameters of KPC-113 were compared with those of KPC-2 by a spectrophotometer. Structure modelling and molecular docking of KPC-2 and KPC-113 were performed using Schrödinger.ResultsR20-14, a sequence type 3903 multidrug-resistant strain, was resistant to carbapenems and ceftazidime-avibactam (CZA) concurrently. S1-nuclease pulsed-field gel electrophoresis and genomic analysis revealed a bla_KPC-113–carrying plasmid pR20-14, which resembled the previously reported type I KPC-encoding P. aeruginosa plasmids and exhibited a high conjugation frequency. KPC-113, with a glycine residue insertion between Ambler positions 266 and 267 in KPC-2, conferred both carbapenem and CZA resistance in DH5α and PAO1 transformants. Diagnostic tests showed that KPC-113 acted in a similar manner to KPC-2. Compared with KPC-2, KPC-113 presented reduced catalytic ability to carbapenems and ceftazidime, meanwhile responding poorly to avibactam inhibition. Modelling structure revealed that KPC-113 possibly had a more flattened binding pocket than KPC-2, leading to the change of ligand binding modes.ConclusionsKPC-113 is a novel KPC variant mediating both CZA resistance and carbapenem resistance. It is of great concern that bla_KPC-113 could transfer horizontally with great efficiency and inactivate carbapenems and CZA simultaneously. Great efforts should be made to prevent its spread in clinical settings.     

Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes

We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of bla_KPC genes. The TaqMan real-time PCR assay amplified a 399-bp product from the bla_KPC gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum β-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the bla_KPC PCR assay was used to confirm the presence of bla_KPC genes in any meropenem-resistant Klebsiella spp. The PCR assay detected bla_KPC in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of bla_KPC in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two bla_KPC gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with bla_KPC-2 and bla_KPC-3, respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.     

Dissemination of IMP-6-producing Pseudomonas aeruginosa ST244 in multiple cities in China

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for nosocomial infections and is currently reported to be a worldwide nosocomial menace. The aim of this study was to investigate the epidemiological traits and the distribution of metallo-β-lactamases (MBLs)-producing P. aeruginosa clinical isolates in ten cities in China between January 2010 and May 2012. Antimicrobial susceptibility was determined by disc diffusion assay and the minimum inhibitory concentrations (MICs) of imipenem and meropenem were also determined by the Etest according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In addition, polymerase chain reaction (PCR) and DNA sequencing were applied to detect bla _MBL genes, and their epidemiological relationships were investigated by multilocus sequence typing (MLST). Of 368 P. aeruginosa isolates, MLST analysis identified 138 sequence types (STs), including 122 known and 16 novel STs, and the most frequently detected clone was ST244, followed by ST235. Besides, our study revealed that 25 isolates carried the bla _IMP-6 gene and three isolates carried the bla _VIM-2 gene, and a probe specific for both genes could be hybridised to an ~1,125-kb fragment in all isolates. Interestingly, all of the bla _IMP-6-producing isolates shared an identical ST, ST244, and exhibited a higher level of resistance to several antibiotics. Overall, these observations suggest that P. aeruginosa ST244 carrying the chromosomally located bla _IMP-6 gene is widely disseminated in multiple cites in China.     

Emergence of Pseudomonas aeruginosa strains producing metallo-β-lactamases of the IMP-15 and VIM-2 types in Mexico

Eighty-six carbapenem non-susceptible Pseudomonas aeruginosa isolates collected in the National Institute of Respiratory Diseases of Mexico City were screened for the presence of metallo-β-lactamase (MBL) activity using both E-test strips and a microbiological assay with EDTA-imipenem. Genomic comparisons and sequence analyses conducted with these isolates revealed the presence of bla_VIM-2 in two clonally related isolates, and bla_IMP-15 in a clonally unrelated isolate. Both genes were found to be carried by class 1 integrons, and bla_IMP-15 was additionally present on a broad host-range plasmid. This is the first report of co-existing P. aeruginosa strains producing different MBLs in a Mexican hospital, highlighting the necessity of appropriate surveillance to prevent dissemination of carbapenem resistance.     

Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii

Purpose: Carbapenem resistance in Acinetobacter baumannii has become highly rampant, which has been ascribed to the presence of multiple carbapenemases. The objective of the present study was to prospectively investigate the presence of multiple carbapenemase encoding genes in clinical isolates of A. baumannii. Materials and Methods: A total of 30 imipenem resistant, consecutive non-repeat clinical isolates A. baumannii from a Tertiary Care Centre of Delhi were subjected to antimicrobial susceptibility testing (AST), screening for carbapenemase production by modified Hodge test (MHT) and determination of minimum inhibitory concentration for imipenem by E-Test®. These were subjected to Real time PCR for bla_{IMP-1 and 2}, bla_{VIM-1 and 2}, bla_{OXA23, 24, 51 and 58} using SYBR green-I. These were grouped together on the basis of their genotype as each isolate harboured multiple carbapenemases and correlated with their AST profile. Detection of the novel carbapenemase bla_NDM-1 was performed by real time PCR using TaqMan probes on 14 isolates. Results: Colistin appeared to be the most effective drug in vitro, followed by tetracycline and beta lactam/beta lactamase inhibitor combinations. All, but one isolate were positive for the MHT. All 30 isolates were positive for bla_OXA-51 like gene as well as bla_IMP-1 and bla_VIM-1 genes. bla_{OXA 24 and 58} were not detected in any of the isolates. bla_IMP-2, bla_VIM-2, bla_OXA-23 were present in 15, 6 and 14 isolates respectively. Grouping based on the genotypic profile did not correlate with susceptibility pattern. Nine among the 14 isolates also harboured the novel bla_NDM-1 gene. Conclusions: This is the first study from North India, which comprehensively detected the presence of multiple carbapenemases as well the bla_NDM-1 gene. The presence of the novel gene bla_NDM-1indicated ability of A. baumannii to acquire new carbapenemase genes despite the existence of multiple carbapenemase genes. The present study confirmed the presence of multiple genetic mechanisms for carbapenemases production among the clinical isolates of A. baumannii in north India.     

A Case of IMP-4-, OXA-421-, OXA-96-, and CARB-2-Producing Acinetobacter pittii Sequence Type 119 in Australia

An IMP-4-producing Acinetobacter pittii strain coproducing oxacillinases was isolated from a leg wound of a 67-year-old female patient. Identification to the species level by rpoB and gyrB sequencing and multiplex-PCR-based analysis revealed that the isolate was A. pittii. Whole-genome sequencing of this A. pittii isolate determined the presence of bla_OXA-96, bla_CARB-2, and a novel bla_OXA-421 gene. The position of this novel bla_OXA-421 gene was similar to that of bla_OXA-51 in A. baumannii, downstream of the phosphinothricin N-acetyltransferase gene and upstream of fxsA in the chromosome. This A. pittii isolate was found to belong to sequence type 119 (ST119). Here, we report the first isolation of IMP-4-producing A. pittii ST119 with a novel bla_OXA-421 gene from a patient in Australia and characterize its draft genome.     

Regional dissemination of Acinetobacter species harbouring metallo-β-lactamase genes in Japan

Metallo-β-lactamase (MBL) producers have been reported among the various Acinetobacter species worldwide. In this study, the epidemiology and molecular characteristics of carbapenemase-encoding genes and mobile elements were studied to analyse the regional dissemination of MBL genes in Acinetobacter species. From January 2001 to December 2006, 48 Acinetobacter isolates harbouring MBL genes identified from five hospitals in Kyoto and Shiga Prefecture, Japan were collected and analysed. The partial rpoB gene or the 16S-23S ribosomal RNA intergenic spacer region was sequenced to obtain a species-level identification. Molecular typing using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) was performed. Twenty-five Acinetobacter pittii isolates were divided into eight PFGE types and five sequence types (STs) using MLST. Nine Acinetobacter bereziniae isolates belonged to five PFGE types. Five Acinetobacter nosocomialis isolates were divided into two PFGE types and two STs. Three unclassified Acinetobacter species isolates were divided into two PFGE types. Eighteen of the 25 A. pittii isolates belonged to ST119 and were identified from four hospitals. The bla_IMP-19 gene was detected in 41 of 48 isolates, including all of the A. pittii ST119 isolates. The bla_IMP-1 and bla_IMP-11 genes were detected in four and three isolates, respectively. The MBL genes were all embedded within a class 1 integron as a gene cassette array: bla_IMP-19-aac(6′)-31-bla_OXA-21-aadA1, catB8-like/aacA4-bla_IMP-1 and bla_IMP-11. This study is the first report demonstrating the regional dissemination of MBL-producing Acinetobacter species. A. pittii ST119 harbouring bla_IMP-19 was widely spread throughout the Kyoto-Shiga region.     

Emergence and establishment of KPC-2-producing ST11 <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in a general hospital in Shanghai,China

The aim of this study was to investigate the characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) collected during an outbreak in a Chinese teaching hospital and to provide insights into the prevention and control of nosocomial infection. We collected unique CRKP clinical isolates from 2009 to 2013. Antibiotic-resistant genes were identified by polymerase chain reaction (PCR) and sequencing. The isolates were typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmids were classified using a PCR-based incompatibility/replicon typing method and a replicon sequence typing method. Conjugation experiments were performed to evaluate the transferability of carbapenem-resistant genes. Whole genome sequencing (WGS) was conducted to further investigate the genetic background of the isolates. Infection control practices were reviewed throughout the study period. Klebsiella pneumoniae sequence type (ST) 11 emerged in 2010 and acquired the bla _KPC-2 gene by 2011. From 2011 to 2013, ST11 KPC-2-producing CRKP (G type) prevailed as the most common CRKP in our hospital, causing a prolonged outbreak. The majority of these CRKP strains possess an IncFII plasmid, with Tn1721-bla _KPC-2-ΔTn3-IS26 bearing the genetic structure for bla _KPC-2. Infection prevention control measures available at the time contained the initial outbreak, but had no effect on the spread of CRKP later. This study demonstrated the seriousness concerning the spread of KPC-2-producing ST11 CRKP in a Chinese hospital, indicating that current prevention and control strategies for carbapenem-resistant Enterobacteriaceae (CRE) nosocomial infection need to be investigated and adjusted.     

Emergence of ceftazidime/avibactam resistance in carbapenem-resistant Klebsiella pneumoniae in China

ObjectivesThe aim was to investigate the activity of ceftazidime/avibactam (CAZ/AVI) against carbapenem-resistant Klebsiella pneumoniae (CRKP) and identify the resistance mechanisms before CAZ/AVI coming to Chinese market.MethodsClinical CRKP isolates were continuously collected from 36 tertiary hospitals in China from 1 March 2017 to 31 July 2017. CAZ/AVI MICs were determined by agar dilution method. CAZ/AVI resistant isolates were submitted to whole genome sequencing. The copy number and relative expression of bla_KPC were determined by quantitative PCR.ResultsA total of 872 CRKP isolates were collected, and MIC₅₀ and MIC₉₀ of CAZ/AVI were 4 and 8 mg/L. The resistant rate of CAZ/AVI was 3.7% (32/872). Among the resistant isolates, 53.1% (17/32) were metallo-β-lactamase-producing K. pneumoniae (MBL-KP), 40.6% (13/32) were Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) and 6.3% (2/32) produced both MBL and KPC. One of the KPC-KP with high level CAZ/AVI resistance (>128 mg/L) harboured mutated bla_KPC-2 (D179Y). In 12 wild-type bla_KPC-2 isolates, the relative copy number and expression of bla_KPC-2 gene were 2.5-fold and 2.7-fold higher than that in the CAZ/AVI MIC ≤0.5 mg/L group (p < 0.05), and when added avibactam at a fixed concentration of 8 mg/L, 91.7% (11/12) isolates could restore susceptibility.ConclusionsResistance against CAZ/AVI in CRKP emerged before clinical use of CAZ/AVI in China, although most of the CRKP isolates maintained the susceptibility. MBL production, bla_KPC-2 point mutation and high KPC expression played an important role in CAZ/AVI resistance.     

Unusual association of NDM-1 with KPC-2 and armA among Brazilian Enterobacteriaceae isolates

We report the microbiological characterization of four New Delhi metallo-β-lactamase-1 (bla _NDM-1)-producing Enterobacteriaceae isolated in Rio de Janeiro, Brazil. bla _NDM-1 was located on a conjugative plasmid and was associated with Klebsiella pneumoniae carbapenemase-2 (bla _KPC-2) or aminoglycoside-resistance methylase (armA), a 16S rRNA methylase not previously reported in Brazil, in two distinct strains of Enterobacter cloacae. Our results suggested that the introduction of bla _NDM-1 in Brazil has been accompanied by rapid spread, since our isolates showed no genetic relationship.     

Outbreak of KPC-3-producing,and colistin-resistant,Klebsiella pneumoniae infections in two Sicilian hospitals

We report the first outbreak caused by colistin-resistant Klebsiella pneumoniae producing KPC-3 carbapenamase in two Italian hospitals. This spread occurred in 1 month, and was caused by eight colistin-resistant and carbapenem-resistant Klebsiella pneumoniae isolates from eight patients. A further three isolates were obtained from the intestinal tract and pharyngeal colonization. All isolates were multidrug-resistant (MDR), including being resistant to colistin, but they were susceptible to gentamicin and tigecycline. PCR detection showed that all isolates harboured the bla_KPC-3 gene associated with bla_SHV-11, bla_TEM-1 and bla_OXA-9. All K. pneumoniae isolates, genotyped by pulsed-field gel electrophoresis and multilocus sequence typing, belonged to the same sequence type (ST)258 clone. From our data and a review of the international literature, K. pneumoniae ST258 seems to be the most widespread genetic background for KPC dissemination in Europe.     

Genomic characterization of a KPC-23-producing Klebsiella pneumoniae ST258 clinical isolate resistant to ceftazidime-avibactam

ObjectivesWe characterized the first ceftazidime-avibactam-resistant KPC-producing-Klebsiella pneumoniae clinical isolate detected in Greece, before the introduction of ceftazidime-avibactam in clinical practice.MethodsK. pneumoniae KP-90 was isolated from a hospitalized patient in Thessaloniki during a nationwide surveillance study conducted between 2014 and 2016. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing (Ion Torrent TM platform) of the isolate was carried out to identify the acquired resistance genes and mutations that were associated with ceftazidime-avibactam resistance.ResultsThe K. pneumoniae isolate belonged to multilocus sequence type ST258 and harboured bla_KPC-23 as the only carbapenemase gene. The isolate had a minimum inhibitory concentration (MIC) of 16 mg/L to ceftazidime-avibactam and was highly resistant to imipenem, meropenem (MICs, 512 mg/L) and ceftazidime (MIC, >1024 mg/L). bla_KPC-23 was detected on a Tn4401a transposon, located on a pKPQIL-type plasmid. A non-functional outer membrane protein OmpK35 and an OmpK36 variant that had been previously associated with K. pneumoniae isolates of ST258 were detected. Transformation studies with Escherichia coli TOP10 showed that KPC-23 offered similar carbapenem MICs as KPC-2 and KPC-3. However, KPC-23 conferred a four-fold higher ceftazidime MIC (>1024 mg/L), which in the presence of avibactam was reduced (>7-fold) to 8 mg/L, which is just within the limit of the susceptibility breakpoint.ConclusionsCeftazidime-avibactam resistance in a KPC-23- producing K. pneumoniae clinical isolate was due to increased ceftazidime hydrolysis and was likely enhanced by OmpK35 porin deficiency.     

A nosocomial outbreak of KPC-2-producing Klebsiella pneumoniae in a Chinese hospital: dissemination of ST11 and emergence of ST37, ST392 and ST395

In China, Klebsiella pneumoniae carbapenemase (KPC) -producing K. pneumoniae isolates have been identified. However, little is known about the spread and outbreak of KPC-producing enterobacterial pathogens. In this study, 48 non-duplicated KPC-producing isolates were analysed for genetic relatedness by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility by E-test, and sequence type (ST) by multilocus sequence typing. S1-PFGE and Southern blot were used for plasmid profiling, and PCR and subsequent sequencing were performed to determine the effects of genetic background on the blaKPC gene. From December 2011 to June 2012, an outbreak of the KPC-2-producing K. pneumoniae was observed. The 48 isolates of K. pneumoniae are categorized into eight PFGE types (A1, A2, A3, A4, B, C, D and E). The predominant pathogens of the outbreak were strains with PFGE types A1, A2 and A3, which all belong to ST11. Furthermore, ST37, ST392 and ST395 KPC-2-producing K. pneumoniae isolates have also been sporadically identified. The blaKPC-2-carrying plasmids vary in size from 30 to 220 kb. The genetic environments of the blaKPC-2 gene for most strains were consistent with the genetic structure of blaKPC-2 on the plasmid pKP048. In conclusion, the dissemination and outbreak of KPC-2-producing K. pneumoniae isolates in this study appeared to be clonal, and ST11 K. pneumoniae was the predominant clone attributed to the outbreak. This is the first study to report the emergence and spread of KPC-producing K. pneumoniae ST392 and ST395 worldwide. Our findings suggest that horizontal transfer of Tn3-based transposons might mediate the spread of blaKPC-2 gene between different K. pneumoniae clones in China.     

First Descriptions of blaKPC in Raoultella spp. (R. planticola and R. ornithinolytica): Report from the SENTRY Antimicrobial Surveillance Program

Two strains of Raoultella planticola and one of Raoultella ornithinolytica showing carbapenem resistance were recovered from patients hospitalized in New Jersey and Ohio. All patients had received previous antimicrobial treatment, including carbapenems. These strains harbored bla_KPC-2 and bla_KPC-3. Carbapenemase genes were embedded in isoforms of Tn4401 and were plasmidic and chromosomal in location.Raoultella species are gram-negative aerobic bacilli belonging to the Enterobacteriaceae family that are closely related to Klebsiella spp. (7). These environmental organisms, infrequently causing human infections, appear to have pathogenicity similar to that of Klebsiella pneumoniae (8). The first human Raoultella spp. invasive infection was described in 1984, and bloodstream infections have been reported, though rarely (1). Studies have shown that 0.2 to 19.0% of isolates initially identified as Klebsiella spp. were Raoultella spp. by 16S rRNA analysis and that the prevalence of these organisms in clinical settings can vary geographically (8).(This work was presented at the 19th European Conference of Clinical Microbiology and Infectious Diseases, Helsinki, Finland, 2009.)A total of 7,248 Enterobacteriaceae isolates collected in medical centers from North America, Latin America, and Europe during 2008 were susceptibility tested by the reference broth microdilution method and interpretation criteria (3, 4). Isolates displaying imipenem and/or meropenem MICs of ≥2 μg/ml were tested with the modified Hodge test (MHT) using imipenem and meropenem disks (4) and multiplex PCRs for the detection of carbapenemase-encoding genes, including bla_IMP, bla_VIM, bla_KPC, bla_SME, and bla_GES variants and bla_IMI, bla_NMC-A, and bla_OXA-48.Among 134 (1.8% overall) isolates that were nonsusceptible to carbapenems, three (2.2%) Raoultella isolates from bloodstream infections were observed. MHT was positive for all three strains, and PCRs were positive for bla_KPC. Sequencing of 16S rRNA revealed that two isolates were R. planticola (from Ohio and New Jersey) and one was R. ornithinolytica (from New Jersey). The isolates from New Jersey were detected in the same hospital and harbored bla_KPC-3, whereas the strain from Ohio carried bla_KPC-2. The clinical histories of the patients presenting infections with KPC-producing Raoultella spp. are summarized below.     

Multilocus Sequence Types of Carbapenem-Resistant Pseudomonas aeruginosa in Singapore Carrying Metallo-β-Lactamase Genes,Including the Novel blaIMP-26 Gene

Nine imipenem-resistant Pseudomonas aeruginosa isolates were found to contain a variety of metallo-β-lactamase genes, including bla_IMP-1, bla_IMP-7, bla_VIM-2, bla_VIM-6, and the novel bla_IMP-26. Multilocus sequence typing showed a diversity of sequence types. Comparison with isolates from an earlier study showed that the epidemic clones from 2000 have not become established.Carbapenem-resistant Pseudomonas aeruginosa is an increasing problem worldwide. While many underlying mechanisms may account for carbapenem resistance in this species, the possession of metallo-β-lactamase (MBL) genes is of particular concern because these enzymes are able to hydrolyze all β-lactam antimicrobials with the exception of aztreonam. In addition, these genes may be mobilized and transferred between different species of bacteria. We conducted a study in 2008 to investigate if there were any changes in the epidemiology of P. aeruginosa isolates containing MBL genes in our hospital compared to results from an earlier survey carried out in 2000 (3).Of 2,552 nonduplicate P. aeruginosa organisms isolated in 2008, 123 isolates were imipenem resistant. Of these, 11 were positive for MBL production by imipenem-EDTA disk diffusion (5). Nine of these yielded a product by multiplex PCR for MBL genes (2). The individual MBL genes were then amplified and sequenced. The clonal relationship between isolates with MBL genes was determined by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA restricted with SpeI (3). The PFGE band patterns were analyzed with Bionumerics (Applied Maths NV, Sint-Martens-Latem, Belgium), and all strains with more than 85% similarity were considered to belong to the same clone. All strains were further subjected to multilocus sequence typing (MLST) (1). Because it is a nucleic acid sequence-based method, MLST is able to characterize bacterial types in an unambiguous fashion and establish evolutionary relationships between strains better than band-based methods like PFGE. Representative MBL-producing P. aeruginosa isolates from the 2000 survey were also subjected to PFGE and MLST. MLST profiles were submitted to eBURST V3 (http://eburst.mlst.net/) on 10 March 2010. Isolates sharing six out of seven alleles were assigned to the same BURST group and can be considered to belong to the same clonal complex descended from a common founder genotype. The PFGE, MBL gene sequence, and MLST results are summarized in Fig. ​Fig.11.Open in a separate windowFIG. 1.Dendrogram of PFGE patterns of P. aeruginosa isolates with metallo-β-lactamase genes, showing the year of isolation, MLST sequence type, and BURST group.In our previous study, 21 of 2,094 (1.0%) of all nonduplicate P. aeruginosa isolates in our hospital had MBL genes (3). With the exception of one isolate with bla_IMP-7, all other isolates had bla_IMP-1 and belonged to one of two PFGE clones. Isolates belonging to clone A had sequences identical to that of the original bla_IMP-1 first reported in Japan. Four representatives of clone A isolated from our hospital in 2000 had sequence type 964 (ST964) by MLST. Isolates belonging to clone B isolated in 2000 had sequences for variant bla_IMP-1 (bla_IMP-1v) with four silent mutations. Three representatives of this clone from 2000 had ST233 and one had ST742 based on MLST. All four representatives of clone B belong to the same BURST group, which was different from that of clone A.In contrast, in the 2008 survey, 9 of 2,552 (0.35%) nonduplicate P. aeruginosa isolates had MBL genes. Unlike the earlier study, there were no large clonal outbreaks. Two isolates with bla_IMP-1v had similar PFGE patterns and belonged to the same BURST group as representative isolates from clone B in 2000.Two isolates from 2008 with bla_IMP-7 had similar PFGE patterns and shared the same BURST group. The rest of the isolates from 2008 had distinct PFGE patterns.There was a greater diversity of MBL genes compared to the 2000 survey results. In particular, this is the first time that bla_VIM-2 and bla_VIM-6 have been found in P. aeruginosa in Singapore. bla_IMP-26 is a novel MBL gene that differs from bla_IMP-4 at position 145 (G-to-T change). The translated amino acid sequence differs from IMP-4 at residue 49 (phenylalanine for valine). This sequence has been previously deposited in the GenBank database as IMP-4 from an Acinetobacter calcoaceticus isolate from Malaysia (accession number {"type":"entrez-protein","attrs":{"text":"ABC24668.1","term_id":"83583501"}}ABC24668.1).Three of the isolates in this study (separately containing bla_VIM-2, bla_IMP-1, and bla_IMP-7) belonged to ST235. This sequence type has been described in a VIM-producing P. aeruginosa isolate in Belgrade and is the founder of an international clonal complex of isolates bearing MBL genes found in several countries in Europe (6). Recently, an increasing prevalence of IMP-1-producing P. aeruginosa has been found in Hiroshima, Japan. This was due entirely to the clonal expansion of only two lineages, ST235 (BURST group 3) and ST357 (BURST group 108) (4). This is similar to the situation that existed in Singapore in 2000, where only two lineages (BURST groups 29 and 44) accounted for the majority of MBL-producing P. aeruginosa (3).It is noteworthy that the original fear that a clone of MBL-producing P. aeruginosa would become established in Singapore has not been realized. The BURST group 29 and 44 lineages from 2000 were represented by only one to two isolates in 2008. The two P. aeruginosa isolates with bla_IMP-7 in 2008 are unrelated to the solitary isolate with bla_IMP-7 from 2000. It has been suggested that P. aeruginosa displays an epidemic population structure, with a limited number of clones emerging from a large number of unrelated genotypes (7). Although we did not correlate our study with hospital infection control measures, the Japanese data and our own seem to suggest that controlling the prevalence of MBL-producing P. aeruginosa may be achieved by preventing the transmission of specific epidemic clones.While it is reassuring to note that the prevalence of MBL producers in carbapenem-resistant P. aeruginosa has not increased, the increased diversity of MBL genes represents a new cause for concern. We were unable to characterize the gene responsible for the MBL phenotype in two isolates in this study, and these may represent novel resistance determinants. Although clones of MBL-producing P. aeruginosa have not become established, it seems likely, given the variation of MBL genes and MLST types in this study, that MBL-producing P. aeruginosa continues to be introduced to our hospital from diverse sources.