Identification of the First VIM Metallo-β-Lactamase-Producing Multiresistant Aeromonas hydrophila Strain

Abstract

A VIM metallo-β-lactamase-producing Aeromonas hydrophila strain carrying an integron-borne bla_VIM-4 gene was isolated from a cirrhotic patient's fecal sample in a Budapest hospital. The variable region of this integron is identical with that of a previously characterized integron from Pseudomonas aeruginosa clinical isolates in Pécs in southern Hungary.

Aeromonas species are considered food-borne pathogens of emerging importance (2, 7, 15). Most species belonging to this genus, particularly those associated with human infections, are widely distributed in the environment, especially in freshwater, sewage, marine environments, and drinking water, and are also found in a wide range of animal and plant food products (1, 2, 5, 21). These pathogens can cause infections in both immunocompetent and -compromised patients, including gastroenteritis, bacteremia, meningitis, and skin and soft-tissue infections (9). Chromosomally mediated, inducible β-lactamases were recognized as the major mechanism of β-lactam resistance in Aeromonas hydrophila. These enzymes comprise a molecular class C cephalosporinase, a class D penicillinase, and a class B metallo-β-lactamase (MBL) designated CphA, the last being a carbapenemase enzyme with a relatively narrow substrate profile (1, 16, 19, 20). In this report we describe the first VIM MBL-producing A. hydrophila strain carrying an integron-borne bla_VIM-4 gene.

Antimicrobial disk susceptibility tests were performed on Mueller-Hinton agar (Oxoid, Basingstoke, United Kingdom) as recommended by the Clinical and Laboratory Standards Institute (6). Test disks were purchased from Oxoid. MICs were determined by the agar dilution method for β-lactam antibiotics and by the Etest (AB Biodisk, Solna, Sweden) for other antibiotics. To detect MBL production, the MBL Etest (AB Biodisk, Solna, Sweden) and the imipenem-EDTA disk method (23) were used and complemented with the use of ceftazidime, cefepime, cefotaxime, cefoxitin, cefoperazone, piperacillin, and piperacillin-tazobactam disks with or without 750 μg EDTA. To prepare β-lactam-EDTA disks, 750 μg EDTA in the form of a 0.5 M EDTA solution (pH 8.0) was added to a β-lactam disk placed on a Mueller-Hinton agar plate (3, 23).

Detection of bla_VIM genes and class 1 integrons by PCR and sequencing was performed as described earlier (13, 14). Conjugation experiments were performed on Mueller-Hinton agar plates with the Escherichia coli J5-3 Rif^r and Pseudomonas aeruginosa PAO4089Rp strains (12) as recipients. Transconjugants were selected on Mueller-Hinton agar plates containing 300 and 100 μg/ml rifampin, respectively, and 32 μg/ml cefotaxime or 128 μg/ml ticarcillin.

The A. hydrophila isolate MB443 was recovered in October 2005 from a fecal sample from a 68-year-old male patient suffering from alcoholic liver disease (cirrhosis) associated with pathological fluid accumulation within the abdominal cavity. According to the anamnesis recorded in the Budapest hospital, the clinical history of the patient included childhood hepatitis and later appendectomy but no relevant antibiotic treatment. After the drainage of the ascites fluid, the patient was released from the hospital and started on diuretic therapy. A. hydrophila was not isolated from any other sample types.

The antimicrobial susceptibility profile of isolate MB443 is shown in Table 1 together with the corresponding values determined for the control A. hydrophila strain ATCC 7966. While MB443 was resistant to ceftazidime, cefepime, piperacillin-tazobactam, and imipenem, it remained susceptible to aztreonam. This profile is often observed among acquired MBL-producing gram-negative clinical isolates (14).

TABLE 1.

Antibiotic susceptibilities for the A. hydrophila strains MB443 and ATCC 7966

Strain	MIC (μg/ml) ofa:
	AMP	TZP	FOX	CAZ	CTX	CRO	FEP	IPM	ATM	GEN	AMK	TOB	CIP	TET	CHL	POB
MB443	>256	>256	>256	>256	>256	>256	32	32	<0.5	2	8	16	8	>256	2	16
ATCC 7966	>256	2	4	<0.5	<0.5	<0.5	<0.5	0.5	<0.5	2	4	4	<0.5	0.5	<0.5	2

^aAMP, ampicillin; TZP, piperacillin-tazobactam; FOX, cefoxitin; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone, FEP, cefepime; IPM, imipenem; ATM, aztreonam; GEN, gentamicin; AMK, amikacin; TOB, tobramycin; CIP, ciprofloxacin; TET, tetracycline; CHL, chloramphenicol; POB, polymyxin B.

Although Aeromonas spp. possess a chromosomal MBL that hydrolyzes the carbapenems, wild-type strains are usually categorized under standardized in vitro testing conditions as carbapenem susceptible (20, 22). Under selective pressure of certain antibiotics, the emergence of β-lactamase-overexpressing carbapenem-resistant Aeromonas species mutant strains has been observed (8, 10). The MBL Etest does not detect the presence of the chromosomal MBLs in wild-type strains (22). For isolate MB443 and for the control strain the MICs could not be determined by the MBL Etest as the inhibitory ellipse did not reach the calibrated scale on the strip (Fig. 1). However, for isolate MB443, ≥10-mm increases of the inhibitory zones around all β-lactam disks tested after the addition of 750 μg EDTA to the disks were observed (Table 2). Moreover, a typical synergistic zone between the cefoxitin and cefoxitin-EDTA disks was detected (Fig. 2). These observations indicated the presence of an acquired MBL with a much broader substrate profile than that of CphA.

FIG. 1.

Etest MBL strip for the A. hydrophila isolate MB443. IP stands for imipenem concentrations (4 to 256 μg/ml) and IPI for imipenem (1 to 64 μg/ml) plus a constant level of EDTA. IP and IPI MICs should be read where the respective inhibition ellipses intersect the strip (AB Biodisk, Solna, Sweden).

TABLE 2.

Diameters of inhibitory zones measured in the absence and presence of EDTA

Antibiotic	Amt of antibiotic on disk (μg)	Inhibitory-zone diam (mm) for strain:
		MB443		ATCC 7966
		Without EDTA	With EDTA	Without EDTA	With EDTA
Imipenem	10	15	27	22	25
Ceftazidime	30	15	25	30	33
Cefepime	30	16	26	32	33
Cefotaxime	30	6	24	27	27
Cefoxitin	30	6	16	21	23
Cefoperazone	30	6	22	25	25
Piperacillin	100	6	26	26	26
Piperacillin-tazobactam	100/10	6	24	26	25

FIG. 2.

Inhibitory zones for the A. hydrophila isolate MB443 around β-lactam disks with or without 750 μg EDTA. FOX, cefoxitin; CTX, cefotaxime; TZP, piperacillin-tazobactam.

PCR experiments detected a bla_VIM-4 gene located on a class 1 integron. The variable region of this integron was sequenced, and it was found to be identical with that of an integron previously characterized from P. aeruginosa isolates in Pécs in southern Hungary, with GenBank accession number AY702100 (Fig. 3) (14). This integron carries two resistance gene cassettes, an aacA4 gene in the first position followed by a bla_VIM-4 gene cassette that contains a 170-bp duplication. To the best of our knowledge, this is the first example of a VIM MBL-producing isolate within the Aeromonas genus worldwide. Mating assays failed to transfer the bla_VIM gene to the recipient E. coli and P. aeruginosa strains under the experimental conditions applied. This observation is similar to several previous reports of conjugation experiments with VIM-producing clinical isolates as donors (13, 14).

FIG. 3.

The variable region of the integron of the VIM-4-producing A. hydrophila isolate MB443 characterized in this study. The empty ellipse represents the attI1 site, and black circles represent the 59-base elements. The asterisk indicates a bla_VIM-4 cassette with the 170-bp duplication. 5′-CS and 3′-CS, 5′ and 3′ conserved sequences, respectively.

The potential of Aeromonas species carrying antibiotic resistance genes to enter the food chain and subsequently infect or colonize humans deserves attention. Class 1 integrons that harbor trimethoprim-sulfamethoxazole, tetracycline, aminoglycoside, and chloramphenicol resistance determinants and also an IMP MBL were detected in Aeromonas spp. in previous studies (4, 5, 11, 17, 21). In this report we identified an integron-borne bla_VIM-4 gene from A. hydrophila, a previously unreported horizontally acquired resistance mechanism in this genus.

The results of the MBL phenotypic tests may also have implications for the screening protocols. As the MBL Etest failed to detect the VIM enzyme in A. hydrophila, screening by the use of in-house disks with or without EDTA or by PCR could be a recommended strategy for the detection of acquired MBLs in Aeromonas spp.

The origin of acquired MBLs of human pathogens is still unknown (18). Aeromonas species isolates carrying such genes could provide a link between their supposed environmental pool and human-pathogenic bacteria. Based on our observations further molecular and metagenomic studies are needed to identify and characterize integron-containing bacteria from environmental sources that share the same ecological niches with the motile aeromonads.

Nucleotide sequence accession number.

The nucleotide sequence for the bla_VIM-4 allele has been submitted to GenBank under accession number EU581706.