Role of the rapA Gene in Controlling Antibiotic Resistance of Escherichia coli Biofilms

Abstract

By using a high-throughput screening method, a mutant of a uropathogenic Escherichia coli strain affected in the rapA gene was isolated. The mutant formed normal-architecture biofilms but showed decreased penicillin G resistance, although the mutation did not affect planktonic cell resistance. Transcriptome analysis showed that 22 genes were down-regulated in the mutant biofilm. One of these genes was yhcQ, which encodes a putative multidrug resistance pump. Mutants with mutations in this gene also formed biofilms with decreased resistance, although the effect was less pronounced than that of the rapA mutation. Thus, an additional mechanism(s) controlled by a rapA-regulated gene(s) was involved in wild-type biofilm resistance. The search for this mechanism was guided by the fact that another down-regulated gene in rapA biofilms, yeeZ, is suspected to be involved in extra cell wall-related functions. A comparison of the biofilm matrix of the wild-type and rapA strains revealed decreased polysaccharide quantities and coverage in the mutant biofilms. Furthermore, the (fluorescent) functional penicillin G homologue Bocillin FL penetrated the mutant biofilms more readily. The results strongly suggest a dual mechanism for the wild-type biofilm penicillin G resistance, retarded penetration, and effective efflux. The results of studies with an E. coli K-12 strain pointed to the same conclusion. Since efflux and penetration can be general resistance mechanisms, tests were conducted with other antibiotics. The rapA biofilm was also more sensitive to norfloxacin, chloramphenicol, and gentamicin.Bacterial biofilms are differentiated communities of sessile cells. Infections involving biofilms play a major role in such diverse medical conditions as endocarditis (24), cystic fibrosis (29), otitis media (17), and urinary tract infections (3), presenting a significant threat to human health (12, 16). Although this sessile mode of growth has long been known, the fact that it constitutes a normal part of the bacterial life cycle has been recognized only relatively recently. The medical importance of biofilms lies in the fact that in comparison to their free-living (planktonic) counterparts, biofilms possess increased resistance to antimicrobial agents (12, 16), and consequently, diseases in which biofilms have a dominant role tend to be chronic and difficult to eradicate.The focus of this study was uropathogenic Escherichia coli (UPEC), which is primarily responsible for urinary tract infections. These affect almost 1 billion women each year. UPEC biofilms can form on the epithelium of the bladder and upper urinary tract (28), as well as intracellularly, evading host defenses (1, 2) and resulting in a disease that is often chronic.Several genes play a role in biofilm formation (9,11,20), but whether the same genes are involved in the development of biofilm resistance has been relatively little studied. Mah et al. (21) identified nvdB gene mutants whose antibiotic resistance was not affected while they were in the planktonic stat...

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…In a uropathogenic E. coli strain, inactivation of the rapA gene, encoding a helicase-like protein, does not alter biofilm formation but increases susceptibility to penicillin G in biofilm cells. In biofilm cells of the rapA mutant, the expression levels of 22 genes are reduced, including those encoding a putative MDR pump (YhcQ), a putative carbohydrate transport and metabolism protein (YeeZ), and a transcriptional regulator (SdiA) (898). B. fragilis cells show elevated RND pump expression upon induction by bile salts and also increased possibility of biofilm formation (899).…”

Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics

“…High-thoughput screening of E. coli mutants showed that rapA mutants displayed decreased resistance toward penicillin, norfloxacin, chloramphenicol, and gentamicin (83). It was demonstrated that rapA not only regulates yhcQ, a gene encoding a putative multidrug resistance (MDR) pump, but also yeeZ, a gene suspected to be involved in ECM production (83).…”

“…It was demonstrated that rapA not only regulates yhcQ, a gene encoding a putative multidrug resistance (MDR) pump, but also yeeZ, a gene suspected to be involved in ECM production (83). Thus, a dual rapA-mediated action was proposed, with efflux through a pump and reduced penetration through an increase in polysaccharide production (83).…”

Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics

“…However, a recent study suggested that MexAB-OprM and MexCD-OprJ are involved in biofilm resistance to the macrolide azithromycin (14). Furthermore, a putative multidrug efflux pump, YhcQ, has been implicated in Escherichia coli biofilm antibiotic resistance (23).…”