IS 5 Element Integration, a Novel Mechanism for Rapid In Vivo Emergence of Tigecycline Nonsusceptibility in Klebsiella pneumoniae

Abstract

dTigecycline nonsusceptibility is concerning because tigecycline is increasingly relied upon to treat carbapenem-or colistin-resistant organisms. In Enterobacteriaceae, tigecycline nonsusceptibility is mediated by the AcrAB-TolC efflux pump, among others, and pump activity is often a downstream effect of mutations in their transcriptional regulators, cognate repressor genes, or noncoding regions, as demonstrated in Enterobacteriaceae and Acinetobacter isolates. Here, we report the emergence of tigecycline nonsusceptibility in a longitudinal series of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Klebsiella pneumoniae isolates collected during tigecycline therapy and the elucidation of its resistance mechanisms. Clinical isolates were recovered prior to and during tigecycline therapy of a 2.5-month-old Honduran neonate. Antimicrobial susceptibility tests to tigecycline determined that the MIC increased from 1 to 4 g/ml prior to the completion of tigecycline therapy. Unlike other studies, we did not find increased expression of ramA, ramR, oqxA, acrB, marA, or rarA genes by reverse transcription-quantitative PCR (qRT-PCR). Whole-genome sequencing revealed an IS5 insertion element in nonsusceptible isolates 85 bp upstream of a putative efflux pump operon, here named kpgABC, previously unknown to be involved in resistance. Introduction of the kpgABC genes in a non-kpgABC background increased the MIC of tigecycline 4-fold and is independent of a functional AcrABTolC pump. This is the first report to propose a function for kpgABC and identify an insertion element whose presence correlated with the in vivo development of tigecycline nonsusceptibility in K. pneumoniae.

Risk Factors, Outcomes, and Mechanisms of Tigecycline-Nonsusceptible Klebsiella pneumoniae Bacteremia

“…The relative expression of each target gene was then calibrated against the corresponding expression of a tigecycline-susceptible K. pneumoniae KP478, which served as reference strain (expression ϭ 1; tigecycline MIC, 0.25 mg/liter). Specific primers for the aforementioned genes are listed in Table S1 in the supplemental material (20)(21)(22). The experiments were performed in triplicate.…”

Emergence and characterization of tigecycline resistance in multidrug-resistant Klebsiella pneumoniae isolates from blood samples of patients in intensive care units in northern China

“…Moreover, recently showed that rarA upregulates, whereas oqxR downregulates, the expression of the OqxAB efflux pump system, which may also be a determining factor for tigecycline resistance. In addition, Nielsen et al (2014) found an IS5 insertion element in tigecycline-non-susceptible strains and named it the KpgABC efflux pump. Overexpression of kpgABC genes in non-kpgABC K. pneumoniae increased the MIC of tigecycline fourfold, independently of the AcrAB and OqxAB efflux pumps that contribute to tigecycline resistance (Nielsen et al, 2014).…”

“…These results implied that other genetic regulative mechanisms of upregulated efflux pumps might be involved in regulation of gene expression and response to the bacteria exposed to antibiotics (Howden et al, 2013). Nielsen et al (2014) postulated that insertion of the IS5 integron might contribute to the increased expression levels AcrB fold expression change…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…In addition to MDR mediated by the chromosomally encoded OqxAB pump (302)(303)(304), overexpression of the newly identified RND-type KpgABC pump was involved in tigecycline nonsusceptibility due to an IS element insertion in the promoter region of kpgABC (305). The KexD RND pump expressed by cloning provides MDR with the requirement of AcrA accessory and KocC OM proteins (306).…”