title: Underlying mechanisms of carbapenem resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a tertiary care centre in Lebanon: role of OXA-48 and NDM-1 carbapenemases.
abstract: A recent increase in carbapenem resistance among extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae and Escherichia coli isolates at a major tertiary care centre in Lebanon prompted the initiation of this study. Consecutive ESBL-producing isolates were tested for resistance to carbapenems, with initial screening by disk diffusion and Etest using ertapenem. The modified Hodge test was also performed. PCR of β-lactamase-encoding genes, including bla(NDM-1), bla(KPC), bla(OXA-48), bla(CTX-M), bla(TEM), bla(SHV), bla(CMY-2) and bla(OXA-1), as well as outer membrane porin genes (ompC and ompF) was performed. Sequencing, efflux pump inhibitor tests and random amplified polymorphic DNA (RAPD) analysis were performed. In total, 14 (2.45%) of 572 K. pneumoniae and 24 (1.07%) of 2243 E. coli were ertapenem-non-susceptible [minimum inhibitory concentration (MIC) ≥0.25 μg/mL]. Resistance to other carbapenems was variable. PCR and sequencing analysis revealed that isolates harboured different β-lactamase genes, including bla(OXA-1), bla(CTX-M-15), bla(TEM-1), bla(CMY-2), bla(OXA-48) and bla(NDM-1). In addition, K. pneumoniae lacked the outer membrane porin-encoding genes, whilst E. coli harboured them with detected mutations. CTX-M-15 was carried on a 90 kb plasmid, whilst OXA-48 was carried on a 70 kb plasmid. Efflux pump inhibition significantly decreased MICs in E. coli. RAPD analysis demonstrated genomic variability. In conclusion, carbapenem resistance in ESBL-producing K. pneumoniae and E. coli is due to the combined effect of β-lactamases with porin impermeability and/or efflux pump activity observed in these organisms, and in a number of isolates is due to the production of the carbapenemase-encoding genes bla(OXA-48) and the newly emerging bla(NDM-1).
authors.affiliation: Center for Infectious Diseases Research, Faculty of Medicine, American University of Beirut, Riad El-Solh, Beirut, Lebanon.
authors.forename: M
authors.initials: M
authors.lastname: Baroud
authors.forename: I
authors.initials: I
authors.lastname: Dandache
authors.forename: G F
authors.initials: GF
authors.lastname: Araj
authors.forename: R
authors.initials: R
authors.lastname: Wakim
authors.forename: S
authors.initials: S
authors.lastname: Kanj
authors.forename: Z
authors.initials: Z
authors.lastname: Kanafani
authors.forename: M
authors.initials: M
authors.lastname: Khairallah
authors.forename: A
authors.initials: A
authors.lastname: Sabra
authors.forename: M
authors.initials: M
authors.lastname: Shehab
authors.forename: G
authors.initials: G
authors.lastname: Dbaibo
authors.forename: G M
authors.initials: GM
authors.lastname: Matar
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015780:Carbapenems / Q000494:pharmacology*; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D007861:Lebanon; D008826:Microbial Sensitivity Tests; D010957:Plasmids; D019105:Random Amplified Polymorphic DNA Technique; D017422:Sequence Analysis, DNA; D062606:Tertiary Care Centers; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1; C108449:oxacillinase