Membrane Efflux and Influx Modulate both Multidrug Resistance and Virulence of Klebsiella pneumoniae in a Caenorhabditis elegans Model

Abstract

Cross-resistance to cefoxitin (FOX), chloramphenicol (CMP), and quinolones (nalidixic acid [NAL]) related to a putative efflux system overexpression has recently been reported for Klebsiella pneumoniae. The potential impact of this multidrug resistance (MDR) on the virulence of K. pneumoniae was evaluated in the Caenorhabditis elegans model. For 2 of the 3 MDR clinical isolates studied, a significant increase in acrB transcription was found in comparison with their antibiotic-susceptible revertants. ATCC 138821 and MDR, revertant, and derivative strains with altered porin expression were studied. Strains proved or suspected to overexpress an efflux system were significantly more virulent than the ATCC and revertant strains (time to kill 50% of nematodes [LT 50 ] in days: 3.4 to 3.8 ؎ 0.2 versus 4.1 to 4.4 ؎ 0.3, P < 0.001). Inversely, strains with altered porin expression were significantly less virulent, independently of the expression level of efflux system (LT 50 ‫؍‬ 5.4 to 5.6 ؎ 0.2, P < 0.001). Altered porin expression did not change MICs of CMP and NAL but did those of FOX (4 to 16؋ MIC) and ertapenem (16 to 64؋ MIC). The strains with a normally or an overexpressed efflux system that received the ␤-lactamase CTX-M-15 became more widely resistant without modification of their virulence potential, suggesting that balance between resistance and virulence is dependent on the type of resistance mechanisms. In conclusion, this study shows that the expression of both efflux systems and porins is a key factor not only for antibiotic resistance but also virulence potential in K. pneumoniae.Klebsiella pneumoniae, which was recognized over 100 years ago as a cause of community-acquired pneumonia and 20 years ago as a cause of community-acquired pyogenic liver abscesses, is also a common cause of nosocomial infections that range from mild urinary tract infections to severe respiratory tract infections and bacteremia (15,30). Moreover, K. pneumoniae has been found to produce various plasmid-mediated enzymes which confer resistance to most ␤-lactams, particularly the extended-spectrum cephalosporins and more recently carbapenems (2,22,24). Treatment of serious infections caused by extended-spectrum ␤-lactamase (ESBL)-producing K. pneumoniae bacteria is difficult because these organisms are frequently resistant to various other families of antibiotics, including fluoroquinolones (28). Fluoroquinolone resistance in K. pneumoniae arises through specific mutations within the target proteins DNA gyrase and topoisomerase IV and also through a lower uptake of quinolones because of efflux system overexpression (3, 18). Ruzin et al. showed that the resistance to quinolones related to overexpression of the AcrAB efflux pump of K. pneumoniae was associated to resistance to other antibiotics, including chloramphenicol, erythromycin, tetracycline, and also tigecycline, a recently commercialized molecule (32). As for us, we showed that cross-resistance to cefoxitin, chloramphenicol, and quinolones that we observed in bacteremia K. pn...

In Vitro Selection of ramR and soxR Mutants Overexpressing Efflux Systems by Fluoroquinolones as Well as Cefoxitin in Klebsiella pneumoniae

“…We had previously shown in K. pneumoniae clinical isolates that cefoxitin, quinolone, and chloramphenicol cross-resistance was not always associated with an increase in the transcription level of the genes encoding AcrAB, the most frequently investigated efflux pump in Enterobacteriaceae (3). Interestingly, such a feature was also found in one of our 17 mutants, KPBj5 M1 …”

“…The collection studied comprised (i) 3 previously published K. pneumoniae clinical isolates shown to have cross-resistance to cefoxitin, quinolones, and chloramphenicol (KPBj Eϩ), with increased expression of the acrB gene for 2 of them (KPBj1 Eϩ and KPBj3 Eϩ) (3,29); (ii) their spontaneous revertants (KPBj Rev) susceptible to the three antibiotic families (3); and (iii) mutants selected from these 3 revertant strains with cefoxitin, ciprofloxacin, or levofloxacin. For all experiments, K. pneumoniae strain ATCC 138821 was used as a control.…”

Antimicrobial Resistance and Virulence: a Successful or Deleterious Association in the Bacterial World?

“…Padilla et al also showed that a K. pneumoniae acrAB knockout mutant exhibited a lower capacity than the wild-type strain to cause pneumonia in a murine model (90). Bialek et al found that overexpression of the AcrAB efflux system in the Caenorhabditis elegans model is linked to an increased virulence potential in K. pneumoniae (91). Overall, these studies emphasize that the expression of efflux systems is a key factor in both antibiotic resistance and virulence in K. pneumoniae.…”

Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility

“…The plates were incubated at 20 uC and scored daily for dead nematodes using a stereoscope (SMZ168 BN; Motic). Nematodes were agesynchronized with bleach and transferred every 2 days to fresh plates to eliminate overcrowding by progeny until they laid no further eggs (Bialek et al, 2010;Fuursted et al, 2012;Srinivasan et al, 2012). Worm mortality was scored over time for~20 days, with death defined when a worm no longer responded to touch.…”

“…The effect of the observed changes in the extracellular matrix triggered by overproduction of YfiN DGC and cellulose was next evaluated using a C. elegans infection model (Bialek et al, 2010;Fuursted et al, 2012;Srinivasan et al, 2012). Age-synchronized, adult nematodes were fed LM21gfp WT cells or isogenic mutants lacking yfiR, yfiN or yfiB, and mortality was scored over time, as an indirect marker of virulence potential.…”