Isolation of VIM-2-Producing Pseudomonas monteilii Clinical Strains Disseminated in a Tertiary Hospital in Northern Spain

Abstract

We describe here the occurrence of bla_VIM-2 in 10 carbapenem-resistant Pseudomonas monteilii strains isolated from different clinical samples from patients at the University Hospital Marqués de Valdecilla in northern Spain. All the bla_VIM-2-harboring P. monteilii isolates possessed a class 1 integron, with the cassette array [intI1_bla_VIM-2_aac(6′)-Ib_qacEΔ1_sul1]. Our results show the emergence of VIM-2-producing multidrug-resistant species other than Pseudomonas aeruginosa or Pseudomonas putida in a Spanish hospital. P. monteilii, although sporadically isolated, should also be considered an important metallo-β-lactamase (MBL) reservoir.

TEXT

Metallo-β-lactamases (MBLs) have appeared worldwide as an important cause of acquired resistance to β-lactams in Gram-negative bacteria, especially in nonfermenting species such as Pseudomonas aeruginosa. The broad spectrum activities of these enzymes are of great concern for clinicians, as they confer resistance to the majority of available β-lactams (1). Twelve groups of MBLs (IMP, VIM, NDM, SPM-1, SIM-1, KHM-1, GIM-1, DIM-1, TMB-1, SMB-1, FIM-1, and AIM-1) have been identified in a number of nosocomial pathogens (2–6). IMP and VIM enzymes are among the β-lactamases that exhibit the widest range of substrate specificity (7).

IMP- and VIM-type MBLs usually form part of chromosomally or plasmid-encoded integrons. These integrons may be associated with mobile genetics elements such as insertion sequences or transposon-like structures, which contribute to their dispersion among different bacterial species (1). The presence of transferable MBLs is also important in pseudomonads other than P. aeruginosa since they can act as reservoirs and contribute to the spread of these resistance determinants (8).

Pseudomonas monteilii is closely related to Pseudomonas putida, but they can be differentiated by some biochemical tests, such as those for the assimilation of inositol and l- and d-tartrate, degradation of aromatic hydrocarbons, and arylamidase activity. P. monteilii differs from P. aeruginosa in pyocyanin production, lipase activity, denitrification, and some other physiological and biochemical features (9).

The occurrence of VIM and IMP has been documented in two P. monteilii strains of environmental and clinical origins, respectively (10, 11). In this study, we analyzed the presence of MBLs in 10 nonduplicated carbapenem-resistant strains initially identified as P. putida by the Vitek 2 system (bioMérieux, Marcy l'Etoile, France). However, all the isolates were subsequently confirmed as P. monteilii by matrix-assisted laser desorption ionization–time of flight mass spectrometry (Microflex LT; Bruker Daltonics, Leipzig, Germany) and sequencing of the gene encoding 16S rRNA using primers described before (12). The strains were collected during a period of 8 years (2004 to 2012) in different settings of our hospital in northern Spain. The isolates were recovered from a variety of clinical specimens, including urine culture (n = 3), bronchoalveolar lavage fluid (n = 1), blood culture (n = 4), abdominal drainage (n = 1), and a cutaneous vesicle (n = 1).

The MICs of 13 antipseudomonal agents were determined by broth microdilution according to the current Clinical and Laboratory Standards Institute (CLSI) guidelines (13). All the isolates were resistant to cephalosporins, aztreonam, carbapenems, gentamicin, and tobramycin, but they were susceptible to amikacin and colistin. In addition, all but one isolate (P. monteilii strain 04/1103) showed resistance to ciprofloxacin and levofloxacin (Table 1). Double-sided Etest (imipenem/imipenem-EDTA; AB Biodisk, Solna, Sweden) results suggested the presence of an MBL (data not shown). PCR amplification and DNA sequencing using the primers and conditions described before were performed to confirm the existence of bla_VIM-2 (14).

TABLE 1.

Characteristics of the P. monteilii strains isolated in this study

Straina	Sourceb	PFGE	VIM-2	AME	Integron	MIC (μg/ml) ofc:
						CAZ	FEP	PIP	TZP	ATM	IMP	MER	GEN	TOB	AMK	CIP	LVX	COL
04/1103	BAL	1	+	aac(6′)-Ib	Class 1	64	64	256	256	16	>64	>64	>64	32	4	0.06	0.25	0.5
08/3472	AD	2A	+	aac(6′)-Ib	Class 1	128	32	256	256	64	>64	>64	>64	64	4	16	16	0.25
09/1724	BC	3	+	aac(6′)-Ib	Class 1	128	32	256	256	64	>64	>64	>64	64	4	16	16	0.25
10/2871	BC	2A	+	aac(6′)-Ib	Class 1	128	128	>256	>256	128	>64	>64	>64	>64	8	16	32	0.25
11/3751	UC	4	+	aac(6′)-Ib	Class 1	>128	64	256	256	64	>64	>64	>64	64	8	16	16	0.25
12/544	UC	2A	+	aac(6′)-Ib	Class 1	32	32	128	128	64	>64	>64	>64	64	8	8	16	0.5
12/1411	UC	5	+	aac(6′)-Ib ant(2″)-Ia	Class 1	64	32	128	128	64	>64	>64	>64	64	8	8	16	0.5
12/2891	BC	2B	+	aac(6′)-Ib	Class 1	64	64	256	256	64	>64	>64	>64	>64	4	8	16	1
12/3475	CV	2A	+	aac(6′)-Ib	Class 1	32	32	128	128	64	>64	>64	>64	64	8	8	>32	0.5
12/3748	BC	2B	+	aac(6′)-Ib	Class 1	64	32	128	128	64	>64	>64	>64	64	8	8	16	0.5

^aDigits before the slash represent the year of isolation of the strain.

^bBAL: bronchoalveolar lavage; AD: abdominal drainage; BC: blood culture; UC: urine culture; CV: cutaneous vesicle.

^cCAZ: ceftazidime; FEP: cefepime; PIP: piperacillin; TZP: piperacillin-tazobactam; ATM: aztreonam; IMP: imipenem; MEM: meropenem; GEN: gentamicin; TOB: tobramycin; AMK: amikacin; CIP: ciprofloxacin; LVX: levofloxacin; COL: colistin.

The presence of aminoglycoside-modifying enzymes (AMEs) was also assessed by PCR according to previously described protocols (15–17). In all cases, the acetyltransferase gene aac(6′)-Ib was amplified. The nucleotidyltransferase gene ant(2″)-Ia was detected in one isolate (P. monteilii strain 12/1411) (Table 1). The bla_VIM-2-containing integrons were detected by PCR and characterized by DNA sequencing using specific primers targeting the 5′ conserved sequences (CS) and 3′ CS of class 1 integrons (14, 18). All the isolates presented the same class 1 integron cassette arrangement [intI1_bla_VIM-2_aac(6′)-Ib_qacEΔ1_sul1], identical to that of the integron of P. aeruginosa strain 81-11963A (GenBank accession number AJ515707).

Several attempts to conjugate the putative bla_VIM-2-carrying plasmids from P. monteilii strains to Escherichia coli, as described before (19), were unsuccessful. Therefore, we performed an alkaline lysis extraction, and plasmids were transformed by electroporation into P. aeruginosa strain PAO1. In each case, plasmid DNA (>50 kb) was observed. PCRs with VIM-2 and AME-specific primers (13–16) using the total DNA of PAO1 transformants as the template showed that the bla_VIM-2 and aac(6′)-Ib genes were present. No amplification of ant(2″)-Ia was observed, suggesting that this gene might have a chromosomal location. According to PCR-based replicon typing, each P. monteilii strain contained an untypeable plasmid (20).

Finally, the clonal relatedness of the isolates was evaluated using pulsed-field gel electrophoresis (PFGE) analysis as previously described (21). Enzymatic restriction was carried out overnight at 37°C with 10 U of SpeI (TaKaRa). Five different clones were observed among the 10 bla_VIM-2-carrying P. monteilii isolates. Six of the isolates were of clonal origin, as they differed from each other in about 3 bands. As shown in Table 1, pulsotype 2A intermittently appeared during the period of study. The rest of the strains were not clonally related (Table 1, Fig. 1).

FIG 1.

PFGE profiles of the SpeI-digested genomic DNAs of the bla_VIM-2-harboring P. monteilii isolates analyzed in this study.

Pseudomonas monteilii is a versatile bacterium that can be isolated from different natural environments (10, 22). Some strains have been isolated from clinical specimens (9, 11). The presence of VIM-2 was previously described in a P. monteilii isolate from an environmental origin (10). To our knowledge, this is the first study to report the occurrence of bla_VIM-2 in strains of P. monteilii isolated from different clinical settings in a Spanish hospital. According to our results, the persistence and spread of bla_VIM-2-mediated resistance in P. monteilii reflect clonal and horizontal dissemination. Further spread of these carbapenem resistance determinants from P. monteilii to other similar or unrelated species represents an important threat. Moreover, our results reinforce the idea of the relevant role of pseudomonads other than P. aeruginosa or P. putida as nosocomial reservoirs of MBLs as is the case of P. monteilii.