Central role of the ramAR locus in the multidrug resistance in ESBL -Enterobacterales

Abstract

The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the ramAR locus among extended-spectrum β-lactamases-Enterobacterales (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several tetR-associated genes including ramAR operon. Among the 443 ESBL-E strains included, isolates of Escherichia coli (n = 194), Klebsiella pneumoniae (n = 122), and Enterobacter cloacae complex (Ecc) (n = 127) were found. Thirty-one ESBL-E strains (7%), 16 K. pneumoniae (13.1%), and 15 Ecc (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no E. coli presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-β-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the ramAR intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein-substrate interactions. The ramAR IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the ramAR IR represent major genetic events for bacterial antimicrobial resistance.IMPORTANCEMorbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of Klebsiella pneumoniae and Enterobacter cloacae complex strains presented mutations within the ramAR locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as ramAR locus played a major role in antibiotic resistance dissemination and need to be further explored.

Keywords: AcrAB-TolC; RamA; RamR; clinical microbiology; efflux pumps; enterobacteriaceae; extended-spectrum beta-lactamase; genomics; gram-negative bacteria; intensive care units; mechanisms of resistance; regulation pathway.

Fig 1

Diversity of the sequences within the acrAB-tolC regulation pathway. The number of unique sequences and the phenotypical association (see Materials and Methods) found in 12 proteins, RamA, RamR, AcrR, AcrA, AcrB, TolC, SoxR, SoxS, MarA, MarB, MarR, and CsrA, and the ramAR intergenic region are represented in bar plots. (A) represents the sequence diversity from the genomes of 122 ESBL-producing K. pneumoniae; (B) represents the sequence diversity from the genomes of 127 ESBL-producing E. cloacae complexes. Sequences only recovered from susceptible strains are represented in green, sequences that were specific to resistant strains are represented in red, and sequences that were found in both susceptible and resistant isolates are colored yellow.

Fig 2

Organization of the RamR DNA-binding domain found among Enterobacter cloacae complex (A) and Klebsiella pneumoniae (B) strains. Annotation of the region was performed according to the description of Baucheron et al. (16). −10 Tata boxes are represented in blue, nucleotide binding sites are represented in green, and the six nucleotides’ “linkers” are in orange. Nucleotide mutations retrieved among t2c2 strains are represented by circles.

Fig 3

Illustration of RamR associated with Enterobacter cloacae complex t2c2 phenotype. Blue rectangles represent the HTH domain of the RamR. Green rectangles constitute the regions involved in the protein–substrate interactions. Pink rectangles are the ninth alpha helices, which are involved in dimerization with a second RamR protein. (A and B) Amino acid substitutions associated with resistant Enterobacter cloacae complex strains are represented in circles. Consequences of the substitution are organized by colors: steric hindrance (orange), amino acid charge modification (yellow), side chain polarity modification (purple), and no significative modification (gray). (B–D) represent the N-terminal deletions found in several RamR. For each individual modification of RamR, the sequence type of the strains as well as the total of isolates is indicated near the blue boxes.

Fig 4

Illustration of RamR associated with Klebsiella pneumoniae t2c2 phenotype. Blue rectangles represent the HTH domain of the RamR. Green rectangles constitute the regions involved in the protein–substrate interactions. Pink rectangles are the ninth alpha helices, which are involved in dimerization with a second RamR protein. (A) Amino acid substitutions associated with resistant Klebsiella pneumoniae strains are represented in circles. Consequences of the substitution are organized by colors: steric hindrance (orange), amino acid charge modification (yellow), and side chain polarity modification (purple). (B–D) represent the C-terminal deletions found in several RamR. For each individual modification of RamR, the sequence type of the strains as well as the total of isolates is indicated near the blue boxes. ^*One ST405 strain had the W89R substitution while another one had the Y92N.