Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen. In compromised human, it can cause infections in urinary tract, wound and respiratory tract such as pneumonia and progressive lung diseases. Particularly the risk groups are cystic fibrosis individuals, bum victims, cancer patients and patients requiring long stays in intensive care units. P. aeruginosa can cause a number of infections in animals, and plants, vegetables and fruits. Diseases of economical importance include hemorrhagic pneumonia in mink and mastitis in cattle.

Although many vaccine candidates against P. aeruginosa are under active investigations, there is no safe and effective vaccine available yet for public health purposes. The management of P. aeruginosa infection is entirely dependent on antibiotic treatment.

The only available antibiotics for effective oral treatment of P. aeruginosa infection are the fluoroquinolones (FQs) such as norfloxacin and ciprofloxacin. However, for P. aeruginosa and some other bacteria, resistance develops rapidly during FQ therapy, which severely limits the use of these drugs. In addition, if P. aeruginosa is resistant to one antibiotic it is often resistant to one or more chemically unrelated antibiotics. We have found that 15-24% of P. aeruginosa strains are resistant or intermediately resistant to quinolones in Stockholm. Similar findings are also shown in a recent study of 25 European university hospitals. This picture could be worse in developing countries where anyone can buy antibiotics without prescription.

Winning the challenge of Pseudomonas will require detailed knowledge of mechanisms of FQ resistance. Therefore, the aim of this study was to examine the quinolone-target genes: gyrA, gyrB, parC, and parE, and to characterize multidrug resistant efflux: mexAB-oprM, mexCD-oprJ, and mexEF-oprN, which are regulated by mexR, nfxB and mexT respectively. The overall goal was to increase understanding the mechanisms of FQ resistance. In this thesis, we reviewed the genetic mechanisms by which P. aeruginosa develop and disseminate FQ resistance. We also reviewed how the knowledge of bacterial genetics has significantly accelerated the ability to understand the spread of resistance.

We found that the point mutations in quinolone-target genes are the major component of resistance mechanisms in clinical isolates. Efflux-mediated resistance to FQ seems to dominating resistance-mechanism in cystic fibrosis isolates and laboratory mutants. However, high-level resistance is the result of accumulation of mutations in the quinolone targets and efflux systems. In addition, RESA (restriction enzyme site-protection assay) showed that a reduced DNA binding activity of the regulatory protein of multidrug efflux might render the bacterium to become resistant to drugs.

The efflux-mediated resistance mechanisms are widely distributed among the human pathogens and are often multidrug resistant. In addition, the efflux systems are highly homologous within Gram-negatives and also for Gram-positives. Therefore, inhibition of efflux systems with broad-spectrum inhibitors would seem to be a prudent approach to combat and/or prevent FQ resistance or even multidrug resistance.

We introduced NIRCA (Non-isotopic RNase cleavage assay) as a rapid, simple and highly sensitive method for detection of mutations in P. aeruginosa. We also established RESA to assess DNA binding activities of the regulatory protein for the efflux pump in this organism.

List of scientific papers

I. Jalal S, Wretlind B (1998). "Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa" Microb Drug Resist 4(4): 257-61
https://pubmed.ncbi.nlm.nih.gov/9988043

II. Stenhoff J, Jalal S, Wretlind B (2000). "Mutations in gyr(beta) and parE) and hypermutability with resistance to flouroquinolones in Pseudomonas aeruginosa" (Submitted)

III. Jalal S, Ciofu O, Hoiby N, Gotoh N, Wretlind B (2000). "Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients" Antimicrob Agents Chemother 44(3): 710-2
https://pubmed.ncbi.nlm.nih.gov/10681343

IV. Jalal S, Wretlind G, Gotoh N, Wretlind B (1999). "Rapid identification of mutations in a multidrug efflux pump in Pseudomonas aeruginosa" APMIS 107(12): 1109-16
https://pubmed.ncbi.nlm.nih.gov/10660141

V. Jalal S, Flock J-I, Wretlind B (2000). "Cloning and characterization of negative regulator (MexR) of MexAB-OprM Multi-drug efflux system." (The data presented in the 100th American Society for Microbiology General Meeting, May 21-25, 2000, Los Angeles,CA). (Submitted)