LETTER
Over the last decade, the increase of carbapenem resistance in Pseudomonas aeruginosa has been due mostly to impermeability because of OprD loss and the production of metallo-β-lactamases (MBL), including those of the IMP, VIM, SPM, GIM, SIM, AIM-1, FIM-1, and NDM families (1–4); also, active efflux and serine carbapenemases may contribute to carbapenem resistance in P. aeruginosa in some cases. VIM-1 was reported in the Mediterranean area in 1997 in a clinical isolate of P. aeruginosa in Verona, Italy (5), and in a clinical isolate of Pseudomonas mosselii isolated in 1994 in Genoa, Italy (6). A VIM-2 variant appeared in Marseille, France, in 1996 (7). VIM-2 has now spread as the predominant MBL variant among P. aeruginosa in all European Mediterranean countries (1), but in North Africa, the presence of VIM-2 has been reported only recently in Tunisia (2, 8, 9). Here we report the first molecular characterization of VIM-2-producing P. aeruginosa clinical isolates from Algeria that harbored a novel class I integron that also contained two gene cassettes encoding resistance to aminoglycosides (aadB and aacA4).
A total of 17 nonreplicate imipenem-resistant P. aeruginosa clinical isolates (imipenem MIC, >8 μg/ml, as determined by the Etest) recovered from December 2010 to September 2011 in a surgical intensive care unit at the University Hospital of Annaba, Algeria, were screened by PCR for the presence of MBL-encoding genes using primers previously described (10–12). Antibiotic susceptibility testing was performed using disk diffusion with the breakpoints and according to the guidelines of the Antibiogram Committee of the French Society for Microbiology (CA-SFM) (www.sfm-microbiologie.org), and results are summarized in Table 1. An imipenem-EDTA synergy test (13) was positive for 14 out of 17 strains that were PCR positive for VIM-2 carbapenemase using the universal primers VIM-all-F (5′-TGGTCTACATGACCGCGTCT-3′) and VIM-all-R (3′-CGACTGAGCGATTTGTGTG-5′), with an expected PCR size of 766 bp. The characterization of class I integrons reported previously (11) showed a novel class I integron that harbored the VIM-2 gene associated with two gene cassettes encoding aminoglycosides resistance (aadB and aacA4). However, the molecular investigation of the remaining three strains using primers previously described (3) revealed chromosomic mutations that created premature stop codons in the oprD gene. Complete multilocus sequence typing (MLST) (14) revealed that the 17 strains belong to 4 different sequence types (ST), including ST162 (one isolate), ST654 (one isolate), ST1175 (one isolate), and a new ST, ST1420 (14 isolates), recently submitted to the PubMLST database (http://pubmlst.org/paeruginosa) from China (January, 2013).
Table 1.
Phenotypic and genotypic features of the 17 imipenem-resistant P. aeruginosa clinical isolatesa
| Strain | Date of isolation (day/mo/yr) | Patient age | Sex | Source | IMP MIC (μg/ml) | Resistance phenotype |
VIM-2 production | Sequence type | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| TIC | TCC | TZP | CAZ | GEN | CIP | FOS | COL | ||||||||
| 1 | 15/12/2010 | 42 yr | M | Bronchial aspirate | 12 | R | R | R | R | R | S | S | S | + | ST1420 |
| 2 | 15/12/2010 | 18 yr | F | Blood | 32 | R | R | R | R | R | S | S | S | + | ST1420 |
| 3 | 28/12/2010 | 18 mo | M | Bronchial aspirate | 12 | R | R | R | R | R | S | S | S | + | ST162 |
| 4 | 25/02/2011 | 2 mo | M | Bronchial aspirate | 8 | R | R | R | R | R | R | R | S | + | ST1420 |
| 5 | 27/02/2011 | 7 yr | M | Bronchial aspirate | 16 | R | R | R | R | R | R | R | S | + | ST1420 |
| 6 | 08/03/2011 | 7 days | M | Bronchial aspirate | 32 | R | R | R | R | S | S | R | S | + | ST1420 |
| 7 | 22/03/2011 | 57 yr | M | Urinary catheter | 12 | R | R | R | R | R | R | S | S | + | ST1420 |
| 8 | 23/03/2011 | 43 yr | M | Urinary catheter | 32 | R | R | R | R | S | S | R | S | + | ST1420 |
| 9 | 18/04/2011 | 35 yr | M | Urine | 12 | R | R | R | R | S | S | R | S | + | ST1420 |
| 10 | 25/05/2011 | 7 yr | M | Pus | 16 | R | R | R | R | R | S | R | S | + | ST1420 |
| 11 | 19/06/2011 | 46 yr | M | Urine | 16 | R | R | R | R | S | R | S | S | + | ST1420 |
| 12b | 22/06/2011 | 58 yr | F | Bronchial aspirate | 32 | R | R | R | R | R | S | S | S | − | ST1175 |
| 13c | 22/07/2011 | 40 yr | M | Bronchial aspirate | 32 | R | R | R | R | S | S | R | S | − | ST1420 |
| 14 | 18/08/2011 | 54 yr | M | Wound | 16 | R | R | R | R | R | S | S | S | + | ST1420 |
| 15 | 27/08/2011 | 5 yr | M | Wound | 12 | R | R | R | R | S | S | R | S | + | ST1420 |
| 16d | 10/09/2011 | 10 yr | M | Urine | 16 | R | R | R | R | R | R | R | S | − | ST654 |
| 17 | 27/08/2011 | 45 yr | M | Wound | 12 | R | R | R | R | R | S | S | S | + | ST1420 |
CAZ, ceftazidime; CIP, ciprofloxacin; COL, colistin; FOS, fosfomycin; GEN, gentamicin; TCC, ticarcillin-clavulanic acid; TIC, ticarcillin; TZP, piperacillin-tazobactam; M, male; F, female; R, resistant; S, susceptible. MICs were determined by the Etest.
Contains a 529CGA-to-TGA mutation, leading to a stop codon in the oprD gene.
Exhibits a 193TGGA-to-TG·A deletion, leading to a stop codon in the oprD gene.
Contains a 757TCG-to-TAG mutation, leading to a stop codon in the oprD gene.
To the best of our knowledge, in North Africa, VIM-2-MBL-producing P. aeruginosa isolates have been reported only in Tunisia (8, 9, 11). VIM-2-producing P. aeruginosa clinical isolates have been reported in other countries in Africa, including 1 from a Hungarian tourist who was hospitalized in Egypt (15), 1 from a single patient who was transferred to Norway after being hospitalized in Ghana (16), 57 recovered in 2006 and 2007 in Kenya (17), and 15 from South Africa (18).
Our results demonstrate that national surveillance should be urgently implemented in Algeria to monitor and control the emergence and spread of carbapenemase-encoding genes.
Nucleotide sequence accession numbers.
The full sequence of the class 1 integron has been deposited in the GenBank database under accession number JX120362.
ACKNOWLEDGMENTS
We thank Linda Hadjadj and Maxime Pit for technical assistance. We thank American Journal Experts for English corrections.
We have no conflicts of interest to declare.
This work was partly funded by CNRS.
Footnotes
Published ahead of print 4 March 2013
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