Genetic Contexts of bla _NDM-1 in Patients Carrying Multiple NDM-Producing Strains

Abstract

hThe carbapenem resistance determinant bla NDM-1 has been found in various Gram-negative bacteria and upon different plasmid replicon types (Inc). Here, we present four patients within two hospitals in Pakistan harboring between two and four NDM-1-producing Gram-negative bacilli of different species coresident in their stool samples. We characterize the bla NDM-1 genetic contexts of these 11 NDM-1-producing Gram-negative bacilli in addition to other antimicrobial resistance mechanisms, plasmid replicon profiles, and sequence types (STs) in order to understand the underlying acquisition mechanisms of carbapenem resistance within these bacteria. Two common plasmid types (IncN2 and IncA/C) were identified to carry bla NDM-1 among the six different bacterial species isolated from the four patients. Two of these strains were novel Citrobacter freundii ST 20 and ST 21. The same IncN2-type bla NDM-1 genetic context was found in all four patients and within four different species. The IncA/C-type bla NDM-1 genetic context was found in two different species and in two of the four patients. Combining genetic context characterization with other molecular epidemiology methods, we were able to establish the molecular epidemiological links between genetically unrelated bacterial species by linking their acquisition of an IncN2 or IncA/C plasmid carrying bla NDM-1 for carbapenem resistance. By combining plasmid characterization and in-depth genetic context assessment, this analysis highlights the importance of plasmids in antimicrobial resistance. It also provides a novel approach for investigating the underlying mechanisms of bla NDM-1 -related spread between bacterial species and genera via plasmids.

Mechanisms Involved in Acquisition of bla NDM Genes by IncA/C 2 and IncFII Y Plasmids

“…pKP1-NDM-1, sequenced here, has an almost identical ARI-A, except that only 89 bp of ISAba125 is present adjacent to ISKpn14 upstream of bla NDM . This difference was confirmed by reexamining raw reads and has been seen in other partial sequences (17,35), and ISKpn14 is ϳ89% identical to IS1, which is known to cause adjacent deletions (34). All of these type 1 IncA/C 2 plasmids except pNDM-KN have the same cassette array, consisting of a single fused cassette comprised of the first 87 bp of the bla OXA-30 cassette and position 17 to the end of the aacA4 cassette, overlapping by a single A (36).…”

“…We have also recently reported partial sequences of bla NDM contexts matching pKP1-NDM-1 (with the 89-bp ISAba125 fragment) in IncA/C plasmids harbored by isolates from a hospital in Pakistan (17) and those matching pECL3-NDM-1 or pEC4-NDM-6 in IncFII Y plasmids in isolates from multiple Australian health care facilities (16). The other related IncA/C 2 and IncFII Y plasmids harboring bla NDM genes discussed here were also isolated in several different countries (Table 1).…”

“…All isolates were from patients recently returned from India. Transconjugants in sodium azide-resistant E. coli J53Azi r were available and/or were obtained by conjugation on solid medium, as previously described (17).…”

“…1A). In plasmids from the Enterobacteriaceae, bla NDM genes are generally found in this immediate genetic context, with at least a fragment of ISAba125 containing the Ϫ35 promoter region present upstream, within different length fragments matching Acinetobacter plasmids and associated with different mobile elements (3,(16)(17)(18)(19)(20)(21).…”

IncFII Conjugative Plasmid-Mediated Transmission of bla NDM-1 Elements among Animal-Borne Escherichia coli Strains

“…2). The mobile element harboring the bla NDM-1 gene in pHNEC55 and pHNEC46 contained a structure identical to the ones found in an Acinetobacter species (18), Citrobacter freundii (19), Providencia rettgeri (20), and a Klebsiella species (GenBank accession number CP010390.1) of human origin. A 2-nucleotide deletion was observed in the aphA6 gene located in pHNEC55 and pHNEC46, resulting in early termination of the protein and inactivation of this gene product; this observation was therefore consistent with the loss of the aminoglycoside resistance phenotype in E. coli strains carrying these two plasmids (see Table S1 in the supplemental material).…”