An evaluation of the potential of the multiple antibiotic resistance operon (mar) and the multidrug efflux pump acrAB to moderate resistance towards ciprofloxacin in Escherichia coli biofilms

The chromosomal multiple antibiotic resistance operon, mar, is widely repre sented amongst Gram-negative bacteria and has been implicated in resistance towards oxidative stress agents, organic solvents and a large number of st ructurally unrelated antimicrobial agents. The major mechanism associated w ith such increased resistance is an upregulation of the efflux pump acrAB. Growth as a biofilm is often associated with similar generalized reductions in susceptibility to inimical agents. Escherichia coli K12 (AG100), an iso genic mutant of AG100 constitutive for mar expression (AG102) and an isolat e deleted of the mar locus (MCH164) were grown as biofilms in cellulose-fib re depth filters and perfused with a simple salts, minimal medium (CDM) ove r 120 h. Biofilms were exposed to various concentrations of ciprofloxacin ( 0.004, 0.015 and 0.1 mg/L) for 42 h. The numbers of viable cells within the perfusate and within the biofilm were estimated throughout. Whereas no dif ferences were seen between the wild-type and mar-deleted isolates, that con stitutive for mar displayed reduced susceptibility to ciprofloxacin at conc entrations of 0.004 mg/L (MIC for AG100 was 0.0052 mg/L). Similar antibioti c perfusion experiments were conducted using isolates in which the efflux p ump acrAB was either deleted (AG100-A) or constitutively expressed (AG100-B ). Exposure of AG100-A biofilms to ciprofloxacin at 0.004 and 0.1 mg/L show ed similar susceptibilities to those seen in the wildtype (AG100) and mar-d eleted (MCH164) isolates and suggested that acrAB was not induced within th e attached population. On the other hand, constitutive expression of acrAB (AG100-B) protected biofilms against the lower concentration of ciprofloxac in used (0.004 mg/L). This protection was again lost at concentrations of 0 .1 mg/L. Overall, these results show that ciprofloxacin resistance in biofi lms is not mediated by the upregulation of the mar or acrAB operons.