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Emergence of an NDM-5-producing clinical Escherichia coli isolate in Egypt

  • Ahmed M. Soliman
    Affiliations
    Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan

    Department of Microbiology and Immunology, Faculty of Pharmacy and Drug Industries, Kafrelsheikh University, Kafr El-Sheikh, Egypt
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  • Hazim O. Khalifa
    Affiliations
    Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan

    Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafr El-Sheikh, Egypt
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  • Ashraf M. Ahmed
    Affiliations
    Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan

    Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafr El-Sheikh, Egypt
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  • Toshi Shimamoto
    Affiliations
    Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan
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  • Tadashi Shimamoto
    Correspondence
    Corresponding author. Tel.: +81 82 424 7897; fax: +81 82 424 7916.
    Affiliations
    Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan
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Open AccessPublished:May 09, 2016DOI:https://doi.org/10.1016/j.ijid.2016.05.003

      Highlights

      • The first occurrence of New Delhi metallo-β-lactamase 5 (NDM-5), carried on an IncI1-Iγ-type plasmid in a multidrug-resistant clinical Escherichia coli strain in Egypt, is reported.
      • This E. coli was found to belong to sequence type (ST) 5018 and to carry other resistance genes including blaCTX-M-15, blaCMY-42, blaOXA-1, and aac(6′)-Ib-cr and class 1 integron with two gene cassettes (dfrA17–aadA5).
      • Southern blot hybridization showed that blaNDM-5 was located on a plasmid of >93 kb in size.

      Summary

      The first occurrence of New Delhi metallo-β-lactamase 5 (NDM-5), carried on an IncI1-Iγ-type plasmid of >93 kb in a multidrug-resistant Escherichia coli strain in Kafr El-Sheikh, Egypt, is reported. The strain was isolated from a wound pus swab from a patient diagnosed with a fracture of the right femur. This E. coli strain was found to belong to sequence type (ST) 5018 and also to carry other resistance genes, including blaCTX-M-15, blaCMY-42, blaOXA-1, and aac(6′)-Ib-cr.

      Keywords

      1. Introduction

      New Delhi metallo-β-lactamase 1 (NDM-1) has received global attention due first to its high level of resistance to many different β-lactams, except aztreonam, second to its dissemination worldwide to different countries, third to its association with hospital-acquired infections, and fourth to its acquisition by many common Gram-negative bacteria.
      • Zafer M.M.
      • Amin M.
      • El Mahallawy H.
      • Ashoure M.S.
      • Al Agamy M.
      First report of NDM-1-producing Pseudomonas aeruginosa in Egypt.
      NDM-1 was first discovered in Klebsiella pneumoniae in a Swedish patient previously hospitalized in India.
      The discovery of NDM-5 was similar; it was first reported in 2011 in an Escherichia coli isolate recovered from a patient in the UK after a recent hospitalization in India.
      • Hornsey M.
      • Phee L.
      • Wareham D.W.
      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.
      It has since been reported from many other countries, including India, Algeria,
      • Sassi A.
      • Loucif L.
      • Gupta S.K.
      • Dekhil M.
      • Chettibi H.
      • Rolain J.M.
      NDM-5 carbapenemase-encoding gene in multidrug-resistant clinical isolates of Escherichia coli from Algeria.
      Spain, Japan, Australia, the USA, and China.
      • Yang P.
      • Xie Y.
      • Feng P.
      • Zong Z.
      blaNDM-5 carried by an IncX3 plasmid in Escherichia coli sequence type 167.
      NDM-5 differs from NDM-1 by two amino acid substitutions (Val88Leu and Met154Leu), which cause a reduced susceptibility of E. coli TOP10 transformants to extended-spectrum cephalosporins and carbapenems.
      • Hornsey M.
      • Phee L.
      • Wareham D.W.
      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.
      The first NDM-5-producing carbapenem-resistant E. coli isolate identified in Egypt is described herein.

      2. Case report

      A 65-year-old man was admitted to the emergency department of a hospital in the city of Kafr El-Sheikh, Egypt, on May 30, 2014, with a diagnosis of right femur fracture resulting in the formation of a deep wound. After surgery on May 31, 2014, the patient was transferred to the department of orthopedics for the completion of treatment, and empiric intravenous ceftriaxone (1 g twice daily for 13 days) was given to prevent bacterial infection. Unfortunately, the wound began to exude yellow pus on June 13, 2014, and the patient was started on intravenous levofloxacin (0.5 g twice daily for 6 days). On June 16, 2014, a multidrug-resistant E. coli strain, designated EC169, was isolated from the wound pus specimen. In accordance with the drug resistance pattern of EC169, the patient's antibiotic treatment was changed to intramuscular amikacin (1 g twice daily). The patient recovered after 9 days, and a wound pus swab was negative for E. coli.
      EC169 was identified biochemically using the API 20E System (bioMérieux, Marcy l’Etoile, France). The broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of selected antimicrobials; the results were interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Amoxicillin, cefotaxime, ceftazidime, and ceftriaxone powders were supplied by Sigma-Aldrich (Tokyo, Japan), meropenem, gentamicin, nalidixic acid, norfloxacin, and colistin by Wako Pure Chemical Ind. Ltd (Osaka, Japan), imipenem, doripenem, and cefoperazone by LKT Laboratories, Inc. (Minnesota, USA), and tetracycline powder was purchased from Nacalai Tesque (Kyoto, Japan). For all experiments, the purified powder of each antibiotic was diluted following the CLSI recommendations. The reference strain Escherichia coli ATCC 25922 was included as a quality control.
      EC169 showed resistance to imipenem, meropenem, doripenem, aztreonam, amoxicillin, cefotaxime, ceftazidime, cefoperazone, ceftriaxone, gentamicin, nalidixic acid, tetracycline, and norfloxacin, while it was sensitive to amikacin and colistin (Table 1). EC169 was also positive on the modified Hodge test, by carbapenem inactivation method (CIM), and when tested with the MBL method, using two disks containing 30 μg of ceftazidime and one disk containing 3 mg of sodium mercaptoacetic acid (SMA) (Eiken Chemical Co. Ltd, Tokyo, Japan), indicating the production of metallo-β-lactamase. Of note, The CIM consists of two steps: (1) incubation of a meropenem disk with the isolate tested, and (2) incubation of this meropenem disk with the Escherichia coli ATCC 25922 strain. Carbapenemase activity can be detected easily after the second incubation step by the absence of an inhibition zone, which indicates enzymatic hydrolysis of meropenem during the first incubation step.
      Table 1Minimum inhibitory concentrations for the NDM-5-producing Escherichia coli
      AntimicrobialMinimum inhibitory concentration (μg/ml)
      IMP≥32
      MEM≥32
      DOR≥32
      AZM≥512
      AMX≥512
      CTX≥512
      CAZ≥512
      CFP≥512
      CRO≥512
      GEN≥256
      NAL≥512
      TET≥256
      NOR≥128
      CST1
      IMP, imipenem; MEM, meropenem; DOR, doripenem; AZM, aztreonam; AMX, amoxicillin; CTX, cefotaxime; CAZ, ceftazidime; CFP, cefoperazone; CRO, ceftriaxone; GEN, gentamicin, NAL, nalidixic acid; TET, tetracycline; NOR, norfloxacin; CST, colistin.
      Following this, PCR and DNA sequencing were used to screen for carbapenemase-encoding genes,
      • Kaase M.
      • Nordmann P.
      • Wichelhaus T.A.
      • Gatermann S.G.
      • Bonnin R.A.
      • Poirel L.
      NDM-2 carbapenemase in Acinetobacter baumannii from Egypt.
      extended-spectrum β-lactamases (ESBLs), plasmid-mediated quinolone resistance genes, integrons, and 16S rRNA methylases. PCR fragments were purified using a FastGene Gel/PCR Extraction Kit (Nippon Genetics Co., Tokyo, Japan). Both DNA strands of the PCR product were sequenced using a 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
      The results demonstrated that EC169 carried not only blaNDM-5, but also blaCTX-M-15, blaCMY-42, blaOXA-1, aac(6′)-Ib-cr, and a class 1 integron with two gene cassettes (dfrA17–aadA5). Multilocus sequence typing (MLST) was performed using seven housekeeping genes (adk, fumC, icd, purA, gyrB, recA, and mdh), according to the Escherichia coli MLST Database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). The results indicated that EC169 belongs to sequence type (ST) 5018.
      PCR mapping and DNA sequencing were used to analyze the immediate genetic environment of the blaNDM-5 gene using primers specific for the genetic environment of previously published blaNDM genes. The insertion sequence ISAba125 was identified upstream from the blaNDM-5 gene, and the bleomycin resistance gene bleMBL was identified downstream from the blaNDM-5 gene. Southern blot hybridization showed that blaNDM-5 was located on a plasmid of >93 kb. Probe labeling and membrane hybridization were performed according to the protocol of the Amersham ECL Direct Nucleic Acid Labeling and Detection System (GE Healthcare Japan, Tokyo, Japan). Transferability of the blaNDM-5-carrying plasmid was performed by conjugation experiment between the clinical donor isolate (EC169) and azide-resistant E. coli strain J53 as a recipient. The transconjugants were selected on MacConkey agar plates containing 4 μg/ml meropenem and 150 μg/ml sodium azide. Unfortunately, conjugation and mating experiments were unsuccessful after several attempts, indicating that this plasmid is unconjugable.
      PCR-based replicon typing (inc/rep PCR) was applied to determine the Inc type of the NDM-5-carrying plasmid. This plasmid was cut and purified from a low melting point agarose gel using standard agarase treatment (Nippon Gene Co., Ltd, Toyama, Japan) and used as a DNA template for inc/rep PCR. The result revealed that the NDM-5-carrying plasmid belonged to the IncI1-Iγ type.

      3. Discussion

      To date, 16 variants of NDM-type β-lactamases (NDM-1 to NDM-16) have been detected and assigned according to the gene bank available at the Lahey Clinic website (see http://www.lahey.org/studies/other.asp#table1). However, only NDM-1
      • Zafer M.M.
      • Amin M.
      • El Mahallawy H.
      • Ashoure M.S.
      • Al Agamy M.
      First report of NDM-1-producing Pseudomonas aeruginosa in Egypt.
      and NDM-2
      • Kaase M.
      • Nordmann P.
      • Wichelhaus T.A.
      • Gatermann S.G.
      • Bonnin R.A.
      • Poirel L.
      NDM-2 carbapenemase in Acinetobacter baumannii from Egypt.
      have been reported previously in Egypt. The discovery of blaNDM-5 in a clinical E. coli isolate from a patient with no history of travel beyond the Egyptian border probably suggests that blaNDM-5 is an autochthonous genetic determinant in Egypt. The overuse and/or misuse of antimicrobials in Egyptian hospitals and the community may be responsible for the development of high levels of antimicrobial resistance. Due to the poor hygiene conditions in developing countries like Egypt, the food chain and water may comprise one of the possible sources that help the spread of these resistance genes. Moreover, NDM-5 was recently reported in Escherichia coli ST1284 from a rectal swab of a domestic dog in Algeria. Also, cases of NDM-5 have arisen in the community in China without hospitalization (E. coli ST5131) or a travel history (Klebsiella pneumoniae ST14).
      The MLST analysis revealed that E. coli EC169 belongs to ST5018, which is completely unlike the ST types of NDM-5-producing E. coli detected in the UK (ST648),
      • Hornsey M.
      • Phee L.
      • Wareham D.W.
      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.
      India (ST648), Algeria (ST2659),
      • Sassi A.
      • Loucif L.
      • Gupta S.K.
      • Dekhil M.
      • Chettibi H.
      • Rolain J.M.
      NDM-5 carbapenemase-encoding gene in multidrug-resistant clinical isolates of Escherichia coli from Algeria.
      Spain (ST448), Japan (ST540), Australia (ST648), the USA (ST167), and China (ST167).
      • Yang P.
      • Xie Y.
      • Feng P.
      • Zong Z.
      blaNDM-5 carried by an IncX3 plasmid in Escherichia coli sequence type 167.
      This appears to be the first report of an ST5018 E. coli strain expressing NDM-5 β-lactamase. The genetic environment of blaNDM-5 is very similar to that previously described for most NDM-1-producing Enterobacteriaceae.
      • Hornsey M.
      • Phee L.
      • Wareham D.W.
      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.
      • Yang P.
      • Xie Y.
      • Feng P.
      • Zong Z.
      blaNDM-5 carried by an IncX3 plasmid in Escherichia coli sequence type 167.
      Other genetic determinants, such as blaCTX-M-15
      • Hornsey M.
      • Phee L.
      • Wareham D.W.
      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.
      • Yang P.
      • Xie Y.
      • Feng P.
      • Zong Z.
      blaNDM-5 carried by an IncX3 plasmid in Escherichia coli sequence type 167.
      and class 1 integron (dfrA17–aadA5),
      • Hornsey M.
      • Phee L.
      • Wareham D.W.
      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.
      have also been reported in NDM-5-producers.
      In conclusion, the worldwide dissemination of NDM-producing Gram-negative bacteria is of great concern. Medical authorities must implement antimicrobial programs and infection control policies to effectively prevent the rapid spread of these genetic determinants. The increasing bacterial drug resistance is a forceful reminder that our world is very close to the situation in the pre-antibiotic era, especially because there are no new antibiotics in the pipeline.

      Acknowledgements

      A.M.S. is supported by a fellowship from the Ministry of Education, Culture, Sports, Science and Technology of Japan. H.O.K. is supported by a doctoral fellowship from the Ministry of Higher Education, Egypt. A.M.A. is supported by a postdoctoral fellowship (number PU14012) from the Japan Society for the Promotion of Science. This work was supported by Grants-in-Aid for Scientific Research (numbers 25460532 and 26.04912) to Tadashi Shimamoto from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
      Conflict of interest: The authors have no conflicts of interest to declare.

      References

        • Zafer M.M.
        • Amin M.
        • El Mahallawy H.
        • Ashoure M.S.
        • Al Agamy M.
        First report of NDM-1-producing Pseudomonas aeruginosa in Egypt.
        Int J Infect Dis. 2014; 29: 80-81
        • Hornsey M.
        • Phee L.
        • Wareham D.W.
        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: 5952-5954
        • Sassi A.
        • Loucif L.
        • Gupta S.K.
        • Dekhil M.
        • Chettibi H.
        • Rolain J.M.
        NDM-5 carbapenemase-encoding gene in multidrug-resistant clinical isolates of Escherichia coli from Algeria.
        Antimicrob Agents Chemother. 2014; 58: 5606-5608
        • Yang P.
        • Xie Y.
        • Feng P.
        • Zong Z.
        blaNDM-5 carried by an IncX3 plasmid in Escherichia coli sequence type 167.
        Antimicrob Agents Chemother. 2014; 58: 7548-7552
        • Kaase M.
        • Nordmann P.
        • Wichelhaus T.A.
        • Gatermann S.G.
        • Bonnin R.A.
        • Poirel L.
        NDM-2 carbapenemase in Acinetobacter baumannii from Egypt.
        J Antimicrob Chemother. 2011; 66: 1260-1262