Macrocolony of NDM-1 Producing Enterobacter hormaechei subsp. oharae Generates Subpopulations with Different Features Regarding the Response of Antimicrobial Agents and Biofilm Formation

Abstract

Enterobacter cloacae complex has been increasingly recognized as a nosocomial pathogen representing the third major Enterobacteriaceae species involved with infections. This study aims to evaluate virulence and antimicrobial susceptibility of subpopulations generated from macrocolonies of NDM-1 producing Enterobacter hormaechei clinical isolates. Biofilm was quantified using crystal violet method and fimbrial genes were investigated by PCR. Susceptibility of antimicrobials, alone and combined, was determined by minimum inhibitory concentration and checkerboard assays, respectively. Virulence and efficacy of antimicrobials were evaluated in Galleria mellonella larvae. Importantly, we verified that some subpopulations that originate from the same macrocolony present different biofilm production ability and distinct susceptibility to meropenem due to the loss of bla_NDM-1 encoding plasmid. A more in-depth study was performed with the 798 macrocolony subpopulations. Type 3 fimbriae were straightly related with biofilm production; however, virulence in larvae was not statistically different among subpopulations. Triple combination with meropenem-rifampicin-polymyxin B showed in vitro synergistic effect against all subpopulations; while in vivo this treatment showed different efficacy rates for 798-1S and 798-4S subpopulations. The ability of multidrug resistant E. hormaechei isolates in generating bacterial subpopulations presenting different susceptible and virulence mechanisms are worrisome and may explain why these infections are hardly overcome.

Keywords: Enterobacter; Galleria mellonella; antimicrobial treatment; blaNDM-1; macrocolony biofilm.

Figure 1

Macrocolonies of E. hormaechei subsp. oharae clinical isolates. Bacterial macrocolonies were grown in LB with Congo Red and Coomassie brilliant blue for 5 days at 37 °C. The white arrows and numbers indicated areas of macrocolonies, termed subpopulations, used for further experiments. These subpopulations were randomly selected according their morphologies.

Figure 2

Biofilm formation of macrocolony subpopulations of E. hormaechei subsp. oharae clinical isolates. Bacteria were grown at 37 °C in microtiter plates containing Tryptone Soya Broth (TSB) for 24 h and then biofilm formation was quantified using crystal violet assay. The results are presented as the means and standard deviation. Significant differences in biofilm formation, among subpopulations of the same macrocolony, were pointed out when p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****).

Figure 3

(A) Biofilm formation of E. hormaechei subsp. oharae 798 group. Bacteria were grown at 37 °C in microtiter plates containing LB broth, LB broth supplemented with glucose, M9 minimal medium or TSB for 24 h, after which biofilm formation was quantified. The results are presented as the means and standard deviation for three independent experiments. Significant differences in biofilm formation using LB broth + GLU were pointed out: p < 0.05 (*) and p < 0.0001 (****). (B) Fimbrial encoding genes: fimA, fimH (type 1 fimbriae genes), papC, papD (P pili genes), csgA, csgB, csgD (curli genes), and mrkB (type 3 fimbriae gene). Gene present: +; and gene absent: –.

Figure 4

Survival curves of G. mellonella larvae inoculated with different inocula of E. hormaechei subsp. oharae (subpopulations 798-1S, 798-2S, 798-3S, and 798-4S): (A) 5.0 × 10⁵, (B) 2.0 × 10⁶, (C) 5.0 × 10⁶, and (D) 1.0 × 10⁷ CFU/larva.

Figure 5

Survival curves for G. mellonella larvae inoculated with: (A–D) 798-1S and (E–H) 798-4S subpopulations following treatment with (A and E) RIF (20 mg/kg), (B and F) POL (3.0 mg/kg), (C and G) MER (85 mg/kg), or (D and H) RIF­–POL–MER combination. H₂O: water; RIF: Rifampicin, POL: Polymyxin B, and MER: Meropenem.