The effects of sub-MIC ciprofloxacin exposure on antibiotic susceptibility and virulence factors in Pseudomonas aeruginosa ATCC 9027

Abstract

Exposure to antibiotics below their minimum inhibitory concentration (sub-MIC) is known to be a selection pressure for the development of antibiotic resistance. In this work, we aimed to provide more understanding of how antibiotic resistance and cross-resistance developed in Pseudomonas aeruginosa under sub-MIC exposure to Ciprofloxacin. Fully susceptible P. aeruginosa ATCC 9027 was serially exposed to sub-MIC level of ciprofloxacin for 14 days to generate E1 strain, then cultured in antibiotic-free environment for 10 days to obtain E2 strain. Changes in MIC values, susceptibility profile and virulence (protease, elastin, biofilm formation, motility) of these strains were evaluated compared to the original P. aeruginosa ATCC 9027. After antibiotic exposure, P. aeruginosa strains developed resistance to the ciprofloxacin with the MIC values increased by 32 times, and the exposed strains only developed resistance to ciprofloxacin and other fluoroquinolones but not to unrelated antibiotics. The resistant phenotype did not revert fully after 10 days in antibiotic-free environment. In addition, there was noticeable changes in the pathogen’s virulence factors during antibiotic exposure. In summary, sub-MICs exposure of P. aeruginosa provided positive selection for antibiotic resistant phenotype, which did not revert after 10 days in antibiotic- free environment. Exposure to sub-MIC level of antibiotics caused significant changes in virulence and antibiotic susceptibility of the pathogen.

Genomic alterations involved in fluoroquinolone resistance development in Staphylococcus aureus

“…It should be well noted that this cross-resistance development under CIP exposure was well observed in Gram-positive bacteria such as Actinobacillus pleuropneumoniae [ 51 ], S . aureus [ 52 ], Mycobacterium tuberculosis [ 53 ]; but not in Gram-negative, ones like Salmonella enterica [ 54 ]; Pseudomonas aeruginosa [ 55 ].…”

“…The results suggested that serial FQ exposure resulted in cross-resistance to unrelated antibiotics and the emergence of multidrug-resistant phenotypes in S. aureus. It should be well noted that this cross-resistance development under CIP exposure was well observed in Grampositive bacteria such as Actinobacillus pleuropneumoniae [51], S. aureus [52], Mycobacterium tuberculosis [53]; but not in Gram-negative, ones like Salmonella enterica [54]; Pseudomonas aeruginosa [55].…”

Adaptive response of Pseudomonas aeruginosa under serial ciprofloxacin exposure

“…The length of 10 days was selected as, at day 10, in most exposure replications, the bacterium maintained its MIC for at least three consecutive days. The antibiotic susceptibility profile determined via an agar- diffusion assay and virulence of the strains used in this study were reported and are provided in Tables S1, S2 and S3 [11]. All bacteria strains were stored at −80 °C in Tryptic Soya broth (TSB; HiMedia) with 30 % glycerol (TSB/glycerol 7 : 3, v/v).…”

“…Previous work in in vitro -induced ciprofloxacin resistance has found that after 14 days of exposure to ciprofloxacin, P. aeruginosa strains developed resistance to ciprofloxacin with MIC values increased 32-fold [11]. The resistant phenotype and its altered virulence, including protease activity, biofilm formation and motility, did not revert fully after the exposed strain was cultured for 10 days in an antibiotic-free environment [11].…”

“…virulence, including protease activity, biofilm formation and motility, did not revert fully after the exposed strain was cultured for 10 days in an antibiotic-free environment [11].…”