Outbreak of metallo- -lactamase VIM-2-positive strains of Pseudomonas aeruginosa in the Ivory Coast

Abstract

Sir, Acquired metallo-b-lactamases (MBLs) are increasingly recognized as important determinants ofb-lactam resistance in Pseudomonas aeruginosa. To date, seven types of MBL enzyme (IMP, VIM, NDM, SPM, GIM, AIM and FIM) have been identified in this opportunistic pathogen. Since its first description in France in 1996, the epidemiologically successful enzyme VIM-2 has been traced in resistant P. aeruginosa strains from various European countries (e.g. Spain, Italy, Croatia, Greece, the Netherlands and Turkey) and more recently in strains from northern (Tunisia and Algeria) and southern (South Africa) Africa. – 6 One study also mentioned its occurrence in Kenya. Illustrating the spread of the VIM-2-encoding gene, blaVIM-2, in sub-Saharan Africa countries, we found here that P. aeruginosa strains from the Ivory Coast harboured an atypical class I integron previously described in sporadic isolates from other continents. Twelve P. aeruginosa isolates that were non-susceptible to imipenem according to the EUCAST interpretation rules (imipenem MIC .4 mg/L) were recovered from separate patients hospitalized in the intensive care unit of the teaching hospital in Abidjan, Ivory Coast, between February 2009 and November 2011. A double-disc (10 mg of imipenem, 2.5 mmol of EDTA) synergy test and an MBL Etest (imipenem+EDTA; bioMerieux, Craponne, France) were performed and found to be positive with seven of these isolates, suggesting the production of an Ambler class B b-lactamase (MIC of imipenem ≥256 mg/L; MIC of imipenem+EDTA 32 mg/L). PCR and DNA sequencing experiments revealed the presence of the gene blaVIM-2 in these isolates. All were highly resistant to carbapenems (imipenem MIC ≥128 mg/L, meropenem MIC ≥32 mg/L and doripenem MIC ≥32 mg/L), ceftazidime (MIC ≥64 mg/L), aminoglycosides (tobramycin MIC ≥128 mg/L) and ciprofloxacin (MIC ≥16 mg/L), but remained susceptible to aztreonam (MIC ≤1 mg/L) and colistin (MIC ≤4 mg/L). Six isolates were responsible for bloodstream (n1⁄43), urinary tract (n1⁄42) and pulmonary (n1⁄41) infections; the last one was considered to be a cathetercontaminant. Theirgenetic relatednesswas investigatedbymultiple-locusvariablenumber tandem-repeat analysis using eight variable-number tandem repeats, as described previously. All the VIM-2-positive isolates turned out to belong to the same epidemic clone (serotype O6), which was assigned to a new sequence type (ST), ST1488, after multilocus sequence typing (MLST) analysis. Interestingly, the closest ST found in the PubMLST database (http://pubmlst.org/paeruginosa), ST233, relates to a P. aeruginosa strain (serotype O6) identified in Sweden in 2006 from a patient repatriated from Ghana. Nucleotide sequence alignments demonstrated that this strain isolated from Ghana carried the same blaVIM-2-containing integron, In559, as ST1488 (Figure 1). Since Ghana and the Ivory Coast are neighbouring countries, this suggests that closely related multidrug-resistant genotypes are present in this region of the African continent. Screening for additional b-lactamase genes by PCR (blaTEM, blaSHV, blaPER, blaGES, blaVEB, blaIMP, blaNDM, blaSPM, blaPSE and blaOXA including blaOXA-198) revealed that the seven isolates except one (12.871) harboured blaOXA-4, which codes for the narrow-spectrum oxacillinase OXA-4. The gene was located on a class 1 integron upstream of the aadA2 and cmlA1 cassettes, which code for the aminoglycoside-modifying enzyme ANT(3′′) (streptomycin and spectinomycin resistance) and for an active efflux pump accommodating chloramphenicol, respectively. PCR experiments using primers specific to 5′ and 3′ conserved sequences (5′CS and 3′CS) failed to detect a class 1 integron associated with blaVIM-2 in the strains. To characterize the genetic environment of blaVIM-2, a BamHI library of strain 12.871 DNA was cloned into the BamHIrestricted plasmid pK18 (Km). Transformants of E. coli DH5a were selected on Mueller–Hinton agar plates containing 100 mg/L ampicillin and 30 mg/L kanamycin. Compared with strain DH5a, carrying the empty pK18 vector, all the recombinant clones tested displayed high levels of resistance to b-lactams except aztreonam. Sequence analysis of the 6224 kb insert from one of these clones led to the identification of a class 1 integron with a 5′CS sequence and gene tniCat its 3′ end. This genetic structure wasfound to carry the following gene cassettes downstream of the integrase gene intI1: aacA7 (encoding acetyltransferase AAC6′-Il, which confers amikacin resistance), blaVIM-2, dhfrB5 (encoding a dihydrofolate reductase, providing trimethoprim resistance) and aacC5b (encoding an uncharacterized aminoglycoside acetyltransferase). Interestingly, this genetic structure was identical to that of blaVIM-2-containing integrons characterized in P. aeruginosa from the USA (AY943084), Taiwan, Russia (AM749810), India (HQ005291) and the Indian Ocean (AM296017), but was different from integrons reported so far in African countries other than Ghana (Figure 1). Interestingly, upstream of the intI1 gene, we could identify the transposase gene tnpA from ISPa58 (ISfinder database, http://www-is.biotoul.fr). The sequence was 100% identical to that of tnpA belonging to the Tn3/ Tn21 family (JX194160). Repeated assays to visualize a plasmid by