To the Editor: The metallo-β-lactamase (MBL) group of enzymes inactivates many β-lactam antimicrobial drugs. First identified from a Klebsiella pneumoniae strain recovered from a patient hospitalized in India, the New Delhi metallo-β-lactamase-1 (NDM-1), particularly in Enterobacteriaceae, is now the focus of worldwide attention (1). Whereas India and Pakistan were considered as the main reservoirs of the bla_NDM-1 gene (2) that produces this MBL, several NDM-1–producing Enterobacteriaceae isolates have been reported from the Balkan states and the Middle East, suggesting that those areas might be secondary reservoirs (2).

Since 2010, 3 NDM-1 point-mutation variants have been described (3–5). The first variant, NDM-2, was identified from an Acinetobacter baumannii isolate collected from a patient transferred from a hospital in Egypt to Germany (4). Subsequently, a clonal dissemination of NDM-2–producing A. baumanni was described in Israel (6). The second variant, NDM-4, which was identified in Escherichia coli from a patient hospitalized in India, possessed a higher carbapenemase activity compared with NDM-1 (5). The most recent variant, NDM-5, was identified in E. coli from a patient who had a history of hospitalization in India (3).

As recommended for the detection of carbapenemase producers (7), a rectal swab specimen was collected from a patient transferred from Cameroon to France. The E. coli strain FEK was isolated from the specimen. He had been hospitalized for 1 month in Douala for an inflammatory syndrome associated with a kidney failure before his transfer to Paris. No history of travel in India was reported for this patient. Susceptibility testing was performed by disk diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, France), and results were interpreted according to the updated guidelines of the Clinical and Laboratory Standards Institute (Wayne, PA, USA; www.clsi.org). The MICs were determined by using Etest (bioMérieux, La Balmes-Les-Grottes, France) on Mueller-Hinton agar at 37°C.

E. coli FEK was fully resistant to all β-lactam antimicrobial drugs, including imipenem, meropenem, ertapenem, and doripenem (MICs >32 mg/L for all carbapenems). This isolate was also resistant to aminoglycosides, except amikacin, and to fluoroquinolones. We performed PCR amplification followed by sequencing on whole-cell DNA, as described (8). We identified the bla_NDM-4, bla_CTX-M-15, and bla_OXA-1 genes. E. coli FEK also harbored the aacA4 gene encoding the AAC(6′)-Ib acetyltransferase that confers high-level resistance to aminoglycosides, except amikacin. Results of multilocus sequence typing analysis performed as described (5) showed that the isolate belonged to sequence type (ST) ST405. Identification of this ST type among NDM-producing E. coli, compared with NDM-4– and NDM-5–producing E. coli in ST648, demonstrated that the spread of NDM-4 occurred among unrelated E. coli clonal backgrounds (3,5).

Plasmid DNA of E. coli FEK was extracted and analyzed as described (5). A single, ≈120-kb plasmid was identified. Direct transfer of the β-lactam resistance marker into E. coli J53 was attempted by liquid mating-out assays at 37°C. With the exception of the aminoglycoside amikin, transconjugants from E. coli were resistant to β-lactam antimicrobial drugs. MICs of imipenem, meropenem, ertapenem, and doripenem were 6, 3, 6, and 4 mg/L, respectively. The transconjugants harbored an ≈120-kb plasmid carrying bla_NDM-4 and the bla_CTX-M-15, bla_OXA-1, and aacA4 genes. We performed PCR-based replicon typing as described (5) and showed that this bla_NDM-4–positive plasmid belonged to the IncFIA incompatibility group. The IncF incompatibility group was previously reported to be associated with bla_NDM-4 and bla_NDM-5 (3,5).

By analyzing genetic structures surrounding the bla_NDM-4 gene, performed by PCR mapping as described (8), we identified insertion sequence ISAba125 upstream and the bleomycin resistance gene ble_MBL downstream of the bla_NDM-4 gene. The same genetic environment has been observed for most NDM-1–positive enterobacterial isolates (8). We showed in previous research that expression of ble_MBL conferred high-level resistance to bleomycin and bleomycin-like molecules (9); accordingly, the E. coli clinical isolate and its transconjugant were highly resistant to bleomycin (MIC >512 µg/mL) (9).

The patient had a history of Hodgkin lymphoma treated by 8 sessions of bleomycin chemotherapy 1 year before his hospitalization. This anticancer drug is widely distributed throughout the body following intravenous administration, and plasmatic concentrations increase in proportion with the increase of the dose (10). Because the patient was successively treated with 30 mg of bleomycin, the serum levels achieved (≈2–5 mg/mL) might have contributed to selection of the ble_MBL gene. Similarly, the multiple courses of antibacterial drug therapy administered in Cameroon (including carbapenems) could have contributed to selection of the bla_NDM-4 gene.

By culturing rectal swab samples from the patient, we identified fecal carriage of E. coli carrying a plasmid-encoded bla_NDM-1 gene. That strain had a distinct ST type (ST5) compared with the index strain. The plasmid carrying the bla_NDM-1 gene with the bla_OXA-1 and aacA4 genes belonged to the IncFIA incompatibility group.

β-Lactamase NDM-4 displaying increased carbapenemase activity compared with NDM-1 was described in a patient hospitalized in India (5). This study shows that NDM-4 producers are also present in Africa; specifically, in the highly populated city of Douala, providing an environment that may promote the dissemination of those strains. We showed that the same patient was carrying strains expressing 2 NDM variants, possibly indicating ongoing evolution of NDM variants.

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