In vitro emergence of carbapenem resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae clinical isolates

Klebsiella pneumoniae is thought to be the most common species producing ESBLs, and almost 50% of Klebsiella pneumoniae isolates produce ESBL in some countries [1]. Furthermore, antimicrobial co-resistance within ESBL-producing isolate communities limits the number of drugs that are effective against these strains, leaving carbapenems as the most reliable agents [2-3]. Carbapenem resistance can arise through the production of acquired metallo-β-lactamases such as VIM and IMP or from production of non-metallocarbapenemases of the IMI/NMC, SME, OXA, or KPC families. Resistance may also be due to a combination of impermeability caused by porin loss and ESBL or AmpC β-lactamase production. This impermeability was documented in several cases in which carbapenem-resistant K. pneumoniae emerged in vivo in response to ertapenem [4-5], meropenem [67], and less frequently to imipenem exposure [8]. Among the several factors that contribute to the appearance and spread of acquired antibiotic resistance, the selection of high-level resistant mutants is especially important. In the current study, we examined a collection of ESBL-producing K. pneumoniae with the aim of evaluating the ability of carbapenem exposure to select single-step resistant mutants. Clinical K. pneumoniae isolates (n = 35) were obtained from inpatients admitted to the Sanatorio San Lucas, Buenos Aires, Argentina (n = 18) and adult male outpatients (n = 17) who attended the Laboratorio Hidalgo, Buenos Aires, Argentina. The API 20 E system (bioMerieux, Marcy L’Étoile, France) was used for biochemical identification of all strains. Antibiotic minimal inhibitory concentrations (MICs) were determined by the epsilometric test (Etest; bioMerieux) and data was interpreted in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [9]. Susceptibility to cefoxitin was determined by disc diffusion. For phenotypic detection of ESBL, an overnight culture suspension of the test isolate, adjusted to 0.5 McFarland’s standard, was inoculated onto the surface of a Mueller-Hinton agar plate. Cefotaxime (30 μg) and cefotaxime-clavulanic acid (30 μg/10 μg) discs were placed 20 mm apart on the agar. Similarly, ceftazidime (30 μg) and ceftazidime-clavulanic acid (30 μg/10 μg) discs were also placed 20 mm apart. An increase of ≥ 5 mm in the zone diameter for an antimicrobial agent tested in combination with clavulanic acid versus the zone when tested alone was considered positive for ESBL production. AmpC βlactamases were phenotypically detected using inhibitor-based assays with cefoxitin discs (30 μg) and boronic acid (300 μg) (Laboratorios Britania, Buenos Aires, Argentina). The modified Hodge test (MHT) was performed as described previously, with a 10-μg imipenem disk [9]. The frequency of spontaneous single-step mutation was determined by spreading cultures (~10 7 –10 9