VIM-2 metallo- -lactamase-producing Pseudomonas aeruginosa causing an outbreak in South Africa

Abstract

Sir, Pseudomonas aeruginosa is an opportunistic pathogen that has been implicated in nosocomial outbreaks in immunocompromised patients worldwide. We present an outbreak caused by a multidrug-resistant P. aeruginosa (MRPA) clone in a haematology unit of a tertiary academic hospital in Cape Town, South Africa. Fifteen MRPA isolates were recovered from separate patients between January 2010 and April 2011, including 10 from blood, 2 from stool, 1 from bile, 1 from urine and 1 from a catheter tip. The majority of the patients were severely neutropenic following stem cell transplants and eight of them died. The genetic relatedness of the isolates was investigated using PFGE according to a previously published protocol with minor amendments. Whole genomic DNA was digested in situ with SpeI (New England Biolabs, Inc., UK) and the resulting restriction fragments were separated on a 1% agarose gel using a CHEF-DRII GeneNavigator apparatus (GE Healthcare, Piscataway, NJ, USA). Restriction profiles were analysed using GelCompar II version 5.1 (Applied Maths, St-Martens-Latem, Belgium). A dendrogram indicating the levels of similarity between the isolates was created using the Dice similarity coefficient. The band tolerance and optimizations were set at 1%, and a similarity threshold of 80% was used to define clusters. The profiles of 10 of the 15 isolates were indistinguishable and were assigned to cluster A. One isolate, with 77% similarity to cluster A, was assigned subtype A1. The remaining four isolates were all unique. To further characterize the genetic background of this MRPA clone, multilocus sequence typing (MLST) of three representative isolates from cluster A, spanning the defined outbreak period, were selected. MLST indicated that these three isolates belong to sequence type (ST) 233. Identification and susceptibility testing was performed on Vitek 2 (bioMerieux, Marcy l’Etoile, France). Imipenem and meropenem MICs were determined by Etests, according to the manufacturer’s specification (bioMerieux). Fourteen of the isolates expressed high levels of resistance to imipenem (MIC ≥128 mg/L) and meropenem (MIC ≥128 mg/L). The 15th strain was susceptible to both imipenem (MIC ≤1 mg/L) and meropenem (MIC ≤2 mg/L). As metallo-b-lactamases (MBLs) are considered a major mechanism of carbapenem resistance in P. aeruginosa, PCR assays were carried out using primers for the detection of the MBL-encoding genes blaIMP (IMPF 5′-ATTGACACTCCATTTAC-3′/ IMPR 5′-AACAACCAGTTTTGC-3′) and blaVIM (VIMF 5′-GTGAGTATCC GACAGTC-3′/VIMR 5′-GAGCAAGTCTAGACCG-3′). Although a PCR product of the expected size was obtained from a control P. aeruginosa strain carrying blaIMP-1 (gift from Y. Hirakata, Tohuku University Graduate School of Medicine, Japan), no products were obtained from the 15 Cape Town P. aeruginosa isolates. However, amplicons of the expected size for the blaVIM gene were obtained for the 10 strains in PFGE cluster A and the closely related strain, A1, as well as the blaVIM-2 positive control (gift from P. Nordmann, Hopital de Bicetre, France). No PCR products were obtained from the four unrelated P. aeruginosa isolates. PCR products from the 11 strains were purified (QIAquick PCR purification kit, QIAGEN, Germany) and sequencing analysis revealed the blaVIM-2 gene in all 11 strains. Additional PCR screening for the NDM, SPM, KPC and GES b-lactamase genes for all 15 isolates revealed that the single isolate, sensitive to both imipenem and meropenem and cultured from a catheter tip, carried a blaGES-2 gene, which has previously been implicated in a South African outbreak. MLST typing indicated that this carbapenem-susceptible strain belonged to ST625, a new genotype not previously described. Relatively little is known regarding the prevalence of carbapenem-hydrolysing enzymes in African countries. This outbreak of MBL blaVIM-2-carrying P. aeruginosa highlights the urgent need for the development of more active surveillance systems in South Africa and the importance of molecular epidemiology in a hospital outbreak situation.