Complete Sequencing of the blaNDM-1-Positive IncA/C Plasmid from Escherichia coli ST38 Isolate Suggests a Possible Origin from Plant Pathogens

Tsuyoshi Sekizuka,Kunikazu Yamane,Makoto Kuroda,Mari Matsui,Fumihiko Takeuchi,Makoto Ohnishi,Akira Hishinuma,Yoshichika Arakawa,Niyaz Ahmed

doi:10.1371/journal.pone.0025334

Tsuyoshi Sekizuka, Kunikazu Yamane + Show 7 more

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https://doi.org/10.1371/journal.pone.0025334

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Abstract

The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla CMY-2-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla NDM-1 gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla NDM-1 gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla NDM-1-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla NDM-1 gene. The complete sequence of pNDM-1_Dok01 suggests that the bla NDM-1 gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.

Highlights

Gram-negative bacteria have acquired mobile genetic elements associated with multiple resistance determinants for most antibiotic classes
De novo shotgun sequencing of the transconjugant DH10B strain, which harbors the plasmid transferred by filter-mating conjugation, was performed and revealed the plasmid to be composed of 225 predicted coding sequences (CDSs) of 195,560 bp with a guanine-cytosine content (GC) of 51.0% (Fig. 1)
The IncA/C incompatibility group of pNDM1_Dok01 can be determined by in silico polymerase chain reaction (PCR) using the PCR-based replicon typing (PBRT) primers described by Carattoli et al [21]; the primer A/C-RV sequence has 2 nucleotide mismatches with the corresponding sequence in pNDM-1_Dok01, suggesting that the PCR assay might fail due to such variation in primer sequence

Summary

Introduction

Gram-negative bacteria have acquired mobile genetic elements associated with multiple resistance determinants for most antibiotic classes. In Gram-negative bacteria, the most common b-lactam resistance mechanism involves b-lactamasemediated hydrolysis, which leads to inactivation of antibiotics [2]. Metallo-b-lactamase (MBL) genes, which hydrolyze all b-lactams including carbapenems (except aztreonam), are increasing in frequency among Gram-negative organisms such as multidrugresistant Enterobacteriaceae [3]. In 2008, a novel MBL, New Delhi metallo-b-lactamase (NDM-1), was identified in K. pneumoniae (strain 05-506) and Escherichia coli isolates from a Swedish patient who was transferred from India [4]. A recent surveillance study showed that NDM-1-positive isolates were circulating in New Delhi as early as 2006, and it was two years before the first European case was reported in 2008 [9]

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