Combined effect of Polymyxin B and Tigecycline to overcome Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae

Abstract

We assessed the prevalence of polymyxin B (PMB)- and tigecycline (TGC)-heteroresistant Klebsiella pneumoniae isolates and investigated the combined effect of PMB and TGC against dual-heteroresistant K. pneumoniae. Ninety-five nonduplicated carbapenem-resistant K. pneumoniae (CRKP) clinical isolates were collected from a tertiary-care teaching hospital in China. PCR was used to detect the resistant genes among the CRKP isolates. Population analysis profiling (PAP) was carried out to evaluate the existence of heteroresistance. A time-kill assay of PMB combined with TGC was conducted against heteroresistant K. pneumoniae strains. Real-time PCR was performed to determine the <i>pmrA</i>, <i>phoP</i>, and <i>acrB</i> expression levels. Among them, 74 isolates (77.9%) were susceptible to TGC, and 90 isolates (94.7%) were susceptible to PMB. In addition, of the TGC-susceptible isolates, 49 strains (66.2%) exhibited heteroresistant phenotypes. All of the PMB-susceptible isolates showed heteroresistant phenotypes. Forty-six isolates (48.4%) were heteroresistant to both TGC and PMB. All of the isolates carried the <i>bla</i>_KPC gene, and one strain carried both <i>bla</i>_KPC and <i>bla</i>_NDM genes. The time-kill assay revealed in four isolates that early bactericidal activity could be triggered by the combination of PMB and TGC, and there was no regrowth, even at a relatively lower concentration (0.125 mg/liter PMB with 1 mg/liter TGC). Upregulated expression of <i>pmrA</i>, <i>phoP</i>, and <i>acrB</i> indicated that heteroresistance could be related to two-component systems and the AcrAB-TolC efflux pump. The combination of PMB and TGC may be a treatment strategy for those infected with CRKP heteroresistant to PMB and/or TGC. <b>IMPORTANCE</b> Tigecycline and colistin are two of the last treatment options remaining for carbapenem-resistant <i>Enterobacteriaceae</i>. Unfortunately, tigecycline resistance and colistin heteroresistance are also increasing rapidly. In the current study, we identified a high prevalence of heteroresistance to both PMB and TGC among clinical isolates of carbapenem-resistant K. pneumoniae (CRKP). The resistant subpopulations could survive pressure from TGC or PMB but were killed by the combination at a relatively low dose. It is proposed that the combination of PMB and TGC may be a treatment strategy for patients who are infected with CRKP heteroresistant to PMB or TGC.