Genetic variants of AcrAB and OqxAB and the impact of efflux pump inhibition on ciprofloxacin and amikacin resistance in clinical isolates of Klebsiella pneumoniae from Lima, Peru

Background: Klebsiella pneumoniae is a World Health Organization priority pathogen because of its rapidly accumulating resistance to multiple drug classes. Over-expression of the AcrAB and OqxAB Resistance-Nodulation-Division (RND) efflux pumps is a key mechanism, yet genomic data on these systems in Peruvian strains are scarce. Methods: Sixteen non-duplicate K. pneumoniae isolates obtained from hospitalised patients in Lima (2019–2020) were tested for ciprofloxacin and amikacin susceptibility by broth microdilution, with and without the efflux-pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and phenylalanine-arginine β-naphthylamide (PAβN). AcrA, AcrB, OqxA and OqxB were detected by PCR, and the resulting amplicons were sequenced on the Oxford Nanopore platform. Reads were analysed with Epi2me (Amplicon workflow); single nucleotide polymorphisms (SNPs) were identified by aligning to reference sequences in MEGA, and all sequences were deposited in GenBank. Results: Resistance to ciprofloxacin and amikacin was evaluated in all isolates; 93.8% were resistant to ciprofloxacin and 18.8% to amikacin. PAβN produced a ≥ 4-fold MIC reduction in 18.8% (ciprofloxacin) and 100% (amikacin) of resistant strains; CCCP yielded a ≥ 3-fold decrease in 75% of ciprofloxacin resistant isolates. AcrB and AcrA were detected in 56.3% and 43.8% of isolates, while OqxB and OqxA occurred in 25.0% and 31.3%, respectively. Nanopore sequencing con-firmed gene identity and revealed 12 SNPs detected, OqxB c.56 G > T (V19F) and c.356 C > A (P119H), which may modulate efflux efficiency. Conclusion: Efflux-mediated resistance to ciprofloxacin and amikacin in clinical isolates of K. pneumoniae is underpinned by genetic variants in the AcrAB and OqxAB pumps. The incorporation of long-read amplicon sequencing exposes SNP-level variation missed by conventional PCR studies and underscores the value of routine genomic surveillance. The MIC reversals observed with PAβN and CCCP against these two antibiotics highlight efflux pump inhibition as a promising complementary strategy against resistant K. pneumoniae.