·Ciprofloxacin heteroresistance in 227 clinical PA strains was initially identified by disk diffusion. Meanwhile, susceptibilities of these strains was classified according to the diameter of the inhibition zone. The stability of the identified heteroresistant strains was tested by measuring the sensitivity after serial passage under antibiotic-free conditions. Mechanisms mediating heteroresistance were analyzed by whole-genome sequencing. Susceptibilities of resistant subpopulations to different antibiotics were also detected.

·Based on disk diffusion, 142 (62.6%), 26 (11.5%), and 59 (26.0%) of 227 isolates were classified as susceptible, intermediate and resistant to ciprofloxacin respectively. Eighteen putative heteroresistant strains were detected through primary disk diffusion selection, and 11 among them were further verified as stable heteroresistant strains by population analysis profiling (PAP). By whole-genome sequencing, 8 and 2 among 11 resistant subpopulations carried mutations in mexS and mutations in gyrA individually. In addition, mutations in fleQ, PA2632 or PAKAF_02255 were detected respectively. Both mexS and gyrA mutants led to moxifloxacin resistance, and some mexS mutants also showed resistance to chloramphenicol and imipenem.

·Ciprofloxacin heteroresistance rate inPA is 4.8% (11/227). As a screening method for heteroresistance of PA, the disk diffusion has low sensitivity and has a certain false negative rate. The mutation mexS is the predominant mutation type in resistant subpopulations and it might enhance the overexpression of efflux pump MexEF-OprN, thus mediating resistance to different antibiotics including ciprofloxacin.