Enteric bacteria producing acquired carbapenemases have been increasingly reported worldwide. Verona integron-encoded metallo-β-lactamase (VIM) -producing Enterobacteriaceae have been isolated in several countries with high prevalence noted in the Mediterranean region . The expression of blaVIM genes confers resistance to all β-lactams except aztreonam. The single amino acid variant of the first reported VIM-1 enzyme, VIM-4 was described in a Pseudomonas aeruginosa strain isolated in Greece in 2002 . Subsequently, VIM-4 has been identified in Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter spp., Aeromonas hydrophyla and Escherichia coli [3–9]. VIM-4-producing Enterobacteriaceae strains reportedly also harboured various other β-lactamases, e.g. KPC-2 , CMY-4 [10,11], CTX-M-15 [8,10], SHV-12 , SHV-2a  and TEM-1 , respectively. The blaVIM-4 gene has been described exclusively as part of a class I integron located, in most of the cases, on conjugative plasmids of varying size, which, if tested, all belonged to the A/C incompatibility group (summarized in Table 1).
Table 1. Genotypic characteristics of VIM-4 producing Enterobacteriaceae
|Species||Country||GenBank accession no.||Class I integron structure||VIM plasmid||Co-harboured β-lactamasesa||Reference|
|Gene cassettes (GC)||3′ end|
| Enterobacter cloacae ||UAE|| JX275775 || blaVIM-4 || aacA7|| dfrA1|| ΔaadA1|| smr ||ISPa21|| qacEΔI || sul1 ||175 kb||incA/C|| CMY-4, TEM-1, CTX-M-15||This study|
| Escherichia coli ||Russia||Unknown|| blaVIM-4 || aacA7|| dfrA1|| ΔaadA1|| smr ||ISPa21||40 kb||incA/C||CTX-M-15|||
| E. cloacae, Klebsiella pneumoniae||Italy|| AJ704863 || blaVIM-4 || aacA7|| dfrA1|| ΔaadA1|| smr ||ISPa21||Unknown||incA/C|| CMY-4 |||
| K. pneumoniae ||Tunisia|| AM181293 || blaVIM-4 || aacA7|| dfrA1|| ΔaadA1||ND||ND||>130 kb||NT|| CMY-4, TEM-1, CTX-M-15|||
| K. pneumoniae, Klebsiella oxytoca||Hungary|| GU181265, GU181269|| aacA4|| blaVIM-4 ||–||–||–|| qacEΔI ||90 kb||NT||None|||
| E. cloacae ||Greece|| EF467306 || blaVIM-4 || aacA7|| dfrA1|| aadA1||–|| qacEΔI || sul1 ||40 kb||NT|| SHV-2a |||
| K. pneumoniae ||Greece||unknown|| blaVIM-4 ||–|| – || – ||–||–||175 kb||NT||CMY-4, KPC-2|||
The VIM-type metallo-β-lactamase-producing bacteria are rarely described from the Arabian Peninsula. Acinetobacter baumannii and P. aeruginosa producing VIM-2 were reported from Kuwait and Saudi Arabia [12,13]. However, at the time of writing, a search of the literature did not reveal any description of VIM-producing Enterobacteriaceae from the Gulf region, beyond a conference abstract from Kuwait . Here we report on the isolation and first detailed analysis of a VIM-producing enteric bacterium from the Arabian Peninsula, an Enterobacter cloacae strain carrying the blaVIM-4 gene.
Screening 34 carbapenem non-susceptible clinical Enterobacteriaceae strains isolated in Abu Dhabi Emirate by a multiplex PCR  identified known carbapenemase genes in 22 isolates. blaNDM was found in six K. pneumoniae, one E. cloacae, two Escherichia coli and one Citrobacter freundii, while blaOXA-48-like was identified in eight K. pneumoniae and three Escherichia coli strains, respectively. An E. cloacae strain positive for blaVIM (ABC104) was also identified. This strain was isolated in January 2012 from the urine sample of a 45-year-old Egyptian male, permanent resident of the United Arab Emirates. The man had a spinal cord injury and urinary incontinence since the age of 12 years. Two years before sampling the patient had stayed in Egypt for a short while on family business, but he had never received any medical treatment beyond the United Arab Emirates. He was diagnosed with lumbosacral ependymoma in 2009 and underwent subtotal excision of the mass in June 2011. Because of residual tumour, the surgical wound never healed and was secondarily infected. Superficial wound cultures grew K. pneumoniae, methicillin-resistant Staphylococcus aureus and normal skin flora. Over the ensuing months, he was treated with multiple courses of intravenous and oral antibiotics including piperacillin–tazobactam, ertapenem, meropenem, amoxicillin–clavulanate and levofloxacin for a cumulative total of 138 Defined Daily Doses. In January 2012, he was admitted with back pain and cloudy urine.
The ABC104 strain was resistant to imipenem (MIC = 16 mg/L), meropenem (MIC = 8 mg/L), ertapenem (MIC = 4 mg/L), ceftazidime (MIC >128 mg/L), cefotaxim (MIC >128 mg/L), cefepime (MIC = 64 mg/L), aztreonam (MIC >128 mg/L), cefoperazone (MIC = 64 mg/L), trimethoprim/sulphamethoxazole (MIC = 8/76 mg/L), chloramphenicol (MIC = 32 mg/L), gentamicin (MIC = 32 mg/L), tobramycin (MIC = 48 mg/L), netilmicin (MIC = 32 mg/L), ciprofloxacin (MIC = 32 mg/L), moxifloxacin (MIC = 12 mg/L), levofloxacin (MIC = 12 mg/L), tetracycline (MIC = 64 mg/L) and minocycline (MIC = 32 mg/L) and exhibited sensitivity to amikacin (MIC = 6 mg/L) and colistin (MIC = 0.125 mg/L), only as tested by microdilution according to the CLSI standards . It carried the blaVIM-4 gene on an incA/C type  plasmid of c.175 kilobases as detected by Southern blotting of the S1-digested genomic DNA (Fig. 1). Attempts to conjugally transfer the plasmid into an azid-resistant derivative of Escherichia coli J53 failed at 30°C, as well as at 37°C. By PCR mapping and sequencing using primers designed based on previously published sequences (GenBank accession numbers. AJ704863 and AY339625) the blaVIM-4 gene was identified as part of a class I integron (Table 1). The integron was similar to other blaVIM-4-containing integrons identified in various Enterobacteriaceae isolates in Italy, Russia, Tunisia and Greece (Table 1) [4,8,10,11]. However, unlike in the Italian isolate VA-416/02 , in ABC104 the conserved 3′ end of the class 1 integron was identified downstream of the ISPa21 (GenBank accession number JX27577). ABC104 also harboured other β-lactamase genes: blaCTX-M-15, blaTEM-1 and blaCMY-4; blaCMY-4 being localized on the same plasmid as blaVIM-4 by Southern blotting. The blaCTX-M-15 gene was carried on a plasmid of c.300 kilobases, whereas a probe for blaTEM-1 hybridized with both plasmids (Fig. 1).
Figure 1. Southern blot of Enterobacter cloacae strain ABC104 S1-digested genomic DNA. Gel, left lane: λ concatamer. Oligonucleotide probes of the respective genes (see above the membrane strips) were PCR-generated and labelled with a DIG-DNA labelling kit (Roche Diagnostics GmbH, Mannheim, Germany). After hybridization and colour development the probe was removed and the membrane was re-hybridized with the next probe. Detection was achieved by an alkaline-phosphatase-based reaction according to the manufacturer’s instructions.
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This first description of a VIM-4-producing enteric bacterium in the Arabian Peninsula adds a further resistance mechanism to the list of carbapenemases, mostly of the NDM and OXA-types, in Enterobacteriaceae already reported from the region [18–20]. The nationality and travel history of the patient, and the fact that the other β-lactamases, the plasmid, and the class I integron containing the blaVIM-4 of ABC104 were all highly similar to those previously described from Italy  and North Africa  suggests the possibility of these resistance genes, or the organism itself, spreading from the Mediterranean region to the Gulf.