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

Abstract

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.

TEXT

The genetic context of the bla_NDM-1 gene has been extensively investigated, mostly among members of the family Enterobacteriaceae (1, 2, 3, 4, 5, 6, 7, 8). From a genetic perspective, the key finding is that the bla_NDM-1 gene can be located on plasmids belonging to different incompatibility groups. Although bla_NDM-1 genes have the same flanking regions, in a broader sequence context, they reside in diverse genetic environments (6). Although the bla_NDM-1 gene is typically found in a plasmid, in certain Escherichia coli and Providencia stuartii isolates, it was chromosome borne (6). The bla_NDM-1 gene is considered endemic to the Balkan region, and this gene has been found not only in Enterobacteriaceae (9) but also in Pseudomonas aeruginosa (10). Considering the role of P. aeruginosa in the development of nosocomial infections worldwide and the subsequent complicated clinical management of patients infected with this pathogen, the emergence of NDM-1-positive strains is alarming. This is why the analysis of the genetic context of the bla_NDM-1 gene, not only in Enterobacteriaceae but also in all other NDM-1-positive isolates, is crucial and could reveal associations with other genes that confer antimicrobial resistance and potential routes of dissemination. The objective of our study was to identify the genetic location and genetic context of the bla_NDM-1 gene in P. aeruginosa MMA83, one of the NDM-1-positive clinical isolates from Serbia.

To determine the genetic location of the bla_NDM-1 gene in P. aeruginosa MMA83, transformation, mating assays, and DNA-DNA hybridization were performed. Heat shock transformation of E. coli DH5α was performed with total DNA isolated from P. aeruginosa MMA83 (selection based on 100 μg/ml ampicillin). Triparental mating assays with P. aeruginosa MMA83 as a donor, E. coli(pRK2013) as a helper, and two recipients, E. coli DH5α (selection based on 100 μg/ml ampicillin) and an imipenem-sensitive isolate of Pseudomonas mendocina (selection based on 32 μg/ml imipenem), did not result in any transconjugant (11). Counterselection for recipients was based on glycerol for P. mendocina and sucrose for E. coli DH5α as the sole carbon sources. This result suggested that the gene(s) conferring resistance to β-lactams was not present on a conjugative plasmid(s) in MMA83. To find out if MMA83 is a plasmid-free strain, total DNA was isolated in agarose blocks and treated with S1 nuclease, which is used to visualize large, supercoiled plasmids (12). The results obtained confirmed that MMA83 does not carry any plasmid (see Fig. S1 in the supplemental material). Also, pulsed-field gel electrophoresis and subsequent DNA-DNA hybridization of a bla_NDM-1 gene probe (1,059-bp EcoRI fragment) (11) with native (not digested) and S1 nuclease-treated total MMA83 DNA revealed the location of the signal in the region where chromosomal DNA migrates (see Fig. S1). These results strongly suggest that the bla_NDM-1 gene is chromosome borne rather than plasmid located in MMA83. This is an unusual but not unique hallmark, since chromosomally located bla_NDM-1 genes have been described previously (6). To uncover the genetic context of the bla_NDM-1 gene, two cosmid libraries of MMA83 were constructed with Gigapack III Gold packaging extract (Stratagene, Amsterdam, Netherlands). Total DNA was partially digested with either BamHI or EcoRI and cloned into the pLAFR3 cosmid (11). Cosmid libraries were screened for the presence of β-lactamases by plating on tetracycline (20 μg/ml, selection for cosmid) and ampicillin (100 μg/ml). The cosmid selected from the BamHI cosmid library conferring resistance to β-lactams was designated pLAFRBamNDM, and the cosmid selected from the EcoRI library was designated pLAFREcoNDM. pLAFRBamNDM encompasses only one large BamHI fragment, so the order of genes in pLAFRBamNDM is the same as in the genome of strain MMA83, from which it originated. Afterwards, pLAFRBamNDM was digested with BamHI and XbaI, in which it produced three fragments of 8,292 bp (BamHI-XbaI fragment), 6,451 bp (XbaI-XbaI fragment), and 4,541 bp (XbaI-BamHI fragment). These fragments were cloned into a pBluescript vector (Kan^r) digested with the same enzymes. The clones obtained were confirmed by restriction enzyme analysis and subsequently sequenced by primer walking. Cosmid pLAFREcoNDM was digested with EcoRI and PstI, and plasmid libraries were constructed in pBluescript (Kan^r). The resulting constructs were sequenced and used as a control in the process of aligning and stacking sequences derived from BamHI-XbaI, XbaI-XbaI, and XbaI-BamHI fragments of pLAFRBamNDM. The order of the open reading frames in fragments from pLAFREcoNDM was the same as that in pLAFRBamNDM, which confirmed that the order of genes in pLAFRBamNDM was the same as in the genome of strain MMA83. Surprisingly, these results revealed that P. aeruginosa MMA83 carries two copies of the bla_NDM-1 gene (Fig. 1). Moreover, hybridization of total DNA digested with different restriction enzymes with a bla_NDM-1 gene probe revealed the presence of signals at positions predicted based on the sequence of a cloned BamHI fragment of 19.2 kb (see Fig. S1 in the supplemental material). Studies published thus far describe the presence of only one copy of the bla_NDM-1 gene in the chromosome or plasmids of bacteria. To our knowledge, ours is the first observation and report of two bla_NDM-1 gene copies. Amplification of genes conferring resistance to antibiotics, as a common adaptive mechanism in bacteria, could be related to a gene dosage effect (13). The genetic environment of the bla_NDM-1 gene in the genome of MMA83 showed certain similarities to but, in general, was different from the nucleotide sequences of the other bacterial strains analyzed. ISAba125 insertion sequences were identified immediately upstream of the bla_NDM-1 genes (Fig. 1). As observed in other bla_NDM-1 gene environments, ISAba125 provides the −35 promoter sequence for the bla_NDM-1 gene (6). The ISCR1 mobile element lies upstream of both copies of the bla_NDM-1 gene and is followed by the gene encoding type VI aminoglycoside phosphotransferase. Since the regions downstream of both ISCR1 elements are identical, it is possible that the two copies of the bla_NDM-1 gene were the result of genetic duplication via ISCR1-mediated gene mobilization. ISCR1 undergoes rolling-circle transposition, in which a single IS element can mobilize the sequences to which it is attached and subsequent homologous recombination may result in gene duplication (14). The genetic organization downstream of the bla_NDM-1 genes in MMA83 is the same, encompassing the fusion of a bleomycin resistance gene and the qacEΔ1 gene, followed by the sul1 gene. The regions upstream of both bla_NDM-1 gene copies in MMA83 are also identical and show similarity to the regions upstream of the bla_NDM-1 gene in pNDM-BJ01 and pMR0211 (4, 5). In addition, regions downstream of the bla_NDM-1 genes in MMA83 show some similarity to regions of pMR0211 and pNDM-BJ01 downstream from the bla_NDM-1 gene but are not identical to them (Fig. 1). The first ISCR1 insertion sequence was preceded by a class 1 integron harboring aacA7, aadA6, orfD, and qacEΔ1 and sul1 resistance genes. This is not atypical, since ISCR1 is commonly associated with class 1 integrons (14). Moreover, although the bla_NDM-1 gene is associated with other bla genes, in the case of P. aeruginosa MMA83, no other β-lactamase-encoding gene was found in the vicinity of two bla_NDM-1 genes.

Fig 1.

Comparative schematic representations of the bla_NDM-1 gene's genetic context in the genomes of Enterobacteriaceae and Acinetobacter and the genetic environment of the bla_NDM-1 gene in P. aeruginosa MMA83. Insertion sequence transposase genes are drawn as arrows filled with different patterns. The box in the scheme of the MMA83 bla_NDM-1 gene's genetic context represents the left end of ISAba14. The selection of the genetic environments represented was based on the presence of ISCR1 and relationships to different bacterial genera.

This study provides new data on the complexity and diversity of genetic features associated with the bla_NDM-1 gene, which are of great importance for further analyses of dissemination routes and mechanisms, including clonal dispersions of the bla_NDM-1 gene within clinical isolates.

Nucleotide sequence accession number.

The nucleotide sequence of the BamHI fragment (19,272 bp) obtained from the pLAFRBamNDM cosmid is available in GenBank/EMBL under accession number HF546976.