Uncovering the Important Genetic Factors for Growth during Cefotaxime-Gentamicin Combination Treatment in blaCTX-M-1 Encoding Escherichia coli

Abstract

Due to the rapid spread of CTX-M type ESBLs, the rate of resistance to third-generation cephalosporin has increased among Gram-negative bacteria, especially in Escherichia coli, and there is a need to find ways to re-sensitize ESBL E. coli to cephalosporin treatment. A previous study showed that genes involved in protein synthesis were significantly up-regulated in the presence of subinhibitory concentration of cefotaxime (CTX) in a CTX-M-1-producing E. coli. In this study, the interaction between CTX and gentamicin (GEN), targeting protein synthesis, was evaluated in MG1655/pTF2, and the MIC of CTX was strongly reduced (128-fold) in the presence of this combnation therapy. Since the underlying mechanism behind this synergy is not known, we constructed a saturated transposon mutant library in MG1655/pTF2::blaCTX-M-1 containing 315,925 unique transposon insertions to measure mutant depletion upon exposure to CTX, GEN, and combination treatment of CTX and GEN by Transposon Directed Insertion-site Sequencing (TraDIS). We identified 57 genes that were depleted (log2FC ≤ −2 and with q.value ≤ 0.01) during exposure to CTX, 18 for GEN, and 31 for combination treatment of CTX and GEN. For validation, we deleted eight genes that were either uniquely identified in combination treatment, overlapped with monotherapy of GEN, or were shared between combination treatment and monotherapy with CTX and GEN. Of these genes, we found that the inactivation of dnaK, mnmA, rsgA, and ybeD increased the efficacy of both CTX and GEN treatment, the inactivation of cpxR and yafN increased the efficacy of only CTX, and the inactivation of mnmA, rsgA, and ybeD resulted in increased synergy between CTX and GEN. Thus, the study points to putative targets for helper drugs that can restore susceptibility to these important drugs, and it indicates that genes involved in protein synthesis are essential for the synergy between these two drugs. In summary, the study identified mutants that sensitize ESBL-producing E. coli to CTX and a combination of CTX and GEN, and it increased our understanding of the mechanism behind synergy between β-lactam and aminoglycoside drugs. This forms a framework for developing new strategies to combat infections caused by resistant bacteria.

Genome-wide identification of fitness-genes in aminoglycoside-resistant Escherichia coli during antibiotic stress

“…Genes categorized as ‘essential’ in TraDIS are either essential for survival or their interruption is associated with a severe fitness-cost under the given conditions 40 . This number of ‘essential’ genes is in a similar range as previously published counts of ‘essential’ genes in E. coli that were determined by TraDIS analysis 16 , 40 , which provides strong assurance in the quality of our Tn-libraries enabling us to obtain meaningful conclusions.…”

“…Deletion of the hflCK cluster confirmed that the cluster was indeed important for growth in the STREP and GEN-resistant background in the presence of the corresponding aminoglycosides. In previous studies, disruption of hflK (and hflC 30 ) has been associated with increased sensitivity to GEN in GEN-susceptible E. coli MG1655 30 , MG1655/pTF2 16 and E. coli BW25113 45 allowing us to confide in the reliability of our data.…”

“…We compared the fitness-genes from our aminoglycoside Tn-libraries with fitness-genes identified in other antibiotic-resistant Tn-libraries to gain an insight into which fitness-genes were antibiotic specific or in general advantageous during antibiotic stress (Supplementary Table S10 online). Among 35 fitness-genes in a multidrug-resistant K. pneumoniae challenged with colistin 19 , six were shared with our list of aminoglycoside fitness-genes, and among 57 genes fitness-genes during CTX exposure in ESBL E. coli 16 six were predicted as fitness-genes in the current study. Four genes namely lpp , pal , cpxR and envC were identified in all three studies.…”

“…TraDIS has been established as a powerful tool to identify sets of fitness-genes that are advantageous during antibiotic exposure, including exposure to colistin 19 and CTX 16 in antibiotic-resistant bacteria, as well as fosfomycin 31 , trimethoprim and sulphonamide 32 in antibiotic-sensitive bacteria. Using TraDIS, we identified 106 conditionally essential genes (STREP (n = 56), GEN (n = 39) and NEO (n = 32)) which could represent targets for helper-drugs to re-sensitize resistant E. coli to aminoglycoside treatment.…”

“…Extended Spectrum Beta-Lactamase (ESBL)-producing E. coli undergo modifications in gene expression patterns when exposed to the antibiotic cefotaxime (CTX), and affected pathways were suggested as novel drug targets to re-sensitize resistant bacteria to treatment 15 . This idea has recently been followed up on by a more direct approach, where conditionally essential or fitness-genes i.e., a set of non-essential genes which becomes important for growth specifically in the presence of CTX, were identified using Transposon Directed Insertion-site Sequencing (TraDIS) 16 . TraDIS is a high-throughput technique that combines the advantages of random transposon mutagenesis with next-generation sequencing 17 , 18 .…”