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

Abstract

In Gram-negative bacteria, the active efflux is an important mechanism of antimicrobial resistance, but little is known about theEnterobacter cloacaecomplex (ECC). It is mediated primarily by pumps belonging to the RND (resistance-nodulation-cell division) family, and only AcrB, part of the AcrAB-TolC tripartite system, was characterized in ECC. However, detailed genome sequence analysis of the strainE. cloacaesubsp.cloacaeATCC 13047 revealed to us that 10 other genes putatively coded for RND-type transporters. We then characterized the role of all of these candidates by construction of corresponding deletion mutants, which were tested for their antimicrobial susceptibility to 36 compounds, their virulence in the invertebrateGalleria mellonellamodel of infection, and their ability to form biofilm. Only the ΔacrBmutant displayed significantly different phenotypes compared to that of the wild-type strain: 4- to 32-fold decrease of MICs of several antibiotics, antiseptics, and dyes, increased production of biofilm, and attenuated virulence inG. mellonella In order to identify specific substrates of each pump, we individually expressed intransall operons containing an RND pump-encoding gene into the ΔacrBhypersusceptible strain. We showed that three other RND-type efflux systems (ECL_00053-00055, ECL_01758-01759, and ECL_02124-02125) were able to partially restore the wild-type phenotype and to superadd to and even enlarge the broad range of antimicrobial resistance. This is the first global study assessing the role of all RND efflux pumps chromosomally encoded by the ECC, which confirms the major role of AcrB in both pathogenicity and resistance and the potential involvement of other RND-type members in acquired resistance.

FIG 1

Genetic organization of the operons containing one gene coding for a putative RND efflux pump transporter homologous to AcrB.

FIG 2

Effect of the deletions of efflux pump-encoding genes on virulence. Percent survival of G. mellonella larvae 24 h (black bars), 48 h (white bars), and 72 h (gray bars) after infection with around 4 × 10⁶ CFU of E. cloacae bacterial cells per larva. Experiments were repeated at least three times, and the results represent the means ± standard deviations from live larvae.

FIG 3

AcrB is involved in the biofilm formation of E. cloacae. The ability of E. cloacae ECL13047 and different mutant strains to form biofilm on polystyrene surfaces after 24 h (black bars), 48 h (white bars), and 1 week (gray bars) of incubation at 37°C is shown. OD values at 580 nm are from three independent experiments, and the means ± standard deviations are presented.