Plasmid Carriage of blaNDM-1 in Clinical Acinetobacter baumannii Isolates from India

NDM-1 probably emerged in Acinetobacter species prior to its dissemination among Enterobacteriaceae, and NDM-1-like enzymes are increasingly reported in Acinetobacter species. Here, we report on the genetic context of bla_NDM-1 in the earliest known NDM-1-producing organisms, clinical isolates of Acinetobacter from India in 2005. These strains harbor bla_NDM-1 plasmids of different sizes. The gene is associated with the remnants of the Tn125 transposon normally associated with bla_NDM-1 in Acinetobacter spp. The transposon has been disrupted by the IS26 insertion and subsequent movement events.     

Characterization of Tn3000, a Transposon Responsible for blaNDM-1 Dissemination among Enterobacteriaceae in Brazil,Nepal, Morocco,and India

In Enterobacteriaceae, the bla_NDM genes have been found in many different genetic contexts, and a wide diversity of plasmid scaffolds bearing those genes has been found. In August 2013, we identified NDM-1-producing Escherichia coli and Enterobacter hormaechei strains from a single rectal swab sample from a patient hospitalized in Rio de Janeiro, Brazil, who had no history of travel abroad. Complete DNA sequencing using the Illumina platform and annotation of the two plasmids harboring the bla_NDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFII_K and IncX3 and harbored a novel transposon named Tn3000. Similar genetic structures have been identified among other isolates in Brazil but also on plasmids from other continents. Our findings suggest that the bla_NDM-1 gene may be transmitted by Tn3000 in different parts of the world.     

First Report of an OXA-23 Carbapenemase-Producing Acinetobacter baumannii Clinical Isolate Related to Tn2006 in Spain

A carbapenem-resistant Acinetobacter baumannii clinical isolate belonging to European clone II and sequence type 2 was recovered from a patient in the Son Espases hospital in Mallorca, Spain. Genetic analysis showed the presence of the bla_OXA-23 gene in association with the widely disseminated transposon Tn2006. This is the first reported identification of A. baumannii carrying bla_OXA-23 in Spain.     

Molecular Characterization of blaNDM-1 in a Sequence Type 235 Pseudomonas aeruginosa Isolate from France

An NDM-1 carbapenemase-producing Pseudomonas aeruginosa isolate was recovered from a patient hospitalized in France after a previous hospitalization in Serbia. Genetic studies revealed that the bla_NDM-1 gene was surrounded by insertion sequence ISAba125 and a truncated bleomycin resistance gene. This bla_NDM-1 region was a part of the variable region of a new complex class 1 integron bearing IS common region 1 (ISCR1). The presence of ISPa7 upstream of this integron suggests insertion in a chromosomally located Tn402-like structure.     

Complete Sequence of p07-406, a 24,179-base-pair plasmid harboring the blaVIM-7 metallo-beta-lactamase gene in a Pseudomonas aeruginosa isolate from the United States

An outbreak involving a Pseudomonas aeruginosa strain that was resistant to all tested antimicrobials except polymyxin B occurred in a hospital in Houston, TX. Previous studies on this strain showed that it possesses a novel mobile metallo-β-lactamase (MBL) gene, designated bla_VIM-7, located on a plasmid (p07-406). Here, we report the complete sequence, annotation, and functional characterization of this plasmid. p07-406 is 24,179 bp in length, and 29 open reading frames were identified related to known or putatively recognized proteins. Analysis of this plasmid showed it to be comprised of four distinct regions: (i) a region of 5,200 bp having a Tn501-like mercuric resistance (mer) transposon upstream of the replication region; (ii) a Tn3-like transposon carrying a truncated integron with a bla_VIM-7 gene and an insertion sequence inserted at the other end of this transposon; (iii) a region of four genes, upstream of the Tn3-like transposon, possessing very high similarity to plasmid pXcB from Xanthomonas campestris pv. citri commonly associated with plants; (iv) a backbone sequence similar to the backbone structure of the IncP group plasmid Rms149, pB10, and R751. This is the first plasmid to be sequenced carrying an MBL gene and highlights the amelioration of DNA segments from disparate origins, most noticeably from plant pathogens.     

A case of NDM-1-producing Acinetobacter baumannii transferred from India to Japan

A 52-year-old male Japanese businessman with massive cerebral bleeding was transferred from India to Japan and was admitted to our hospital. Multidrug-resistant Acinetobacter baumannii was isolated from his sputum. The minimum inhibitory concentrations for this strain were as follows: imipenem, 64 μg/ml; meropenem, 32 μg/ml; ciprofloxacin, 16 μg/ml; amikacin, 16 μg/ml; aztreonam, 16 μg/ml; colistin, <1 μg/ml. This A. baumannii strain had both bla _NDM-1 and bla _OXA-23 by polymerase chain reaction analysis. In Japan, NDM-1-producing bacteria are extremely rare in clinical specimens. To date, three NDM-1-positive cases have been detected in Japan, and this is the first case of A. baumannii-producing NDM-1 in Japan. Our case suggests that NDM-1-producing bacteria could be introduced into our country easily. There is concern that various resistant bacteria may be transferred from epidemic countries as a result of international medical care.     

Genomic Characterization of Enterobacter cloacae Isolates from China That Coproduce KPC-3 and NDM-1 Carbapenemases

Here, we report two Enterobacter cloacae sequence type 231 isolates coproducing KPC-3 and NDM-1 that have caused lethal infections in a tertiary hospital in China. The bla_NDM-1-harboring plasmids carry IncA/C₂ and IncR replicons, showing a mosaic plasmid structure, and the bla_NDM-1 is harbored on a novel class I integron-like element. bla_KPC-3 is located on a Tn3-Δbla_TEM-1-bla_KPC-3-ΔTn1722 element, flanked by two 9-bp direct-repeat sequences and harbored on an IncX6 plasmid.     

The Clinical Isolate Pseudomonas aeruginosa MMA83 Carries Two Copies of the blaNDM-1 Gene in a Novel Genetic Context

The genetic context of the bla_NDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was investigated. Sequencing of the cosmid selected for the bla_NDM-1 gene revealed the presence of two bla_NDM-1 copies in the genome of P. aeruginosa MMA83 in a unique genetic environment. Additionally, mating assays, DNA-DNA hybridization, and an S1 nuclease assay strongly suggest that the bla_NDM-1 gene in P. aeruginosa MMA83 is chromosome borne.     

Characterization of an Enterobacter cloacae Strain Producing both KPC and NDM Carbapenemases by Whole-Genome Sequencing

A carbapenem-resistant Enterobacter cloacae strain, WCHECl-14653, causing a fatal bloodstream infection, was characterized by genome sequencing and conjugation experiments. The strain carried two carbapenemase genes, bla_NDM-1 and bla_KPC-2, on separate IncF plasmids. The coexistence of bla_NDM-1 and bla_KPC-2 conferred slightly higher-level carbapenem resistance compared with that of bla_NDM-1 or bla_KPC-2 alone, and the coexistence of two IncF plasmids may generate new platforms for spreading carbapenemase genes.     

Whole-Genome Assembly of Klebsiella pneumoniae Coproducing NDM-1 and OXA-232 Carbapenemases Using Single-Molecule,Real-Time Sequencing

The whole-genome sequence of a carbapenem-resistant Klebsiella pneumoniae strain, PittNDM01, which coproduces NDM-1 and OXA-232 carbapenemases, was determined in this study. The use of single-molecule, real-time (SMRT) sequencing provided a closed genome in a single sequencing run. K. pneumoniae PittNDM01 has a single chromosome of 5,348,284 bp and four plasmids: pPKPN1 (283,371 bp), pPKPN2 (103,694 bp), pPKPN3 (70,814 bp), and pPKPN4 (6,141 bp). The contents of the chromosome were similar to that of the K. pneumoniae reference genome strain MGH 78578, with the exception of a large inversion spanning 23.3% of the chromosome. In contrast, three of the four plasmids are unique. The plasmid pPKPN1, an IncHI1B-like plasmid, carries the bla_NDM-1, armA, and qnrB1 genes, along with tellurium and mercury resistance operons. bla_NDM-1 is carried on a unique structure in which Tn125 is further bracketed by IS26 downstream of a class 1 integron. The IncFIA-like plasmid pPKPN3 also carries an array of resistance elements, including bla_CTX-M-15 and a mercury resistance operon. The ColE-type plasmid pPKPN4 carrying bla_OXA-232 is identical to a plasmid previously reported from France. SMRT sequencing was useful in resolving the complex bacterial genomic structures in the de novo assemblies.     

Colistin-Nonsusceptible Pseudomonas aeruginosa Sequence Type 654 with blaNDM-1 Arrives in North America

This study describes 3 different bla_NDM-1 genetic platforms in 3 different species obtained from the same patient who was directly transferred to an institution in Calgary, Alberta, Canada, following a prolonged hospital stay in India. The bla_NDM-1 in the Escherichia coli isolate was located on a 176-kb IncA/C plasmid contained within an ISCR1 region. The bla_NDM-1 in the Providencia rettgeri isolate was located on a 117-kb IncT plasmid contained within Tn3000, while the bla_NDM-1 in the Pseudomonas aeruginosa isolate was located on the chromosome within an ISCR3 region. This report highlights the plasticity of the genetic regions and environments associated with bla_NDM-1. To the best of our knowledge, this is the first report of P. aeruginosa with bla_NDM-1 identified in North America and the first report of bla_OXA-181 in P. rettgeri. The P. aeruginosa isolate belonged to the international high-risk sequence type 654 clone and was nonsusceptible to colistin. This case emphasizes the need for the use of appropriate infection prevention and control measures and vigilant screening for carbapenem-resistant Gram-negative bacteria in patients with a history of travel to areas of endemicity, such as the Indian subcontinent.     

pKBuS13, a KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 833, Carrying Tn4401b Inserted into an Xer Site-Specific Recombination Locus

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla_KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla_KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported.     

Comparative Sequence Analysis of a Multidrug-Resistant Plasmid from Aeromonas hydrophila

Aeromonas hydrophila is a pathogenic bacterium that has been implicated in fish, animal, and human disease. Recently, a multidrug resistance (MDR) plasmid, pR148, was isolated from A. hydrophila obtained from a tilapia (Oreochromis niloticus) farm in Thailand. pR148 is a 165,906-bp circular plasmid containing 147 coding regions showing highest similarity to pNDM-1_Dok1, an MDR plasmid isolated from a human pathogen. pR148 was also very similar to other IncA/C plasmids isolated from humans, animals, food, and fish. pR148 contains a mercuric resistance operon and encodes the complete set of genes for the type 4 secretion system. pR148 encodes a Tn21 type transposon. This transposon contains the drug resistance genes qacH, bla_OXA-10, aadA1, and sul1 in a class 1 integron; tetA and tetR in transposon Tn1721; and catA2 and a duplicate sul1 in a locus showing 100% similarity to IncU plasmids isolated from fish. The bla_OXA-10 and aadA1 genes showed 100% similarity to those from the Acinetobacter baumannii AYE genome. The similarity of pR148 to a human pathogen-derived plasmid indicates that the plasmids were either transferred between different genera or that they are derived from a common origin. Previous studies have shown that IncA/C plasmids retain a conserved backbone, while the accessory region points to lateral gene transfer. These observations point out the dangers of indiscriminate use of antibiotics in humans and in animals and the necessity of understanding how drug resistance determinants are disseminated and transferred.     

Globally Expanding Carbapenemase Finally Appears in Spain: Nosocomial Outbreak of Acinetobacter baumannii Producing Plasmid-Encoded OXA-23 in Barcelona,Spain

Resistance of Acinetobacter baumannii clinical isolates to carbapenems is on the rise worldwide mainly in association with the production of OXA-23. Until recently, however, OXA-23 was absent in Spain. In this work, we report the molecular characterization of a hospital outbreak of OXA-23-producing A. baumannii in Barcelona caused by a multidrug-resistant (MDR) clone belonging to international clone IC-II/sequence type ST85 between October 2010 and May 2011. bla_OXA-23 was carried in a plasmid of 90 kb and located within the composite transposon Tn2006.     

Complete Nucleotide Sequence of the First KPC-2- and SHV-12-Encoding IncX Plasmid,pKpS90, from Klebsiella pneumoniae

We report the complete nucleotide sequence of the pKpS90 plasmid, carrying the bla_KPC-2 and bla_SHV-12 genes. This plasmid was isolated from a sequence type 258 (ST258) Klebsiella pneumoniae strain responsible for an outbreak in a French university hospital in 2009. pKpS90 is a 53,286-bp plasmid that belongs to the IncX incompatibility group. pKpS90 consists of a backbone from IncX plasmids, in which the KPC-2-encoding Tn4401 transposon and a bla_SHV-12-encoding region have been inserted.     

First Report of a Clinical,Multidrug-Resistant Enterobacteriaceae Isolate Coharboring Fosfomycin Resistance Gene fosA3 and Carbapenemase Gene blaKPC-2 on the Same Transposon,Tn1721

In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain {"type":"entrez-nucleotide","attrs":{"text":"HS102707","term_id":"312647496","term_text":"HS102707"}}HS102707 isolate and an Enterobacter aerogenes strain {"type":"entrez-nucleotide","attrs":{"text":"HS112625","term_id":"313253202","term_text":"HS112625"}}HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the bla_KPC-2 and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain {"type":"entrez-nucleotide","attrs":{"text":"HS092839","term_id":"312447864","term_text":"HS092839"}}HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that bla_KPC-2 was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored bla_KPC-2. Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and bla_KPC-2 colocated in the same Tn1721-Tn3–like composite transposon on a novel IncP group plasmid.     

Characterization of Plasmids in Extensively Drug-Resistant Acinetobacter Strains Isolated in India and Pakistan

The bla_NDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with bla_NDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring bla_NDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring bla_NDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near bla_NDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of bla_NDM-1 into Enterobacteriaceae.     

High Incidence and Endemic Spread of NDM-1-Positive Enterobacteriaceae in Henan Province,China

The emergence and spread of New Delhi metallo-β-lactamase 1 (NDM-1)-producing carbapenem-resistant Enterobacteriaceae (CRE) present an urgent threat to human health. In China, the bla_NDM-1 gene has been reported mostly in Acinetobacter spp. but is rarely found in Enterobacteriaceae. Here, we report a high incidence and endemic spread of NDM-1-producing CRE in Henan Province in China. Sixteen (33.3%) of the 48 CRE isolates obtained from patients during June 2011 to July 2012 were positive for bla_NDM-1, and the gene was found to be carried on plasmids of various sizes (∼55 to ∼360 kb). These plasmids were readily transferrable to recipient Escherichia coli by conjugation, conferred resistance to multiple antibiotics, and belonged to multiple replicon types. The bla_NDM-1-positive CRE isolates were genetically diverse, and six new multilocus sequence typing (MLST) sequence types were linked to the carriage of NDM-1. Five of the isolates were classified as extensively drug-resistant (XDR) isolates, four of which also carried the fosA3 gene conferring resistance to fosfomycin, an alternative drug for treating infections by CRE. In each bla_NDM-1-positive CRE isolate, the bla_NDM-1 gene was downstream of an intact ISAba125 element and upstream of the ble_MBL gene. Furthermore, gene environment analysis suggested the possible transmission of bla_NDM-1-containing sequences from Acinetobacter spp. to Klebsiella pneumoniae and Klebsiella oxytoca. These findings reveal the emergence and active transmission of NDM-1-positive CRE in China and underscore the need for heightened measures to control their further spread.     

Dissemination of blaOXA-23 in Acinetobacter spp. in China: Main Roles of Conjugative Plasmid pAZJ221 and Transposon Tn2009

Production of the OXA-23 carbapenemase is the most common reason for the increasing carbapenem resistance in Acinetobacter spp. This study was conducted to reveal the genetic basis of bla_OXA-23 dissemination in Acinetobacter spp. in China. A total of 63 carbapenem-resistant OXA-23-producing Acinetobacter sp. isolates, representing different backgrounds, were selected from 28 hospitals in 18 provinces for this study. Generally, two patterns of plasmids carrying bla_OXA-23 were detected according to S1-nuclease pulsed-field gel electrophoresis and Southern blot hybridization. A ca. 78-kb plasmid, designated pAZJ221, was found in 23 Acinetobacter baumannii and three Acinetobacter nosocomialis isolates, while a novel ca. 50-kb plasmid was carried by only two other A. baumannii isolates. Three of these isolates had an additional copy of bla_OXA-23 on the chromosome. Transformation of the two plasmids succeeded, but only pAZJ221 was conjugative. Plasmid pAZJ221 was sequenced completely and found to carry no previously known resistance genes except bla_OXA-23. The bla_OXA-23 gene of the remaining 35 isolates was chromosome borne. The bla_OXA-23 genetic environments were correlated with Tn2009 in 57 isolates, Tn2008 in 5 isolates, and Tn2006 in 1 isolate. The MIC values for the carbapenems with these isolates were not significantly associated with the genomic locations or the copy numbers of bla_OXA-23. Overall, these observations suggest that the plasmid pAZJ221 and Tn2009 have effectively contributed to the wide dissemination of bla_OXA-23 in Acinetobacter spp. in China and that horizontal gene transfer may play an important role in dissemination of the bla_OXA-23 gene.