Colistin heteroresistance in carbapenemase-producing Klebsiella pneumoniae.

Sir, Klebsiella pneumoniae is an important pathogen that causes severe and life-threatening infections. Most isolates demonstrate resistance to various antimicrobial agents. Carbapenems have been widely used to treat serious infections with multidrug-resistant K. pneumoniae. However, the increasing use of these compounds has led to the emergence of carbapenem-resistant isolates via acquired genes encoding carbapenem-hydrolysing enzymes that inactivate carbapenems. Colistin was among the last-resort therapy for treating infections caused by these pathogens. In the present study we report on colistin heteroresistance in carbapenemaseproducing apparently colistin-susceptible K. pneumoniae isolates recovered from patients with and without prior exposure to this drug. A total of 20 K. pneumoniae clinical isolates harbouring carbapenem-hydrolysing enzymes that were classified as colistin-susceptible by routine susceptibility testing were consecutively collected between March and August 2009 from separate patients with nosocomial infections hospitalized in several wards of our hospital. Eight isolates were obtained from patients who had a previous treatment with colistin for more than 3 days, whereas 12 isolates were from patients who did not receive colistin. The identification of isolates and initial susceptibility testing were performed by the VITEK 2 automated system (bioMérieux, Marcy l’Étoile, France). Phenotypic and molecular techniques demonstrated that 13 isolates were KPC producers whereas 7 were VIM producers. The MICs of colistin were determined by the broth microdilution method according to CLSI recommendations and interpreted using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for Enterobacteriaceae (susceptible≤2 mg/L; resistant.2 mg/L). K. pneumoniae isolates were assessed for heteroresistance by spreading a 50 mL aliquot from a 24 h culture serially diluted in saline to a turbidity equivalent to that of a 0.4–0.5 McFarland standard (approximately 10 cfu/mL) on Mueller– Hinton agar plates containing 0, 0.5, 1, 2, 3, 4, 5, 6 and 8 mg/ L colistin sulphate. The plates were incubated at 358C for 48 h and colonies were counted. The frequency of resistant subpopulations at the highest drug concentration was calculated by dividing the number of colonies grown on an antibioticcontaining plate by the colony counts from the same bacterial inoculum plated onto antibiotic-free plates. The MIC for isolates grown at the highest colistin concentration was reassessed after serial daily subcultures on antibiotic-free medium for 2 weeks in order to evaluate whether this resistance was stable. Macrorestriction profiles of K. pneumoniae isolates were investigated by PFGE. Sixteen of the study strains were colistin susceptible (MIC≤2 mg/L) while four strains (4, 5, 7 and 13) were shown to be resistant by broth microdilution (colistin MIC 4 mg/L) and were not studied further for heteroresistance. Results of population analysis profiles for colistin are reported in Table 1. Four strains (11, 14, 15 and 16), derived from patients without colistin treatment, did not exhibit any heteroresistance (no growth in 2 mg/L colistin in population analyses). In population analyses, 12 strains yielded heteroresistant subpopulations that grew in up to 8 mg/L colistin. The proportion of these subpopulations was 3.5×10 to 4.4×10 for isolates exposed to colistin and 1.5×10 to 3.2×10 for isolates not exposed to colistin. When heteroresistant colonies obtained from population analyses were retested for colistin MICs after daily passages on colistin-free medium, all strains demonstrated MICs.8 mg/L except one (strain 8, Table 1). The MIC for this strain, which was recovered from a patient who had previously been treated with colistin, initially increased from 1 mg/L to 4 mg/L, however, after subculture of the resistant colonies onto antibiotic-free medium, its MIC decreased to the susceptible range (1 mg/L). PFGE showed heterogeneity among the study strains that were classified into five distinct clones; two clones among VIM-producing isolates, two clones among KPC-producing isolates and one clone that comprised two KPC producers and one VIM producer (Table 1). Colistin resistance among carbapenemase-producing K. pneumoniae is increasing in Greece, and stable heteroresistance might play a role in resistance development. In fact, in our hospital, resistance rates rose from 0% in 2007 to 8.13% in 2008 and to 24.3% in 2009. Colistinand carbapenem-resistant isolates have been involved in hospital outbreaks with high mortality rates. Furthermore, the increasing reports of colistin-susceptible isolates harbouring resistant subpopulations are of great concern. Commercial automated systems may fail to detect most heteroresistant isolates, and Etest results depend on the medium used. As this species constitutes the majority of carbapenemaseproducing Enterobacteriaceae isolated from clinical specimens worldwide, it is very important to develop a reliable and easy-to-use susceptibility testing method to detect resistant subpopulations. This study was based on relatively few strains, therefore the clinical significance of heteroresistance remains unclear. Although the selection of resistant isolates during colistin treatment could not be excluded, further experiments with additional strains are required to determine the potential role of colistinresistant subpopulations in therapeutic failures. Research letters