Detection and Prevalence of Active Drug Efflux Mechanism in Various Multidrug-Resistant Klebsiella pneumoniae Strains from Turkey

Abstract

The prevalence of active drug efflux pump and porin alterations was investigated in Turkish nosocomial strains of Klebsiella pneumoniae exhibiting a multidrug-resistant phenotype. MICs of various antibiotics, including quinolones, chloramphenicol, tetracycline, and ␤-lactams, for those strains were determined either with or without the efflux pump inhibitor phenylalanine arginine ␤-naphthylamide (PA␤N). Thirty-nine percent of the strains exhibited a PA␤N-modulated resistance for quinolones, chloramphenicol, and tetracycline. In these strains, a significant increase of chloramphenicol accumulation was gained in the presence of the efflux pump inhibitor PA␤N or with the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone. Moreover, high-level expression of the membrane fusion protein AcrA, which was immunodetected in most of those isolates, suggests that the AcrAB/TolC efflux machinery contributed to their antibiotic resistance. Studies of K. pneumoniae porins indicated that the majority of the strains, including extended-spectrum ␤-lactamase producers and efflux-positive ones, presented an alteration in their sorbitol-sensitive porin (OmpK35) expression. This is the first report showing the prominent role of active drug efflux in the antibiotic resistance of nosocomial K. pneumoniae strains from Turkey.

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

“…a study of Klebsiella pneumoniae strains collected from different patients undergoing treatment indicated that the isolates exhibited modified outermembrane permeability 22 . In most of these isolates, OmpK35, which belongs to the OmpF porin group and has a large channel size, was replaced with OmpK36, which belongs to the OmpC porin group and possesses a smaller channel size.…”

Occurrence of Efflux Mechanism and Cephalosporinase Variant in a Population of Enterobacter aerogenes and Klebsiella pneumoniae Isolates Producing Extended-Spectrum β-Lactamases

“…The test results confirmed the existence of a PA␤N-susceptible efflux mechanism in all E. aerogenes and many K. pneumoniae strains, with marked decreases in the MICs of fluoroquinolones and chloramphenicol in the presence of PA␤N (Table 1). Efflux pump activity was identified when the PA␤N addition induced a threefold decrease in the MIC of an antibiotic molecule (4,9). For the two fluoroquinolones, the PA␤N addition had better efficiency in decreasing the MICs of sparfloxacin than those of norfloxacin.…”

“…We used the twofold broth dilution method with Mueller-Hinton broth according to the CLSI guidelines: the tests were carried out with or without the broad-spectrum efflux pump inhibitor (EPI) phenylalanine arginine ␤-naphthylamide (PA␤N). This diamine compound, used at a low concentration (26.3 mg/liter), can increase the activities of chloramphenicol, tetracycline, norfloxacin, and sparfloxacin against various enterobacterial isolates which overproduce efflux pumps such AcrAB-TolC (4,9,16). The test results confirmed the existence of a PA␤N-susceptible efflux mechanism in all E. aerogenes and many K. pneumoniae strains, with marked decreases in the MICs of fluoroquinolones and chloramphenicol in the presence of PA␤N (Table 1).…”

“…AcrA-TolC overproduction in E. aerogenes isolates was noted. An analysis of four E. aerogenes isolates for which cefepime MICs were high revealed no modification in porin expression but a new specific mutation in the AmpC ␤-lactamase.Enterobacter aerogenes and Klebsiella pneumoniae, two of the most prevalent nosocomial enterobacterial species, can frequently express a multidrug resistance (MDR) phenotype by the acquisition or high-level production of ␤-lactamases in combination with the overproduction of efflux pumps, associated or not with porin alterations in the outer membrane (1,3,7,9,13). Such modifications of envelope permeability, including efflux and influx (porin), in 28 Enterobacter aerogenes and 16 Klebsiella pneumoniae clinical isolates from Nîmes University Hospital, previously studied for extended-spectrum ␤-lactamase (ESBL) profiling and clonal identification, were investigated (11).…”

“…It was reported previously that the overproduction of AcrA and TolC pump elements is involved in the resistance levels of clinical isolates (4,8,9). The membrane proteins of selected isolates were extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection with polyclonal antibodies directed against AcrA and TolC components (9,18). Immunoblots were stained by colorimetric detection with alkaline phosphatase-conjugated secondary antibodies.…”