Giuseppe
Emeritus
[Source: US Centers for Disease Control and Prevention, Emerging Infectious Diseases Journal, full page: (LINK). Edited.]
DOI: 10.3201/eid1809.120011
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Volume 18, Number 9?September 2012
Letter
New Delhi Metallo-β-Lactamase 4?producing Escherichia coli in Cameroon
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) 822822, particularly in Enterobacteriaceae, is now the focus of worldwide attention (1). Whereas India and Pakistan were considered as the main reservoirs of the bla<SUB>NDM-1</SUB> 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 (bioM?rieux, 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<SUB>NDM-4</SUB>, bla<SUB>CTX-M-15</SUB>, and bla<SUB>OXA-1</SUB> 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<SUB>NDM-4</SUB> and the bla<SUB>CTX-M-15</SUB>, bla<SUB>OXA-1</SUB>, and aacA4 genes. We performed PCR-based replicon typing as described (5) and showed that this bla<SUB>NDM-4</SUB>?positive plasmid belonged to the IncFIA incompatibility group.
The IncF incompatibility group was previously reported to be associated with bla<SUB>NDM-4</SUB> and bla<SUB>NDM-5</SUB> (3,5).
By analyzing genetic structures surrounding the bla<SUB>NDM-4</SUB> gene, performed by PCR mapping as described (8), we identified insertion sequence ISAba125 upstream and the bleomycin resistance gene ble<SUB>MBL</SUB> downstream of the bla<SUB>NDM-4</SUB> 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<SUB>MBL</SUB> 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 ?g/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<SUB>MBL</SUB> gene. Similarly, the multiple courses of antibacterial drug therapy administered in Cameroon (including carbapenems) could have contributed to selection of the bla<SUB>NDM-4</SUB> gene.
By culturing rectal swab samples from the patient, we identified fecal carriage of E. coli carrying a plasmid-encoded bla<SUB>NDM-1</SUB> gene. That strain had a distinct ST type (ST5) compared with the index strain. The plasmid carrying the bla<SUB>NDM-1</SUB> gene with the bla<SUB>OXA-1</SUB> 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.
Laurent Dortet, Laurent Poirel, Nadia Anguel, and Patrice Nordmann
Author affiliations: Institut National de la Sant? et de la Recherche M?dicale Paris, France (L. Dortet, L. Poirel, P. Nordmann); and H?pital de Bic?tre?Assistance Publiques des H?pitaux de Paris, Le Kremlin-Bic?tre, France (L. Dortet, N. Anguel, P. Nordmann)
Acknowledgment
This work was partially funded by a grant from the INSERM (U914), the Minist?re de l'Education Nationale et de la Recherche (UPRES-EA3539), Universit? Paris XI, France, and by grants from the European Community (TEMPOtest-QC, HEALTH-2009-241742, and R-GNOSIS, HEALTH-2009-24174).
References
Letter
New Delhi Metallo-β-Lactamase 4?producing Escherichia coli in Cameroon
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) 822822, particularly in Enterobacteriaceae, is now the focus of worldwide attention (1). Whereas India and Pakistan were considered as the main reservoirs of the bla<SUB>NDM-1</SUB> 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 (bioM?rieux, 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<SUB>NDM-4</SUB>, bla<SUB>CTX-M-15</SUB>, and bla<SUB>OXA-1</SUB> 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<SUB>NDM-4</SUB> and the bla<SUB>CTX-M-15</SUB>, bla<SUB>OXA-1</SUB>, and aacA4 genes. We performed PCR-based replicon typing as described (5) and showed that this bla<SUB>NDM-4</SUB>?positive plasmid belonged to the IncFIA incompatibility group.
The IncF incompatibility group was previously reported to be associated with bla<SUB>NDM-4</SUB> and bla<SUB>NDM-5</SUB> (3,5).
By analyzing genetic structures surrounding the bla<SUB>NDM-4</SUB> gene, performed by PCR mapping as described (8), we identified insertion sequence ISAba125 upstream and the bleomycin resistance gene ble<SUB>MBL</SUB> downstream of the bla<SUB>NDM-4</SUB> 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<SUB>MBL</SUB> 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 ?g/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<SUB>MBL</SUB> gene. Similarly, the multiple courses of antibacterial drug therapy administered in Cameroon (including carbapenems) could have contributed to selection of the bla<SUB>NDM-4</SUB> gene.
By culturing rectal swab samples from the patient, we identified fecal carriage of E. coli carrying a plasmid-encoded bla<SUB>NDM-1</SUB> gene. That strain had a distinct ST type (ST5) compared with the index strain. The plasmid carrying the bla<SUB>NDM-1</SUB> gene with the bla<SUB>OXA-1</SUB> 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.
Laurent Dortet, Laurent Poirel, Nadia Anguel, and Patrice Nordmann
Author affiliations: Institut National de la Sant? et de la Recherche M?dicale Paris, France (L. Dortet, L. Poirel, P. Nordmann); and H?pital de Bic?tre?Assistance Publiques des H?pitaux de Paris, Le Kremlin-Bic?tre, France (L. Dortet, N. Anguel, P. Nordmann)
Acknowledgment
This work was partially funded by a grant from the INSERM (U914), the Minist?re de l'Education Nationale et de la Recherche (UPRES-EA3539), Universit? Paris XI, France, and by grants from the European Community (TEMPOtest-QC, HEALTH-2009-241742, and R-GNOSIS, HEALTH-2009-24174).
References
- Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Characterization of a new metallo-β-lactamase gene, bla<SUB>NDM-1</SUB>, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother. 2009;53:5046?54. DOI PubMed
- Nordmann P, Poirel L, Walsh TR, Livermore DM. The emerging NDM carbapenemases. Trends Microbiol. 2011;19:588?95. DOI PubMed
- Hornsey M, Phee L, Wareham DW. A novel variant, NDM-5, of the New Delhi metallo-β-lactamase in a multidrug-resistant Escherichia coli ST648 isolate recovered from a patient in the United Kingdom. Antimicrob Agents Chemother. 2011;55:5952?4. DOI PubMed
- Kaase M, Nordmann P, Wichelhaus TA, Gatermann SG, Bonnin RA, Poirel L. NDM-2 carbapenemase in Acinetobacter baumannii from Egypt. J Antimicrob Chemother. 2011;66:1260?2. DOI PubMed
- Nordmann P, Boulanger A, Poirel L. NDM-4 metallo-β-lactamase with increased carbapenemase activity from Escherichia coli. Antimicrob Agents Chemother. 2012;56:2184?6. DOI PubMed
- Espinal P, Fugazza G, Lopez Y, Kasma M, Lerman Y, Malhotra-Kumar S, Dissemination of an NDM-2?producing Acinetobacter baumannii clone in an Israeli rehabilitation center. Antimicrob Agents Chemother. 2011;55:5396?8. DOI PubMed
- Nordmann P, Poirel L, Carr?r A, Toleman MA, Walsh TR. How to detect NDM-1 producers? J Clin Microbiol. 2011;49:718?21. DOI PubMed
- Poirel L, Dortet L, Bernabeu S, Nordmann P. Genetics features of bla<SUB>NDM-1</SUB>?positive Enterobacteriaceae. Antimicrob Agents Chemother. 2011;55:5403?7. DOI PubMed
- Dortet L, Nordmann P, Poirel L. Association of the emerging carbapenemase NDM-1 with a bleomycin resistance protein in Enterobacteriaceae and Acinetobacter baumannii. Antimicrob Agents Chemother. 2012;56:1693?7. DOI PubMed
- Teale JD, Clough JM, Marks V. Radioimmunoassay of bleomycin in plasma and urine. Br J Cancer. 1977;35:822?7. DOI PubMed
DOI: 10.3201/eid1809.120011