Retrospective Molecular Analysis of DIM-1 Metallo-β-Lactamase Discovered in Pseudomonas stutzeri from India in 2000

Abstract

Among 220 clinical isolates of Gram-negative bacilli collected in India during 2000, 22 strains showing elevated imipenem MICs were evaluated for carbapenemase production. One DIM-1-producing Pseudomonas stutzeri isolate was detected, and no other carbapenemase-encoding genes were identified. This detection of a DIM-1-producing P. stutzeri isolate from India predating the finding of this gene in the index Dutch strain and the very recent detection of DIM-1 in Africa suggest an unidentified environmental source of this metallo-β-lactamase gene.

TEXT

Numerous types of metallo-β-lactamases (MBL) have been discovered and characterized to date, and the IMP and VIM types and a few NDM variants seem to possess the most global distribution. In contrast, some other MBL-encoding genes are limited to a single report or to certain geographic locations, including genes encoding SPM-1, GIM-1, SIM-1, AIM-1, KHM-1, DIM-1, BIC-1, and TMB-1 (1). The NDM-1 (New Delhi Metallo-β-lactamase)-encoding gene was detected in a diabetic Swedish patient of Indian origin who traveled to New Delhi and acquired a urinary tract infection (2). Since this initial report, NDM-producing isolates have been reported in several countries across nearly all continents, and in many instances these reports were related to patient travel to the Indian subcontinent. Although much has been studied about NDM producers, the timeline for the emergence of the NDM-encoding gene was not clearly established before 2006 (3).

(This study was presented at the 22nd European Congress of Clinical Microbiology and Infectious Diseases, London, United Kingdom, 31 March to 3 April 2012.)

This study was initiated to determine the presence of bla_NDM and other carbapenemase genes among a collection of Gram-negative bacilli cultured during 2000 at hospitals located in India (4). Among 220 Gram-negative clinical isolates available from five hospitals (one each in the cities Mumbai, Vellore, New Delhi, Lucknow, and Indore), 10.0% (22 strains) demonstrated imipenem MICs of ≥0.5 μg/ml when tested by reference broth microdilution methods (5). These organisms included five Enterobacter cloacae isolates, four Pseudomonas aeruginosa isolates, four Pseudomonas fluorescens isolates, two isolates each of Klebsiella pneumoniae, Acinetobacter baumannii, Citrobacter freundii, and Pseudomonas stutzeri, and one Proteus vulgaris isolate. Enterobacteriaceae strains exhibited the lowest imipenem MICs (0.5 to 1 μg/ml), and higher carbapenem MIC results were noted for Pseudomonas spp. (1 to 8 μg/ml). The two A. baumannii isolates displayed imipenem MICs of 1 μg/ml (Table 1).

TABLE 1.

Susceptibility profiles of the Gram-negative isolates displaying elevated imipenem MICs collected in Indian hospitals in 2000^a

Isolate	Organism	City of isolation	MIC (μg/ml)
			Imipenem	Ceftazidime	Ceftriaxone	Piperacillin- tazobactam	Ciprofloxacin	Gentamicin	Tetracycline	Tigecycline	Trimethoprim- sulfamethoxazole
10627	P. stutzeri	New Delhi	2	16	>8	64	>4	>8	8	0.12	>4
10532	P. stutzeri	Lucknow	1	0.25	2	16	0.12	≤1	1	0.25	>4
10526	P. aeruginosa	Lucknow	8	2	>8	4	0.12	≤1	>8	4	4
10417	P. aeruginosa	Vellore	2	2	>8	16	>4	>8	>8	>4	>4
10550	P. aeruginosa	Lucknow	8	2	>8	4	0.12	≤1	>8	4	4
10552	P. aeruginosa	Lucknow	8	2	>8	4	0.12	≤1	>8	4	4
10528	P. fluorescens	Lucknow	8	4	>8	4	0.06	≤1	1	0.25	≤0.5
10540	P. fluorescens	Lucknow	8	4	>8	4	0.12	>8	2	0.12	2
10543	P. fluorescens	Lucknow	8	4	>8	8	0.12	>8	2	0.12	4
10545	P. fluorescens	Lucknow	4	4	>8	4	≤0.03	≤1	1	0.12	1
10430	A. baumannii	Vellore	1	>32	>8	≤0.5	>4	>8	>8	0.25	>4
10456	A. baumannii	Indore	1	32	>8	>64	>4	>8	>8	0.5	≤0.5
10606	C. freundii	New Delhi	0.5	>32	>8	>64	>4	>8	1	0.25	≤0.5
10431	C. freundii	Indore	1	>32	>8	2	2	4	>8	0.5	>4
10525	E. cloacae	Lucknow	0.5	>32	>8	>64	2	>8	>8	0.5	>4
10418	E. cloacae	Vellore	0.5	>32	>8	32	≤0.03	>8	2	1	>4
10433	E. cloacae	Indore	0.5	>32	>8	>64	≤0.03	≤1	2	0.25	≤0.5
10476	E. cloacae	Mumbai	0.5	>32	>8	>64	0.06	>8	>8	0.5	>4
10488	E. cloacae	New Delhi	0.5	>32	>8	32	0.5	>8	>8	0.25	≤0.5
10447	K. pneumoniae	Indore	1	>32	>8	>64	>4	>8	4	0.25	>4
10480	K. pneumoniae	Mumbai	0.5	0.06	≤0.06	2	≤0.03	≤1	1	0.25	≤0.5
10432	P. stuartii	Vellore	1	>32	>8	2	2	4	>8	0.5	>4

^aThe data for the DIM-1-producing strain are set in boldface.

The candidate isolates were further evaluated for the presence of genes encoding carbapenemases by the use of PCRs targeting the following genes/groups: bla_IMP, bla_VIM, bla_SPM-1, bla_KPC, bla_SME, bla_IMI, bla_NMC-A, bla_GES, bla_KHM-1, bla_DIM-1, bla_BIC-1, bla_NDM, and bla_OXA-48 by multiplex PCR using custom primers (3, 6). Acinetobacter spp. were also screened for the genes encoding OXA-23, OXA-24/OXA-40, OXA-51, and OXA-58 (7). Enterobacteriaceae isolates were screened by the modified Hodge test (MHT) using imipenem and meropenem as the substrates (8).

None of the isolates were positive for bla_NDM, but one P. stutzeri strain yielded positive PCR results for bla_DIM primers. No other carbapenemase genes were detected, and all Enterobacteriaceae strains yielded negative MHT phenotypic results. The two A. baumannii strains were positive only for bla_OXA-51-like genes.

DIM-1 was first identified in a Pseudomonas stutzeri strain collected in 2007 from a Dutch patient (9). This enzyme shares 45 to 52% amino acid identity with IMP types and GIM-1 and only 30 to 38% identity with SPM-1, VIM, and NDM types. The greatest amino acid sequence identity (63.7%) is shared with TMB-1, a new MBL identified in Achromobacter spp. (10). Similar to other MBLs, DIM-1 hydrolyzes broad-spectrum cephalosporins and carbapenems but not monobactams such as aztreonam.

The MBL-positive P. stutzeri was nonsusceptible to cephalosporins, gentamicin, ciprofloxacin, piperacillin-tazobactam, and tetracycline, but the imipenem MIC result was only marginally elevated (MIC, 2 μg/ml) (Table 1) and this isolate was inhibited by 0.25 μg/ml of tigecycline. The MBL genetic environment was amplified using primers annealing to the 5′ and 3′ conserved sequence (CS) regions of class 1 integrons in combination with the bla_DIM-1 primers. Sequences were analyzed using the Lasergene software (DNASTAR, Madison, WI) and compared to others available via internet sources (http://www.ncbi.nlm.nih.gov/blast/). P. stutzeri carried bla_DIM-1, and integron sequencing identified the MBL gene located in the second position of a class 1 integron with aadB located upstream. No 3′-end CS was detected using multiple primers. The comparison of the genetic environment of this strain and that of the index DIM-1-producing strain demonstrated different arrays of genes in the integron structures. The index strain carried bla_DIM-1 in the first position, followed by aadB and qacH, and no 3′-end CS (9).

Clonality among the DIM-1-producing P. stutzeri isolate from India and the index strain from the same bacterial species detected in the Netherlands (kindly supplied by L. Poirel, Bicetre Hospital, France) was evaluated by pulsed-field gel electrophoresis (PFGE), as described elsewhere (9). The Indian DIM-1-producing isolate was genetically distinct from the Dutch P. stutzeri index strain (Fig. 1). Southern blot and hybridization of S1 endonuclease and I-CeuI digests of agarose-embedded DNA using a digoxigenin-labeled (Roche Diagnostics GmbH, Mannheim, Germany) bla_DIM-1-specific probe showed one copy of the DIM-1-encoding gene located on a plasmid of approximately 240 kb, in contrast to the index strain, which carried bla_DIM-1 on a much smaller plasmid (70 kb). Plasmid sizes were estimated by linear regression using a high-molecular-weight lambda ladder (Lonza, Rockland, ME).

FIG 1.

PFGE comparison of the DIM-1-producing P. stutzeri isolate from India and the DIM-1-producing P. stutzeri index isolate from the Netherlands.

P. stutzeri is considered a low-frequency opportunistic pathogen that causes infections mainly in patients with serious underlying diseases (11). The most frequent infection sites have included the bloodstream, wounds, the respiratory tract, and urine. However, in recent years this species has emerged as a pathogen capable of causing serious invasive infections, such as indolent endocarditis, community-acquired (CA) meningitis, neonatal septicemia, and community-acquired pneumonia. Furthermore, P. stutzeri has been sporadically reported to carry MBLs, primarily of the VIM types (12–14).

Genes encoding NDM or other carbapenemases (VIM types among P. aeruginosa and OXA-181 among Enterobacteriaceae) that have been reported from India (3, 15) were not detected in this collection dating from 2000. This suggests that these enzymes emerged in India after 2000 and before 2006, when NDM-1-, OXA-181-, and VIM-producing isolates became more commonly reported. The finding of DIM-1 in two P. stutzeri isolates separated geographically and temporally may imply that the DIM-1-encoding gene has emerged in the Indian subcontinent; however, DIM-1 has not been detected in any other carbapenem-resistant isolate of Indian origin screened to date in our collection (2006-2007 and 2010-2011 sample years; data not shown) or cited in the literature. Additionally, DIM-1 has been very recently detected in an Enterobacter species from Sierra Leone, West Africa (16), and the emergence of the DIM-1-encoding gene on different continents with no apparent worldwide dissemination might indicate that bla_DIM-1 was acquired independently from an unknown natural reservoir. The protein similarity of DIM-1 and TMB-1 characterized from an environmental sample of Achromobacter species (10) also suggests that the possible origin of DIM-1 is as an intrinsic β-lactamase in an environmental species.

Although the potential of the DIM-1-encoding gene to proliferate in clinical isolates seems low, its appearance on three continents warrants screening for the corresponding resistance genotype among isolates with elevated carbapenem MICs.