Sequence of Closely Related Plasmids Encoding bla NDM-1 in Two Unrelated Klebsiella pneumoniae Isolates in Singapore

Background

Spread of the bla _NDM-1 gene that encodes the New Delhi metallo-β-lactamase (NDM-1) in Enterobacteriaceae is a major global health problem. Plasmids carrying bla _NDM-1 from two different multi-drug resistant Klebsiella pneumonia isolates collected in Singapore were completely sequenced and compared to known plasmids carrying bla _NDM-1.

Methodology/Principal Findings

The two plasmids, pTR3 and pTR4, were transferred to Escherichia coli recipient strain J53 and completely sequenced by a shotgun approach using 3-kb paired-end libraries on 454. Although the K. pneumoniae strains were unrelated by molecular typing using PFGE and MLST, complete sequencing revealed that pTR3 and pTR4 are identical. The plasmid sequence is similar to the E. coli NDM-1-encoding plasmid p271A, which was isolated in Australia from a patient returning from Bangladesh. The immediate regions of the bla _NDM-1 gene in pTR3/4 are identical to that of p271A, but the backbone of our plasmid is much more similar to another IncN2 plasmid reported recently, pJIE137, which contained an additional 5.2-kb CUP (conserved upstream repeat) regulon region in comparison to p271A. A 257-bp element bounded by imperfect 39-bp inverted repeats (IR) and an incomplete version of this element flanking the 3.6-kb NDM-1-encoding region were identified in these plasmids and are likely to be the vestiges of an unknown IS.

Conclusions

Although the hosts are not epidemiologically linked, we found that the plasmids bearing the bla _NDM-1 gene are identical. Comparative analyses of the conserved NDM-1-encoding region among different plasmids from K. pneumoniae and E. coli suggested that the transposable elements and the two unknown IR-associated elements flanking the NDM-1-encoding region might have aided the spreading of this worrisome resistance determinant.     

Molecular Characterization of an Atypical IncX3 Plasmid pKPC-NY79 Carrying bla KPC-2 in a Klebsiella pneumoniae

The IncX family of plasmids has recently been expanded to include at least four subtypes, IncX1–IncX4. The revised classification provides an opportunity for improving our understanding of the sequence diversity of the IncX plasmids and the resistance genes they carried. We described the complete nucleotide sequence of a novel IncX3 plasmid, pKPC-NY79 (42,447 bp) from a sequence-type 258 Klebsiella pneumoniae strain that was isolated from a patient who was hospitalized in New York, United States. In pKPC-NY79, the plasmid scaffold and genetic load region were highly similar to homologous regions in pIncX-SHV (IncX3, JN247852) and the bla _KPC carrying pKpQIL (IncFII_k, GU595196), respectively, indicating that it has possibly arisen through recombination of plasmids. The bla _KPC-2 gene, as part of a transposon Tn4401a, was found within the genetic load region. The backbone of pKPC-NY79 differs from pIncX-SHV by a deletion involving the gene tandem hns–topB (encoding H-NS protein and topoisomerase III, respectively) and a putative ATPase gene. Unexpectedly, the impact of the hns–topB deletion on host fitness and plasmid stability was found to be small. In conclusion, the findings contribute to a better understanding of the plasmid platforms carrying bla _KPC and of variations in the backbone of the IncX3 plasmids.     

Carbapenem-Nonsusceptible Enterobacteriaceae in Taiwan

A total of 1135 carbapenem-resistant (nonsusceptible) Enterobacteriaceae (CRE) isolates were recovered between November 2010 and July 2012 (517 from 2010-2011 and 618 from 2012) from 4 hospitals in Taiwan. Carbapenemase-producing Enterobacteriaceae (CPE) comprised 5.0% (57 isolates), including 17 KPC-2 (16 Klebsiella pneumoniae and 1 Escherichia coli), 1 NDM-1 (K. oxytoca), 37 IMP-8 (26 Enterobacter cloacae, 4 Citrobacter freundii, 4 Raoultella planticola, 1 K. pneumoniae, 1 E. coli and 1 K. oxytoca), and 2 VIM-1 (1 E. cloacae, 1 E. coli). The KPC-2-positive K. pneumoniae were highly clonal even in isolates from different hospitals, and all were ST11. IMP-8 positive E. cloacae from the same hospitals showed higher similarity in PFGE pattern than those from different hospitals. A total of 518 CRE isolates (45.6%) were positive for bla _ESBL, while 704 (62.0%) isolates were bla _AmpC-positive, 382 (33.6% overall) of which carried both bla _ESBL and bla _AmpC. CTX-M (414, 80.0%) was the most common bla _ESBL, while DHA (497, 70.6%) and CMY (157, 22.3%) were the most common bla _AmpC. Co-carriage of bla _ESBL and bla _AmpC was detected in 31 (54.4%) and 15 (26.3%) of the 57 CPE, respectively. KPC-2 was the most common carbapenemase detected in K. pneumoniae (2.8%), while IMP-8 was the most common in E. cloacae (9.7%). All KPC-2-positive CRE were resistant to all three tested carbapenems. However, fourteen of the 37 IMP-8-positive CRE were susceptible to both imipenem and meropenem in vitro. Intra- and inter-hospital spread of KPC-2-producing K. pneumoniae and IMP-8-producing E. cloacae likely occurred. Although the prevalence of CPE is still low, careful monitoring is urgently needed. Non-susceptibility to ertapenem might need to be considered as one criterion of definition for CRE in areas where IMP type carbapenemase is prevalent.     

Emergence of OXA-48-Producing Klebsiella pneumoniae in Taiwan

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four bla _OXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured bla _CTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the bla _OXA-48 gene in all four isolates was identical to pKP_OXA-48N1, a bla _OXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify bla _OXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of bla _OXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern.     

pIMP-PH114 Carrying bla IMP-4 in a Klebsiella pneumoniae Strain is Closely Related to Other Multidrug-Resistant IncA/C2 Plasmids

The IncA/C plasmids are broad host-range vehicles which have been associated with wide dissemination of CMY-2 among Enterobacteriaceae of human and animal origins. Acquired metallo-β-lactamases (MBLs) such as the IMP-type enzymes are increasingly reported in multidrug-resistant Gram-negative bacteria worldwide, particularly in Enterobacteriaceae. We described the complete sequence of the first IMP-4-encoding IncA/C₂ plasmid, pIMP-PH114 (151,885 bp), from a sequence type 1 Klebsiella pneumoniae strain that was recovered from a patient who was hospitalized in the Philippines. pIMP-PH114 consists of a backbone from the IncA/C₂ plasmids, with the insertion of a novel Tn21-like class 1 integron composite structure (containing the cassette array bla _IMP-4-qacG-aacA4-catB3, followed by a class C β-lactamase bla _DHA-1 and the mercury resistance operon, merRTPCADE) and a sul2-floR encoding region. Phylogenetic analysis of the IncA/C repA sequences showed that pIMP-PH114 formed a subgroup with other IncA/C plasmids involved in the international spread of CMY-2, TEM-24 and NDM-1. Identical bla _IMP-4 arrays have been described among different Enterobacteriaceae and Acinetobacter spp. in China, Singapore and Australia but the genetic context is different. The broad host range of IncA/C plasmids may have facilitated dissemination of the bla _IMP-4 arrays among different diverse groups of bacteria.     

Copy Number Change of the NDM-1 Sequence in a Multidrug-Resistant Klebsiella pneumoniae Clinical Isolate

The genetic features of the antimicrobial resistance of a multidrug resistant Klebsiella pneumoniae strain harboring bla _NDM-1 were investigated to increase our understanding of the evolution of NDM-1. The strain, KPX, came from a Taiwanese patient with a hospitalization history in New Delhi. Complete DNA sequencing was performed; and the genes responsible for antimicrobial resistance were systematically examined and isolated by library screening. KPX harbored two resistance plasmids, pKPX-1 and pKPX-2, which are 250-kb and 141-kb in size, respectively, with bla _NDM-1 present on pKPX-1. The plasmid pKPX-1 contained genes associated with the IncR and IncF groups, while pKPX-2 belonged to the IncF family. Each plasmid carried multiple antimicrobial resistance genetic determinants. The gene responsible for resistance to carbapenems was found on pKPX-1 and that for resistance to aztreonam was found on pKPX-2. To our surprise, we discovered that bla _NDM-1 exists on pKPX-1 as multiple copies in the form of tandem repeats. Amplification of bla _NDM-1 was found to occur by duplication of an 8.6-kb unit, with the copy number of the repeat varying from colony to colony. This repeat sequence is identical to that of the pNDM-MAR except for two base substitutions. The copy number of bla _NDM-1 of colonies under different conditions was assessed by Southern blotting and quantitative PCR. The bla _NDM-1 sequence was maintained in the presence of the antimicrobial selection; however, removal of antimicrobial selection led to the emergence of susceptible bacterial populations with a reduced copy number or even the complete loss of the bla _NDM-1 sequence. The dynamic nature of the NDM-1 sequence provides a strong argument for judicious use of the broad-spectrum antimicrobials in order to reduce the development and spread of antimicrobial resistance among pathogens.     

Co-Carriage of bla KPC-2 and bla NDM-1 in Clinical Isolates of Pseudomonas aeruginosa Associated with Hospital Infections from India

Global spread of KPC poses to be a serious threat complicating treatment options in hospital settings. The present study investigates the genetic environment of bla _KPC-2 among clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital of India. The study isolates were collected from different wards and clinics of Silchar Medical College and Hospital, India, from 2012–2013. The presence of bla _KPC was confirmed by genotypic characterization followed by sequencing. Cloning of the bla _KPC-2 gene was performed and the genetic environment of this gene was characterized as well. Transferability of the resistance gene was determined by transformation assay and Southern hybridization. Additionally, restriction mapping was also carried out. Two isolates of P. aeruginosa were found to harbor bla _KPC-2, were resistant towards aminoglycosides, quinolone and β-lactam-β-lactamase inhibitor combination. In both the isolates, the resistance determinant was associated with class 1 integron and horizontally transferable. Both the isolates were co-harboring bla _NDM-1. The first detection of this integron mediated bla _KPC-2 coexisting with bla _NDM-1 in P. aeruginosa from India is worrisome, and further investigation is required to track the gene cassette mediated bla _KPC-2 in terms of infection control and to prevent the spread of this gene in hospitals as well as in the community.     

Closely Related NDM-1-Encoding Plasmids from Escherichia coli and Klebsiella pneumoniae in Taiwan

Objective

Two plasmids carrying bla _NDM-1 isolated from carbapenem-resistant Klebsiella pneumoniae (CR-KP) and carbapenem-resistant Escherichia coli (CR-EC) were sequenced. CR-KP and CR-EC were isolated from two Taiwanese patients without travel histories.

Methods

Complete sequencing of the plasmids (pLK75 and pLK78) was conducted using a shotgun approach. Annotation of the contigs was performed using the RAST Server, followed by manual inspection and correction.

Results

These similar plasmids were obtained from two patients with overlapping stays at the same hospital. The pLK75 and pLK78 plasmids were 56,489-bp and 56,072-bp in length, respectively. Plasmid annotation revealed a common backbone similar to the IncN plasmid pR46. The regions flanking the bla _NDM-1 genes in these plasmids were very similar to plasmid pNDM-HU01 in Japan, which contains a complex class 1 integron located next to an ISCR1 element. The ISCR1 element has been suggested to provide a powerful mechanism for mobilising antibiotic resistance genes.

Conclusion

Two indigenous NDM-1-producing Enterobacteriaceae cases were identified for the first time in Taiwan, highlighting the alarming introduction of NDM-1-producing Enterobacteriaceae in this region.     

Characterization of an IncFII plasmid encoding NDM-1 from Escherichia coli ST131

Background

The current spread of the gene encoding the metallo-ß-lactamase NDM-1 in Enterobacteriaceae is linked to a variety of surrounding genetic structures and plasmid scaffolds.

Methodology

The whole sequence of plasmid pGUE-NDM carrying the bla _NDM-1 gene was determined by high-density pyrosequencing and a genomic comparative analysis with other bla _NDM-1-negative IncFII was performed.

Principal Findings

Plasmid pGUE-NDM replicating in Escherichia coli confers resistance to many antibiotic molecules including β-lactams, aminoglycosides, trimethoprim, and sulfonamides. It is 87,022 bp in-size and carries the two β-lactamase genes bla _NDM-1 and bla _OXA-1, together with three aminoglycoside resistance genes aacA4, aadA2, and aacC2. Comparative analysis of the multidrug resistance locus contained a module encompassing the bla _NDM-1 gene that is actually conserved among different structures identified in other enterobacterial isolates. This module was constituted by the bla _NDM-1 gene, a fragment of insertion sequence ISAba125 and a bleomycin resistance encoding gene.

Significance

This is the first characterized bla _NDM-1-carrying IncFII-type plasmid. Such association between the bla _NDM-1 gene and an IncFII-type plasmid backbone is extremely worrisome considering that this plasmid type is known to spread efficiently, as examplified with the worldwide dissemination of bla _CTX-M-15-borne IncFII plasmids.     

Population structure and pangenome analysis of Enterobacter bugandensis uncover the presence of blaCTX-M-55, blaNDM-5 and blaIMI-1, along with sophisticated iron acquisition strategies

Enterobacter bugandensis is a recently described species that has been largely associated with nosocomial infections. We report the genome of a non-clinical E. bugandensis strain, which was integrated with publicly available genomes to study the pangenome and general population structure of E. bugandensis. Core- and whole-genome multilocus sequence typing allowed the detection of five E. bugandensis phylogroups (PG-A to E), which contain important antimicrobial resistance and virulence determinants. We uncovered several extended-spectrum β-lactamases, including bla_CTX-M-55 and bla_NDM-5, present in an IncX replicon type plasmid, described here for the first time in E. bugandensis. Genetic context analysis of bla_NDM-5 revealed the resemblance of this plasmid with other IncX plasmids from other bacteria from the same country. Three distinctive siderophore producing operons were found in E. bugandensis: enterobactin (ent), aerobactin (iuc/iut), and salmochelin (iro). Our findings provide novel insights on the lifestyle, physiology, antimicrobial, and virulence profiles of E. bugandensis.     

Epidemiological Characteristics of bla NDM-1 in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii Complex in China from 2011 to 2012

Objectives

The study aimed to investigate the prevalence and epidemiological characteristics of bla _NDM-1 (encoding New Delhi metallo-β-lactamase 1) in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ABC) in China from July 2011 to June 2012.

Methods

PCR was used to screen for the presence of bla _NDM-1 in all organisms studied. For bla _NDM-1-positive strains, 16S rRNA analysis and Analytical Profile Index (API) strips were used to identify the bacterial genus and species. The ABCs were reconfirmed by PCR detection of bla _OXA-51-like. Antibiotic susceptibilities of the bacteria were assessed by determining minimum inhibitory concentration (MIC) of them using two-fold agar dilution test, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot hybridization were conducted to ascertain the gene location of bla _NDM-1. Conjugation experiments were conducted to determine the transmission of bla _NDM-1-positive strains.

Results

Among 2,170 Enterobacteriaceae and 600 ABCs, seven Enterobacteriaceae strains and two A. calcoaceticus isolates from five different cities carried the bla _NDM-1 gene. The seven Enterobacteriaceae strains comprised four Klebsiella pneumoniae, one Enterobacter cloacae, one Enterobacter aerogen and one Citrobacter freundii. All seven were non-susceptible to imipenem, meropenem or ertapenem. Two A. calcoaceticus species were resistant to imipenem and meropenem. Three K. pneumoniae showed the same PFGE profiles. The bla _NDM-1 genes of eight strains were localized on plasmids, while one was chromosomal.

Conclusions

Compared with previous reports, the numbers and species containing the bla _NDM-1 in Enterobacteriaceae have significantly increased in China. Most of them are able to disseminate the gene, which is cause for concern. Consecutive surveillance should be implemented and should also focus on the dissemination of bla _NDM-1 among gram-negative clinical isolates.     

Transmission and characterization of <Emphasis Type="Italic">bla</Emphasis><Subscript>NDM-1</Subscript> in <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> at a teaching hospital in Yunnan,China

Background

In recent years, New Delhi metallo-beta-lactamases 1 (bla _NDM-1) has been reported with increasing frequency and become prevalent. The present study was undertaken to investigate the epidemiological dissemination of the bla _NDM-1 gene in Enterobacter cloacae isolates at a teaching hospital in Yunnan, China.

Methods

Antimicrobial susceptibility testing was performed using VITEK 2 system and E test gradient strips. The presence of integrons and insertion sequence common region 1 were examined by PCR and sequencing. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Conjugation experiments and Southern blot hybridization were performed to determine the transferability of plasmids.

Results

Ten E. cloacae isolates and their Escherichia coli transconjugants were exhibited similar resistant patterns to carbapenems, cephalosporins and penicillins. 8 (80%) of E. cloacae isolates carried class 1 integron and 1 (12.5%) carried class 2 integron. Integron variable regions harbored the genes which encoded resistance to aminoglycosides (aadA1, aadA2, aadA5, aadB, aac(6′)-Ib-cr), sulfamethoxazole/trimethoprim (dfrA17, dfrA12, dfrA15) and Streptozotocin (sat2). Six E. cloacae isolates belonged to ST74 and exhibited highly similar PFGE patterns. Each isolate shared an identical plasmid with ~33.3 kb size that carried the bla _NDM-1 gene, except T3 strain, of which the bla _NDM-1 gene was located on a ~50 kb plasmid.

Conclusions

Our findings suggested that plasmid was able to contribute to the dissemination of bla _NDM-1. Hence, more attention should be devoted to monitor the dissemination of the bla _NDM-1 gene due to its horizontal transfer via plasmid. In addition, nosocomial surveillance system should actively monitor the potential endemic clone of ST74 to prevent their further spread.

     

Salmonella Typhi acquires diverse plasmids from other Enterobacteriaceae to develop cephalosporin resistance

BackgroundRecent reports have established the emergence and dissemination of extensively drug resistant (XDR) H58 Salmonella Typhi clone in Pakistan. In India where typhoid fever is endemic, only sporadic cases of ceftriaxone resistant S. Typhi are reported. This study aimed at elucidating the phylogenetic evolutionary framework of ceftriaxone resistant S. Typhi isolates from India to predict their potential dissemination.MethodsFive ceftriaxone resistant S. Typhi isolates from three tertiary care hospitals in India were sequenced on an Ion Torrent Personal Genome Machine (PGM). A core genome single-nucleotide-polymorphism (SNP) based phylogeny of the isolates in comparison to the global collection of MDR and XDR S. Typhi isolates was built. Two of five isolates were additionally sequenced using Oxford Nanopore MinION to completely characterize the plasmid and understand its transmission dynamics within Enterobacteriaceae.ResultsComparative genomic analysis and detailed plasmid characterization indicate that while in Pakistan (4.3.1 lineage I) the XDR trait is associated with bla_CTX-M-15 gene on IncY plasmid, in India (4.3.1 lineage II), the ceftriaxone resistance is due to short term persistence of resistance plasmids such as IncX3 (bla_SHV-12) or IncN (bla_TEM-1B + bla_DHA-1).ConclusionConsidering the selection pressure exerted by the extensive use of ceftriaxone in India, there are potential risks for the occurrence of plasmid transmission events in the predominant H58 lineages. Therefore, continuous monitoring of S. Typhi lineages carrying plasmid-mediated cephalosporin resistant genes is vital not just for India but also globally.     

National Surveillance Study on Carbapenem Non-Susceptible Klebsiella pneumoniae in Taiwan: The Emergence and Rapid Dissemination of KPC-2 Carbapenemase

Objectives

The global spread and increasing incidence of carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) has made its treatment difficult, increasing the mortality. To establish nationwide data on CnSKP spread and carbapenem-resistance mechanisms, we conducted a national surveillance study in Taiwanese hospitals.

Methods

We collected 100 and 247 CnSKP isolates in 2010 and 2012, respectively. The tests performed included antibiotic susceptibility tests; detection of carbapenemase, extended-spectrum β-lactamases (ESBL), and AmpC β-lactamases genes; outer membrane porin profiles; and genetic relationship with pulsed-field gel electrophoresis and multilocus sequence type.

Results

The resistance rate of CnSKP isolates to cefazolin, cefotaxime, cefoxitin, ceftazidime, and ciprofloxacin was over 90%. Susceptibility rate to tigecycline and colistin in 2010 was 91.0% and 83.0%, respectively; in 2012, it was 91.9% and 87.9%, respectively. In 2010, carbapenemase genes were detected in only 6.0% of isolates (4 bla _IMP-8 and 2 bla _VIM-1). In 2012, carbapenemase genes were detected in 22.3% of isolates (41 bla _KPC-2, 7 bla _VIM-1, 6 bla _IMP-8, and 1 bla _NDM-1). More than 95% of isolates exhibited either OmpK35 or OmpK36 porin loss or both. Impermeability due to porin mutation coupled with AmpC β-lactamases or ESBLs were major carbapenem-resistance mechanisms. Among 41 KPC-2-producing K. pneumoniae isolates, all were ST11 with 1 major pulsotype.

Conclusions

In 2010 and 2012, the major mechanisms of CnSKP in Taiwan were the concomitance of AmpC with OmpK35/36 loss. KPC-2-KP dissemination with the same ST11 were observed in 2012. The emergence and rapid spread of KPC-2-KP is becoming an endemic problem in Taiwan. The identification of NDM-1 K. pneumoniae case is alarming.     

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of blaNDM-1

New Delhi metallo-β-lactamase-1 gene (bla_NDM-1) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of β-lactam ring, and provides resistance against major classes of β-lactam antibiotics. Dissemination of the plasmid borne bla_NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla_NDM-1. Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6–25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla_NDM-1 gene.     

Genetic Characterization of Carbapenem-Resistant Enterobacteriaceae and the Spread of Carbapenem-Resistant Klebsiella pneumonia ST340 at a University Hospital in Thailand

Carbapenem-resistant Enterobacteriaceae (CRE) has increasingly spread worldwide in the past decade. The prevalence and characteristics of CRE in Thailand are unknown. In this study, we conducted a 2-year surveillance of CRE among 12,741 clinical isolates of Enterobacteriaceae at the largest university hospital in Thailand with molecular characterization of beta-lactamase (bla) genes, including carbapenemase genes. The CRE prevalence was 1.4%. bla _KPC-13 and bla _IMP-14a were the only carbapenemase genes detected among these CRE isolates. bla _KPC-13 gene was found in a single isolate of Escherichia coli, Enterobacter cloacae and Citrobacter freundii, and bla _IMP-14a was found in four isolates of Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) isolates were resistant to multiple carbapenems at a higher ratio than other CRE species, and thus were further characterized for resistance phenotypes, bla genotypes and molecular epidemiology. Most CRKP isolates harboured multiple bla genes, especially those related to extended-spectrum beta-lactamases. Seven CRKP isolates were resistant to all tested carbapenems, and showed decreased ompK35 and/or ompK36 porin gene expression. Molecular typing of CRKP based on pulsed-field gel electrophoresis (PFGE) demonstrated several unrelated clones. Multilocus sequence typing (MLST) was partially concordant with PFGE results and revealed that ST340, a member of drug-resistant K. pneumoniae clonal complex 258, was the most predominant clone, followed by ST48, ST11 and ST273. The novel ST1645 was identified from this study. ST340 has neither been shown to be predominated among CRKP from other studies, nor been reported in Thailand. Therefore, it emphases a critical concern to monitor and control the spread of CRKP.     

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.     

Virulence of Klebsiella pneumoniae Isolates Harboring bla KPC-2 Carbapenemase Gene in a Caenorhabditis elegans Model

Klebsiella pneumoniae carbapenemase (KPC) is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i) five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii) seven transformants obtained after electroporation of either these natural KPC plasmids or a recombinant plasmid harboring only the bla _KPC-2 gene into reference strains K. pneumoniae ATCC10031/{"type":"entrez-protein","attrs":{"text":"CIP53153","term_id":"878514309","term_text":"CIP53153"}}CIP53153; and (iii) five clinical strains cured of plasmids. The virulence of K. pneumoniae isolates was evaluated in the Caenorhabditis elegans model. The clinical KPC producers and transformants were significantly less virulent (LT50: 5.5 days) than K. pneumoniae reference strain (LT50: 4.3 days) (p<0.01). However, the worldwide spread KPC-2 positive K. pneumoniae ST258 strains and reference strains containing plasmids extracted from K. pneumoniae ST258 strains had a higher virulence than KPC-2 strains belonging to other ST types (LT50: 5 days vs. 6 days, p<0.01). The increased virulence observed in cured strains confirmed this trend. The bla _KPC-2 gene itself was not associated to increased virulence.     

Identification and Characterization of the First Escherichia coli Strain Carrying NDM-1 Gene in China

New Delhi metallo-β-lactamase-1 (NDM-1), an acquired class B carbapenemase, is a significant clinical threat due to its extended hydrolysis of β-lactams including carbapenems. In this study, we identified the first confirmed clinical isolate of Escherichia coli BJ01 harboring bla _NDM-1 in China. The isolate is highly resistant to all tested antimicrobials except polymyxin. bla _NDM-1, bla _CTX-M-57, and bla _TEM-1 were identified in the isolate. bla _NDM-1 was transferable to E. coli EC600 and DH5α in both plasmid conjugation experiments and plasmid transformation tests. BJ01 was identified as a new sequence type, ST224, by multilocus sequence typing. Analysis of genetic environment shows complex transposon-like structures surrounding the bla _NDM-1 gene. Genetic analysis revealed that the region flanking bla _NDM-1 was very similar to previously identified NDM-positive Acinetobacter spp. isolated in China. The findings of this study raise attention to the emergence and spread of NDM-1-carrying Enterobacteriaceae in China.