Quinolone resistance in Bacteroides fragilis and Pseudomonas aeruginosa, two opportunistic pathogens

Abstract

Opportunistic pathogens can cause infections in case of structural or functional defects in the host. Examples of Gram-negative opportunists are the Bacteroides fragilis group, anaerobic, non-spore forming rods and the aerobic, non-fermentative rod Pseudomonas aeruginosa. Both are intrinsically resistant against many antimicrobial agents and are also very prone to become resistant to previously active antibiotics.

Quinolones are synthetic antimicrobial agents that target the bacterial type II topoisomerases: DNA gyrase (encoded by gyrA and gyrB genes) and topoisomerase IV (encoded by parE and parC genes). Newer quinolones have good activity against anaerobic bacteria, including the B. fragilis group. Quinolones are also the only available group of antibiotics for oral treatment of P. aeruginosa infections. Unfortunately, quinolone resistance has developed rapidly among many bacterial species and indications of emerging resistance also among the B. fragilis group and P. aeruginosa to these agents are inevitable. Quinolone resistance mechanisms thus far identified include alterations in the quinolone-resistancedetermining region (QRDR) of drug targets and efflux pumps. Decreased colonization resistance in the normal microflora following antimicrobial therapy might favour overgrowth of already present naturally resistant microorganisms or establishment of new resistant potentially pathogenic bacteria.

The aims of the present study were to investigate the ecological disturbances and a possible emergence of resistant strains caused by the broad-spectrum quinolone clinafloxacin, and to study the mechanisms of quinolone resistance in isolates of the B. fragilis group and P. aeruginosa.

Faecal specimens were collected from healthy volunteers at defined intervals before, during and after oral administration of clinafloxacin. Treatment with clinafloxacin resulted in pronounced ecological disturbances. The aerobic microflora was eradicated in 11 of 12 subjects and the anaerobic microflora was strongly suppressed. There was also a significant emergence of resistant Bacteroides spp. strains. The antimicrobial susceptibilities to four quinolones were determined in selected strains from the above study and in some clinical strains and the gyrA QRDR was amplified and sequenced.

Mutations at hotspot positions 82 (n=15) and 86 (n=8) were found of which Ser82Leu, Ser82Phe and perhaps Tyr86Phe seem to confer quinolone resistance in the B. fragilis group. Hydrophobicity of the replacing amino acid at resistance hotspots might be a determinant for the final level of quinolone resistance. Although newer quinolones have good antimicrobial activity against the B. fragilis group, quinolone resistant strains were readily selected in vivo.

The role of efflux pumps in quinolone resistance for these isolates was investigated using a silicon oil based fluorometric assay. With the exception of two strains, increased resistance to quinolones were correlated either with target mutations, increased efflux or a combination of both factors. Mutational events in the QRDR of gyrA as well as increased efflux activity seem to contribute to quinolone resistance in the Bacteroides fragilis group. Clinical norfloxacin resistant P. aeruginosa strains with known gyrA and parC genotypes were investigated regarding expression of efflux pumps and mutations in gyrB and parE genes. The amount of mRNA for efflux pump proteins were determined as cDNA by realtime PCR. The results indicate that some mutations in gyrB might contribute to quinolone resistance. In addition, enhanced expression of MexB and high-level overexpression of the efflux pump proteins MexF, MexY, and possibly MexD, may contribute to quinolone resistance. Hypermutable strains are specifically prone to develop high-level resistance due to accumulation of multiple mutations in target genes.