In Vitro Emergence of Carbapenem Resistance in Extended-spectrum Β- Lactamase-producing Klebsiella Pneumoniae Clinical Isolates

This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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-metallo-carbapenemases 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 [6-7], 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 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 β-lactamases were phenotypically detected using inhibitor-based assays with cefoxitin discs …

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 [6][7], 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].
Plates were incubated aerobically at 35°C for 48-72 hours.The mutation frequency was calculated as the number of resistant colonies per inoculum.For each isolate, a representative mutant that was stable after three subcultures was conserved for further susceptibility testing.
Clinical K. pneumoniae isolates (n = 35) were subjected to antibiotic testing.All the clinical strains were susceptible to ertapenem, meropenem, and imipenem, according to their CLSI breakpoints.MHTs were negative, indicating the lack of carbapenemase production.Three strains were found to be resistant to cefoxitin by disc diffusion tests, but were negative in synergism tests with boronic acid, indicating the absence of AmpC β-lactamases.All the isolates displayed synergistic activity between third-generation cephalosporins and clavulanic acid, and thus were phenotypically characterized as ESBL producers.The thirty-five K. pneumoniae strains were tested for incidence of single-step mutation by exposure to 0.5 µg/ml ertapenem, meropenem, or imipenem.Singlestep mutants were isolated from 16 strains (45.7%) exposed to ertapenem and 6 strains (17.1%) exposed to meropenem, but no mutants were selected with imipenem exposure.The single-step mutation frequencies were 6.7×10 -7 -1.6×10 -9 with ertapenem and 5.4×10 -7 -1.9×10 -10 with meropenem.One representative stable mutant derived from each isolate was selected for further testing.The characteristics of the ertapenem-selected mutants are displayed in Table 1.When compared with parental strains, the MICs for the ertapenem-selected mutants increased 5.3-fold (LH8) to 500-fold (LH16), 2.7-fold (LH4) to 125-fold (LH3) for meropenem, and no increase (LH11) to 16fold (LH12) for imipenem.Cross-resistance (MIC >1 µg/ml) was observed to both meropenem and imipenem in four strains (LH3 EM, LH6 EM, LH9 EM, and LH12 EM) and to meropenem alone in two strains (LH14 EM and LH16 EM).
The characteristics of the meropenem-selected mutants are displayed in Table 2.When compared with parental strains, the MICs for the meropenemselected mutants increased 64-fold (LH6) to 375-fold (LH7 and LH16) for ertapenem, 16-fold (LH9) to 64fold (LH8) for meropenem, and no increase (LH7) to 8-fold (LH8) for imipenem.Cross-resistance to ertapenem was detected in all mutants and to imipenem in two mutants (LH8 MM and LH9 MM).
Mutant K. pneumoniae isolates that were resistant to carbapenem antibiotics were generated in this study, demonstrating that resistance against ertapenem and meropenem can emerge in clinical ESBL-producing K. pneumonia isolates.In addition, several of the mutants selected with ertapenem or meropenem displayed cross-resistance to imipenem.Previous research indicates that ESBL expression in combination with the loss of porin expression can reduce susceptibility to carbapenems in clinical K. pneumoniae and E. coli isolates [10][11][12].Whilst numerous outbreaks of carbapenem-resistant K. pneumoniae possessing various carbapenemases have been documented; an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant was only recently described [13][14].
The mutants selected as a result of exposure to ertapenem or meropenem exhibited dramatically increased MICs when challenged with either of the two antibiotics, indicating that the mechanisms of uptake are likely to be similar.No mutants were selected when imipenem was used, but some of the ertapenem-and meropenem-selected mutants nevertheless displayed cross-resistance to imipenem.Despite this observation, most of the mutants remained susceptible to imipenem.This suggests that porin loss is more significant for ertapenem resistance and meroperem resistance than for imipenem resistance [15].The current study has some limitations.For example, the selection was performed using a stable carbapenem concentration (0.5 µg/ml) and not related to carbapenem MICs.It was not possible to perform molecular characterization of the relevant bacterially expressed enzymes, to detect isolates expressing multiple enzymes, or to assess outer membrane permeability patterns.Future studies may allow the resistance mechanisms to be elucidated.
It is important to note that single-step mutants showing resistance to ertapenem and meropenem were selected in vitro with relative ease .This is of clinical importance because such resistance could inadvertently make subsequent therapy using imipenem less effective or even ineffective.
This variety of in vitro resistant mutants may reflect the growing number of studies describing treatment failure with ertapenem [4][5] and meropenem [6][7].Additionally, nosocomial outbreaks by carbapenem-resistant strains were recently documented in which resistance was not mediated by carbapenemases [13][14].In conclusion, this study demonstrates the rapid acquisition of decreased carbapenem susceptibility in ESBL-producing K. pneumoniae clinical isolates.The use of ertapenem in high-inoculum infections or in undrained infection foci should therefore be monitored to reduce the risk of resistance selection.

Table 1 .
Minimal inhibitory concentrations of ertapenem, meropenem, and imipenem against K pneumoniae isolates and the corresponding mutants selected with ertapenem.

Table 2 .
Minimal inhibitory concentrations of ertapenem, meropenem, and imipenem against K pneumoniae isolates and the corresponding mutants selected with meropenem P: parent strain, MM: mutant selected with meropenem