Carbapenem-Resistant Klebsiella pneumoniae Exhibiting Clinically Undetected Colistin Heteroresistance Leads to Treatment Failure in a Murine Model of Infection

Abstract

Antibiotic resistance is a growing crisis and a grave threat to human health. It is projected that antibiotic-resistant infections will lead to 10 million annual deaths worldwide by the year 2050. Among the most significant threats are carbapenem-resistant Enterobacteriaceae (CRE), including carbapenem-resistant Klebsiella pneumoniae (CRKP), which lead to mortality rates as high as 40 to 50%. Few treatment options are available to treat CRKP, and the polymyxin antibiotic colistin is often the "last-line" therapy. However, resistance to colistin is increasing. Here, we identify multidrug-resistant, carbapenemase-positive CRKP isolates that were classified as susceptible to colistin by clinical diagnostics yet harbored a minor subpopulation of phenotypically resistant cells. Within these isolates, the resistant subpopulation became predominant after growth in the presence of colistin but returned to baseline levels after subsequent culture in antibiotic-free media. This indicates that the resistance was phenotypic, rather than due to a genetic mutation, consistent with heteroresistance. Importantly, colistin therapy was unable to rescue mice infected with the heteroresistant strains. These findings demonstrate that colistin heteroresistance may cause in vivo treatment failure during K. pneumoniae infection, threatening the use of colistin as a last-line treatment for CRKP. Furthermore, these data sound the alarm for use of caution in interpreting colistin susceptibility test results, as isolates identified as susceptible may in fact resist antibiotic therapy and lead to unexplained treatment failures.IMPORTANCE This is the first report of colistin-heteroresistant K. pneumoniae in the United States. Two distinct isolates each led to colistin treatment failure in an in vivo model of infection. The data are worrisome, especially since the colistin heteroresistance was not detected by current diagnostic tests. As these isolates were carbapenem resistant, clinicians might turn to colistin as a last-line therapy for infections caused by such strains, not knowing that they in fact harbor a resistant subpopulation of cells, potentially leading to treatment failure. Our findings warn that colistin susceptibility testing results may be unreliable due to undetected heteroresistance and highlight the need for more accurate and sensitive diagnostics.

Keywords: Klebsiella; antibiotic resistance; clonal heteroresistance; colistin; heteroresistance.

FIG 1

Carbapenem-resistant Klebsiella pneumoniae can harbor clinically undetected colistin-resistant subpopulations. (A) Colistin-susceptible isolate GA65146 and the colistin-heteroresistant isolates Mu9 and Mu156 were assayed for colistin resistance using the Etest (bioMérieux, Marcy-l'Étoile, France) method. The MIC is represented by the highest concentration along the strip at which bacteria grow. (B) Population analysis profile of GA65146, Mu9, and Mu156. The proportion of total colonies is the number of CFU able to grow at each concentration of colistin on solid medium divided by the number growing on medium without drug. Heteroresistant isolates exhibit a minor subpopulation that is able to grow on concentrations of colistin above 4 µg/ml. (C) Workflow for genomic and transcriptomic analysis of colistin-susceptible and -resistant subpopulations. Cultures of Mu9 or Mu156 were grown for 18 h in MH broth with or without colistin as indicated. (D and E) Quantitative real-time PCR (qRT-PCR) analysis of mgrB (D) and phoP (E) expression in resistant and susceptible subpopulations of Mu9 and Mu156. Resistant and susceptible subpopulations were enriched as shown in panel C. Relative abundance was calculated by normalizing the expression of each gene to the average expression of two housekeeping genes, 23S and rpsL (n = 6). *, P < 0.05; **, P < 0.01; n.s., not significantly different (unpaired t test).

FIG 2

K. pneumoniae isolates lead to in vivo colistin treatment failure. (A) Mice were infected intraperitoneally with 3 × 10⁸ CFU of Mu9 or Mu156, treated with phosphate-buffered saline (PBS) or colistin (20 mg/kg colistin methanesulfonate) at 12 and 18 h, and then sacrificed at 24 h. Peritoneal lavage fluid was collected and plated onto drug-free medium and medium containing 16 µg/ml colistin to assess percentages of colistin-resistant cells of the heteroresistant strains. The preinfection inoculum (input) was plated similarly (n = 5). *, P < 0.05 (Mann-Whitney test). (B to D) Mice were infected with the colistin-susceptible isolate GA65146 (B) or the colistin-heteroresistant isolate Mu9 (C) or Mu156 (D) and then treated with 20 mg/kg colistin methanesulfonate every 6 h starting at 12 h. Mice were monitored for survival and weight loss and were sacrificed if their weight fell below 80% of their starting weight (n = 5). *, P < 0.05 (Gehan-Breslow-Wilcoxon test).