Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

Abstract

Multidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence in Enterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates of E. cloacae were used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). The acrA and tolC genes were deleted in strains EcDC64 and Jc194 to produce, respectively, Ec⌬acrA and Ec⌬tolC and Jc⌬acrA and Jc⌬tolC knockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance of E. cloacae to several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that the acrA and tolC genes both affect the fitness of E. cloacae, as fitness was clearly reduced in the acrA and tolC KO strains. The median CI values obtained in vitro and in vivo were, respectively, 0.42 and 0.3 for EcDC64/Ec⌬acrA, 0.24 and 0.38 for EcDC64/Ec⌬tolC, 0.15 and 0.11 for Jc194/Jc⌬acrA, and 0.38 and 0.39 for Jc194/Jc⌬tolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in both E. cloacae clinical strains when either the acrA or tolC gene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates of E. cloacae.

Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex

“…AcrABTolC is a well-characterized multidrug efflux pump of the RND family in E. cloacae (20,26,27). Based on the sequence of the protein AcrB (designated ECL_01233 in ECL13047), the inner membrane transporter protein of the system, we searched for the presence of other members of this family of transporters.…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…A Salmonella isolate with an inactivated MacAB pump is less virulent, and a mutant deficient in all drug efflux pumps is avirulent in mice (222). In E. cloacae, inactivation of either acrA or tolC led to reduced fitness (both in vitro and in vivo) as well as reduced virulence in a mouse model of systemic infection (264). A deficiency in AcrAB and OqxAB in K. pneumoniae also caused reduced virulence (792).…”

“…Deletion of acrA predictably decreased the MICs of oxacillin, erythromycin, clindamycin, linezolid, ciprofloxacin, chloramphenicol, tetracycline, and tigecycline (264). All multidrug-resistant E. aerogenes strains tested (also resistant to ␤-lactams) were found to overproduce AcrAB (265), but the effect of an EPI on ␤-lactam MICs was not examined.…”

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

“…As indicated in Fig. 4, deletion of either acrA or tolC in two different E. cloacae strains (each hyperexpressing AcrAB-TolC or expressing them at basal level) has a clear impact on fitness (measured in vitro and in vivo) (89). In this family, efflux pumps are also required for the colonization and persistence of bacteria in the host, whether plants, animals, or humans.…”