DivergentIn vitroMIC Characteristics and underlying isogenic mutations in host-specializedPseudomonas aeruginosa

Abstract

Clinical isolates of Pseudomonas aeruginosa (Pa) from patients with cystic fibrosis (CF) are known to differ from those associated with infections of non-CF hosts in colony morphology, drug susceptibility patterns, and genomic hypermutability. Although Pa isolates from CF have long been recognized for their overall higher resistance rate calculated generally by reduced “percent susceptible”, this study takes the approach to compare and contrast Etest MIC distributions between two distinct cohorts of clinical strains (n=224 from 56 CF patients and n=130 from 68 non-CF patients respectively) isolated in 2013. Logarithmic transformed MIC (logMIC) values of 11 antimicrobial agents were compared between the two groups. CF isolates tended to produce heterogeneous and widely dispersed MICs compared to non-CF isolates. By applying a test for equality of variances, we were able to confirm that the MICs generated from CF isolates against 9 out of the 11 agents were significantly more dispersed than those from non-CF (p<0.02-<0.001). Quantile-quantiles plots indicated little agreement between the two cohorts of isolates. Based on whole genome sequencing of 19 representative CF Pa isolates, divergent gain- or loss-of-function mutations in efflux and porin genes and their regulators between isogenic or intra-clonal associates were evident. Not one, not a few, but the net effect all adaptive mutational changes in the genomes of CF Pa, both shared and unshared between isogenic strains, are responsible for the divergent heteroresistance patterns. Moreover, the isogenic variations are suggestive of a bacterial syntrophic lifestyle when “lockedȍ inside a host focal airway environment over prolonged periods.Significance statementBacterial heteroresistance is associated with niche specialized organisms interacting with host species for prolonged period of time, medically characterized by “chronic focal infections”. A prime example is found in Pseudomonas aeruginosa isogenic/non-homogeneous isolates from patient airways with cystic fibrosis. The development of pseudomonal polarizing MICs in vitro to many actively used antimicrobial agents among isogenic isolates and “Eagle-type” heteroresistance patterns are common and characteristic. Widespread isogenic gene lesions were evident for defects in drug transporters, DNA mismatch repair, and many other structural or cellular functions—a result of pseudomonal symbiotic response to host selection. Co-isolation of extremely susceptible and resistant isogenic Pa strains suggests intra-airway evolution of a multicellular syntrophic bacterial lifestyle, which has laboratory interpretation and clinical treatment implications.