Characterization of Multiple NDM-1-Producing Enterobacteriaceae Isolates from the Same Patient

DOI: 10.1128/AAC.04862-14

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FIG 1
Comparison of the NDM plasmids characterized in this study with closely related IncA/C plasmid pNDM-KN (GenBank accession no. JN157804) (9). (A) Regions with high homology are shaded light blue. Arrows indicate the following predicted ORFs: conjugation, stability, and accessory genes (green, yellow), antimicrobial resistance genes (red, bla_NDM-1 is light green), transposon-related genes (blue), and hypothetical proteins (white). The replicase gene is pink. The orange-boxed segments are detailed in panel B. (B) Detail of the bla_NDM-1-flanking regions (those of pNDM-PstGN576 are the same as those of pNDM-EcoGN568). The orange circles represent the 256-bp direct repeats with similarities to MITEs identified in bla_NDM-1 and insA. A third MITE was found ∼11 kb upstream of the bla_NDM-1 gene, between a truncated copy of IS5 and a putative endonuclease III gene.

TABLE 1

Antibacterial drug susceptibility profiles, plasmid types, and resistance genes of clinical isolates and their E. coli transconjugant

Isolate^a	MIC (μg/ml)^b																		Rep type (plasmid size [kb])	PCR and sequencing results^d
Isolate^a	Ampicillin	Cefoxitin	Ceftazidime	Cefotaxime	Cefepime	Aztreonam	Ertapenem	Meropenem	Imipenem	Amikacin	Gentamicin	Tobramycin	Nalidixic acid	Ciprofloxacin	Tetracycline	Tigecycline	Colistin	Co-trimoxazole	Rep type (plasmid size [kb])	bla_NDM-1	bla_CTX-M-15	bla_OXA-1	bla_TEM-1	bla_CMY-6	rmtC
Eco GN568	≥256	≥256	≥256	≥256	≥256	256	16	32	6	≥256	≥1,024	≥256	≥256	≥32	1.5	0.64	0.047	≥32	A/C FIA Frep	+	+	−	+	+	+
Ecl GN574	≥256	≥256	≥256	≥256	128	128	≥32	≥32	≥32	≥256	≥1,024	≥256	64	8	12	3	0.064	≥32	Neg^c	+	+	+	+	−	+
Pst GN576	≥256	≥256	≥256	16	1.5	0.75	≥32	≥32	≥32	≥256	≥1,024	≥256	4	0.12	96	0.5	≥256	0.25	A/C	+	−	−	−	+	+
Eco J-568	≥256	≥256	≥256	256	16	6	≥32	4	8	≥256	≥1,024	≥256	3	0.012	0.75	0.064	0.047	0.094	A/C (∼160)	+	−	−	−	+	+
Eco J-574	≥256	≥256	≥256	≥256	16	0.125	16	6	24	≥256	≥1,024	≥256	3	0.012	1	0.094	0.047	0.064	Neg^c (∼100)	+	−	−	−	−	+
Eco J-576	≥256	≥256	≥256	192	16	6	≥32	4	12	≥256	≥1,024	≥256	3	0.016	0.75	0.094	0.047	0.25	A/C (∼150)	+	−	−	−	+	+
Eco J53	6	8	0.19	0.094	0.064	0.125	0.008	0.023	0.38	1.5	1.5	1	3	0.012	1	0.047	0.047	0.064	NA^e	NA	NA	NA	NA	NA	NA

↵a Eco, E. coli; Ecl, E. cloacae; Pst, P. stuartii; Eco J53, recipient E. coli J53. E. coli J53 transconjugant strains were derived from E. coli GN568 (J-568), P. stuartii GN576 (J-576), and E. cloacae GN574 (J-574).
↵b Susceptibility testing was performed by the Etest (bioMérieux) and agar dilution methods, and results were interpreted according to Clinical and Laboratory Standards Institute guidelines (13), except for colistin and tigecycline, for which the European Committee on Antimicrobial Susceptibility Testing breakpoints were used (http://www.eucast.org/clinical_breakpoints/).
↵c Nontypeable by the PCR-based replicon typing scheme described by Carattoli et al. (5). A new set of primers was designed for detection of this repA allele: FIB F2, 5′-CGTCTATCTCTCGCCAGAGC-3′; FIB R2, 5′-GTTCTCAGCACTCTCATCATGC-3′.
↵d Sequencing of complete genes was performed with samples positive by PCR. PCR included screening for bla_TEM; bla_SHV; bla_OXA-1-like; bla_CTX-M groups 1, 2, and 9; bla_VEB; bla_PER; bla_GES; bla_OXA-48-like; bla_IMP; bla_KPC; bla_NDM; and six groups of bla_AmpC β-lactamase genes, as well as the armA, rmtA to -F, and npmA 16S methylase genes (14, 15). A plus sign indicates a positive result, and a minus sign indicates a negative result.
↵e NA, not applicable.