Resolving colistin resistance and heteroresistance in Enterobacter species

Abstract

Species within the Enterobacter cloacae complex (ECC) include globally important nosocomial pathogens. A three-year study of ECC in Germany identified Enterobacter xiangfangensis as the most common species (65.5%) detected, a result replicated by examining a global pool of 3246 isolates. Antibiotic resistance profiling revealed widespread resistance and heteroresistance to the antibiotic colistin and detected the mobile colistin resistance (mcr)-9 gene in 19.2% of all isolates. We show that resistance and heteroresistance properties depend on the chromosomal arnBCADTEF gene cassette whose products catalyze transfer of L-Ara4N to lipid A. Using comparative genomics, mutational analysis, and quantitative lipid A profiling we demonstrate that intrinsic lipid A modification levels are genospecies-dependent and governed by allelic variations in phoPQ and mgrB, that encode a two-component sensor-activator system and specific inhibitor peptide. By generating phoPQ chimeras and combining them with mgrB alleles, we show that interactions at the pH-sensing interface of the sensory histidine kinase phoQ dictate arnBCADTEF expression levels. To minimize therapeutic failures, we developed an assay that accurately detects colistin resistance levels for any ECC isolate.

Fig. 1. Distribution of Enterobacter species among six university hospitals in Germany.

a A total of 165 Enterobacter isolates were studied. Genome-based species classification showed E. xiangfangensis as the most frequent species at all centers. Values in parentheses denote the % of total Enterobacter (n = 165) isolates. The outline map of Germany is obtained from https://vemaps.com. b Phylogenomic distribution of the Enterobacter isolates obtained between 2011 and 2018 from six different study centers. Whole-genome sequence-based study of 165 Enterobacter isolates revealed 65.4% of isolates to be E. xiangfangensis. Bayesian Analysis of Population Structure (BAPS) in concordance with the multi-phylogenomic approach (concatenate core genes (shown here), core- and whole-genome) clustered E. xiangfangensis isolates into four lineages. A majority (60% of E. xiangfangensis isolates) belonged to Lineage-1, which harbor arnBCADTEF gene cassette associated with colistin heteroresistance. Importantly, 9 E. xiangfangensis and 4 other Enterobacter species isolates carried the recently identified mobile colistin resistance gene mcr-9. The hsp60 types correlated well to the phylogenomic lineages. The lineages were supported by bootstrap values (100). The distribution data for the isolates are provided in the Source data file.

Fig. 2. Analysis of L-Ara4N-modification levels of lipid A and their correlation with heteroresistance frequency and the number of cells that survived after colistin treatment.

a Heat map summarizing the MS-based analysis of L-Ara4N-modification levels of lipid A in selected E. xiangfangensis strains. Corresponding, representative mass spectra are shown in Supplementary Fig. 5, further details are specified in Supplementary Data 4. b General chemical structure of Enterobacter lipid A. c Heat map summarizing the MS-based analysis of L-Ara4N-modification levels of lipid A in 11 other Enterobacter species observed in this study. d Various Enterobacter isolates grown at identical conditions (LB broth, O.D.₆₀₀ 0.8–1.0, without colistin) were analyzed for L-Ara4N-modification of lipid A ((see panels a and c), Supplementary Data 4) and for their correlation with % of heteroresistant cells (on a plate containing 8 and 32 mg/L of colistin), and % of cells survived after colistin treatment (64 mg/L for 15 min). The % of heteroresistance CFUs at 8 and 32 mg/L was positively associated with L-Ara4N-modification levels of lipid A (Pearson’s correlation coefficient, r = 0.88 and 0.73, respectively). This data suggested colistin heteroresistance to be directly associated with such levels. Given that only a fraction of isogenic growing population survives on plates containing 8 and 32 mg/L of colistin, we determine the % of cells that can survive after colistin treatment (64 mg/L for 15 min) by Live/Dead^TM staining assay. This assay showed a positive association (r = 0.86) between % of cells survived and L-Ara4N-modified lipid A. This data indicated that only a certain number of cells from isogenic growing population carried modified lipid A and can survive when exposed to colistin, resulting in the heteroresistance phenomenon. This data show that cells with L-Ara4N-modified lipid A form the founder population of the colistin heteroresistance (also see Supplementary Table 5). Source data are provided as a Source data file.

Fig. 3. Genetic determinants of heteroresistance in Enterobacter species.

Comparative genomics of colistin-resistant and -sensitive isolates revealed that arnBCADTEF is necessary for colistin heteroresistance. In Enterobacterales, arnBCADTEF expression is controlled by the TCS PhoPQ, while the activity of the PhoPQ pathway is inhibited by the small transmembrane protein MgrB. Deletion mutants were created in E. xiangfangensis L-1 isolate RBK-17-0394-1 (WT) and studied for the MIC_LB and HRF (number of cells recovered on LB agar plate containing 8 or 32 mg/L of colistin/number of cells inoculated). MIC_LB was measured by BMD while HRF was calculated by PAP test. Analysis of mutants showed the phoPQ/arnBCADTEF axis is absolutely required for the colistin heteroresistance, while MgrB acts as a negative regulator of arnBCADTEF resulting in colistin susceptibility. As reported previously by Kang et al. for E. cloacae, PmrAB did not contribute to colistin resistance in E. xiangfangensis. Source data are provided as a Source data file. The MIC data is provided in the Source data file.

Fig. 4. Heteroresistance capabilities of phoP-phoQ hybrid and mgrB complemented mutants.

The hybrid complement mutants were created by combining phoP and phoQ genes either of E. xiangfangensis RBK-17-0394-1 (Ex), E. bugandensis RBG-17-0246-1 (Eb), and E. roggenkampii RPB-17-0516-2 (Er). Plasmids harboring phoP-phoQ hybrids were transferred to Ex394ΔphoPQ. Complementation of the phoQ from the isolates exhibiting higher colistin heteroresistance i.e., Eb and Er to the ΔphoPQ mutant of E. xiangfangensis RBK-17-0394-1 resulted in the colistin resistance equivalent to respective parent strains. Complementation of the ΔmgrB mutant of E. xiangfangensis RBK-17-0394-1 with the mgrB from Eb (which has an identical amino acid sequence as of Er) resulted in slightly lesser inhibition due to changes in the binding energy (detailed in Supplementary Fig. 9). The MIC data is provided in the Source Data file.

Fig. 5. Minimum inhibitory concentration (MIC_LB) and heteroresistance frequency (HRF) of different Enterobacter species.

a MIC_LB and b HRF. The MIC_LB and HRF were studied by BMD assay and PAP test for isolates of 12 species observed in this study. The red and blue boxes (in a) depict MIC_LB at pH 5 and 7, respectively. Enterobacter species exhibiting lower HRFs are shown also in the inset (in b). The MICs and HRFs for each individual isolate are provided in the Source data file. Keys for box and whisker plot: The black center line denotes the median value (50th percentile), while the red or blue box contains the 25th to 75th percentiles of dataset. The black whiskers mark the inter-quartile range (1.5× IQR) and outlier points are shown individually.

Fig. 6. Model of mechanistic pathways leading to colistin heteroresistance.

The PhoQ component (of the two component system PhoPQ) senses environmental conditions (for e.g., low pH and cationic antimicrobial peptide (CAMP)), which results in a change in its conformation leading to phosphorylation of PhoP. Phosphorylated PhoP binds to the promoter of many genes, including arnBCADTEF. Expression of arnBCADTEF results in the addition of L-Ara4N to lipid A, which changes the overall charge of the outer membrane causing colistin resistance. a Under normal growth conditions (LB, pH 7) PhoQ of the overall growing population is strongly inhibited by MgrB. As a result of microenvironmental changes experienced during growth, a portion of the isogenic growing population has activated PhoQ with concomitant expression of the arnBCADTEF loci that result in L-Ara4N-modified lipid A. Only these cells survive when exposed to colistin (HRF + ). Overall, levels of modified lipid A are low. b As a result of low pH (pH 5) during growth conditions, MgrB is prevented from interacting with PhoQ, resulting in higher expression of the arnBCADTEF cassette and subsequent L-Ara4N-modified lipid A in a higher number of cells. Quantitative lipid A analysis confirms these observations and these populations exhibit increased survival (HRF++). c In the presence of subinhibitory concentrations of a cationic antimicrobial peptide such as colistin (2 mg/L), the antibiotic penetrates the outer membrane and is sensed by PhoQ strongly weakening interactions with MgrB and thereby releasing expression of arnBCADTEF. This results in L-Ara4N-modified lipid A in a higher number of bacteria. When exposed to colistin, these populations show relatively high survival levels (HRF+++). d As a result of low pH (pH 5) and antimicrobial peptide colistin during growth, PhoQ experiences a cumulative effect, and thus MgrB inhibition is strongly prevented, resulting in heightened expression of arnBCADTEF cassette and subsequent arabinose modified lipid A in a higher number of cells. These populations exhibit increased survival (HRF++++). (Core sugars and fatty acids of the LPS are only drawn symbolically and do not reflect exact structures).