Studies on the molecular mechanisms of resistance to fluoroquinolones and carbapenems in selected bacterial species

The emergence of mutant bacteria with multi-resistant phenotype, no longer responsive to any of the available therapeutic agents is a growing concern. We have investigated the resistance mechanisms to two classes of antibacterials with broad spectrum of activity, carbapenems and fluoroquinolones.

Resistance to quinolones develops as a result of chromosomal mutations in the target genes, DNA topoisomerases, alterations in outer membrane proteins or interference with binding to the target by a protein encoded on a transferable plasmid.

Resistance to the carbapenems involves alterations of the so-called Penicillin BindingProteins (PBPs), the target for these antibiotics. Carbapenem resistance results also from alterations in outer membrane permeability and production of metallo-beta-lactamases. The aims of these studies were to provide a better understanding of the molecular mechanisms underlying resistance to fluoroquinolone and carbapenem antibacterials.

Quinolone resistant clinical isolates of Enterococcus faecium, Bacteroides fragilis group and Pseudomonas aeruginosa were investigated. We analysed the genes encoding the DNA gyrase (gyrA) and topoisomerase IV (parC), and the efflux gene mexB in these strains. We found that ciprofloxacin and trovafloxacin resistance in E. faecium was associated with alteration Ser-80gIle in ParC and substitution at either Ser-83gArg/ Ile/Tyr, or have a Glu 87gLys in GyrA.

In the B. firagilis group, substitutions with profound effect on quinolone susceptibility were observed at residues 82 and 86 of GyrA. These alterations affected mainly susceptibilities to levofloxacin, moxifloxacin and clinafloxacin. However, the effect of gyrA mutations on ciprofloxacin susceptibility was unclear.

In B. fragilis, B. ovatus, B. thetaiotaomicron, and B. uniformis, substitution at residues 82 and/or 86 of gyrA appeared to be associated with raised MIC to one or several of the quinolones. Nevertheless, substitution of Phe-86g Tyr in a B. ovatus isolate did not appear to affect the MIC of any of the quinolones under investigation. For B. vulgatus, however, the role of alterations at residues 82 and 86 was less certain. In P. aeruginosa isolates, our results showed that association between mexB expression and ciprofloxacin MICs was unlikely.

We have also investigated resistance mechanisms to two carbapenems, imipenem and meropenem. In E. faecium stains we found imipenem resistance to be associated with overproduction of PBP-5 with decreased affinity for imipenem. In addition, PBP-5 of these isolates harboured alterations around the active site cavity, which might affect the binding affinity.

In carbapenem resistant P. aeruginosa, the outer membrane protein D (oprD) expression was down regulated in all clinical isolates investigated, both susceptible and resistant. However, the relative amount of OprD mRNA appeared to correlate with imipenem MICs in most of the strains. The association between the expression of mexF, mexB and mexY and imipenem susceptibility was uncertain. Nevertheless, a good correlation was seen between mexB expression and meropenem MICs.

List of scientific papers

I. el Amin NA, Jalal S, Wretlind B (1999). Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium. Antimicrob Agents Chemother. 43(4): 947-9.
https://pubmed.ncbi.nlm.nih.gov/10103206

II. Oh H, El Amin N, Davies T, Appelbaum PC, Edlund C (2001). gyrA mutations associated with quinolone resistance in Bacteroides fragilis group strains. Antimicrob Agents Chemother. 45(7): 1977-81.
https://pubmed.ncbi.nlm.nih.gov/11408211

III. El Amin N, Lund B, Tjernlund A, Lundberg C, Jalakas K, Wretlind B (2001). Mechanisms of resistance to imipenem in imipenem-resistant, ampicillin-sensitive Enterococcus faecium. APMIS. 109(11): 791-6.
https://pubmed.ncbi.nlm.nih.gov/11900060

IV. El Amin N, Sunnerhagen M, Wenger A, Brandt V, Wretlind B (2003). Expression of PBP-5 and modification of its penicillin-binding site in imipenem resistant E. faecium isolates. [Manuscript]

V. El Amin N, Giske C, Keijser B, Wretlind B, Kronvall G (2003). Analysis of oprD and efflux genes expression in carbapenem-resistant Pseudomonas aeruginosa indicates that other resistance mechanisms are involved in clinical isolates. [Manuscript]