Impact of quinolone-resistance acquisition on biofilm production and fitness in Salmonella enterica

Abstract

Objectives: To investigate the potential relationship between quinolone resistance and biofilm production in a collection of Salmonella enterica clinical isolates and in S. enterica serovar Typhimurium serial mutants with increasing resistance to ciprofloxacin.

Methods: Nalidixic acid susceptibility and biofilm formation were assessed in a collection of 122 S. enterica clinical isolates. An in vitro quinolone-resistant mutant, 59-64, was obtained from a biofilm-producing and quinolone-susceptible clinical isolate, 59-wt, in a multistep selection process after increasing ciprofloxacin concentrations. The quinolone resistance mechanisms [target gene and multidrug resistance (MDR) regulatory mutations, MICs of several antibiotics, cell envelope protein analysis, real-time PCR and ciprofloxacin accumulation] were characterized for mutant strains. In addition, analysis of fitness, biofilm formation, rdar morphotype and expression of biofilm-related genes by real-time PCR were also determined.

Results: Nalidixic acid-susceptible S. enterica strains were more prevalent in producing biofilm than the resistant counterparts. Strain 59-64 acquired five target gene mutations and showed an MDR phenotype. AcrAB and acrF overexpression were ruled out, whereas TolC did show increased expression in 59-64, which, in addition, accumulated less ciprofloxacin. Consistently, increased ramA expression was seen in 59-64 and attributed to a mutation within its promoter. Reduced biofilm production related to diminished csgB expression as well as reduced fitness was seen for 59-64, which was unable to form the rdar morphotype.

Conclusions: Quinolone resistance acquisition may be associated with decreased production of biofilm due to lower csgB expression. Efflux, biofilm production and fitness seem to be interrelated.

Keywords: Salmonella Typhimurium; csgB; efflux; ramA; rdar morphotype.