Dataset Information

Development of Resistance in Escherichia coli ATCC25922 under Exposure of Sub-Inhibitory Concentration of Olaquindox.

ABSTRACT: Quinoxaline1,4-di-N-oxides (QdNOs) are a class of important antibacterial drugs of veterinary use, of which the drug resistance mechanism has not yet been clearly explained. This study investigated the molecular mechanism of development of resistance in Escherichia coli (E. coli) under the pressure of sub-inhibitory concentration (sub-MIC) of olaquindox (OLA), a representative QdNOs drug. In vitro challenge of E. coli with 1/100× MIC to 1/2× MIC of OLA showed that the bacteria needed a longer time to develop resistance and could only achieve low to moderate levels of resistance as well as form weak biofilms. The transcriptomic and genomic profiles of the resistant E. coli induced by sub-MIC of OLA demonstrated that genes involved in tricarboxylic acid cycle, oxidation-reduction process, biofilm formation, and efflux pumps were up-regulated, while genes involved in DNA repair and outer membrane porin were down-regulated. Mutation rates were significantly increased in the sub-MIC OLA-treated bacteria and the mutated genes were mainly involved in the oxidation-reduction process, DNA repair, and replication. The SNPs were found in degQ, ks71A, vgrG, bigA, cusA, and DR76_-4702 genes, which were covered in both transcriptomic and genomic profiles. This study provides new insights into the resistance mechanism of QdNOs and increases the current data pertaining to the development of bacterial resistance under the stress of antibacterials at sub-MIC concentrations.

SUBMITTER: Gu Y

PROVIDER: S-EPMC7696260 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Development of Resistance in Escherichia coli ATCC25922 under Exposure of Sub-Inhibitory Concentration of Olaquindox.

Gu Yufeng Y Wang Shuge S Huang Lulu L Sa Wei W Li Jun J Huang Junhong J Dai Menghong M Cheng Guyue G

Antibiotics (Basel, Switzerland) 20201110 11

Quinoxaline1,4-di-N-oxides (QdNOs) are a class of important antibacterial drugs of veterinary use, of which the drug resistance mechanism has not yet been clearly explained. This study investigated the molecular mechanism of development of resistance in Escherichia coli (E. coli) under the pressure of sub-inhibitory concentration (sub-MIC) of olaquindox (OLA), a representative QdNOs drug. In vitro challenge of E. coli with 1/100× MIC to 1/2× MIC of OLA showed that the ...[more]

PMID: 33182563

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Project description:Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by Gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth new peptidoglcyan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how Gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the β-lactam antibiotics mecillinam and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all of the PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to β-lactam antibiotics. We used microarrays to identify changes in gene expression resulting from treatment of Escherichia coli with the β-lactam antibiotics cefsulodin, mecillinam, or the combination. Keywords: Dose response, stress response

Project description:Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by Gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth new peptidoglcyan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how Gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the Î²-lactam antibiotics mecillinam and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all of the PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to Î²-lactam antibiotics. We used microarrays to identify changes in gene expression resulting from treatment of Escherichia coli with the Î²-lactam antibiotics cefsulodin, mecillinam, or the combination. Experiment Overall Design: E. coli was treated with two b-lactam antibiotics, cefsulodin and mecillinam, alone at the minimum inhibitory concentration to identify the expression changes in response to antibiotic-induced peptidoglycan stress. E. coli was grown to an OD of 0.2 at which time cefsulodin or mecillinam was added. Samples of the bacteria were collected at 5 and 10 minutes after treatment with cefsulodin and 60 minutes after treatment with mecillinam. Untreated cells were also collected at the same time points as controls. Three biological replicates were collected for all time points and samples, however, only one of the 5 minute untreated samples was hybridized to the arrays. In addition, a single sample from cells treated with the combination of cefsulodin and mecillinam was hybridized to allow for comparison with samples hybridized to Affymetrix antisense genechips.

Project description:ObjectiveTo determine the effect of exposure to remnants of a phagemid-containing E. coli, killed by treatment with a propanol-based hand rub, on antimicrobial resistance in E. coli isolates.MethodsAn in vitro model was developed in which a clinical E. coli isolate (EUR1) was exposed to remnants of an E. coli K-12 strain containing a phagemid (pBS-E12) strain treated with Sterillium®. A series of 200 experiments was performed using this in vitro model. As a control, a series of 400 experiments was performed where the EUR1 was exposed either to the remnants of an E. coli K-12 strain (not containing a phagemid) (E12) treated with Sterillium® (n = 200) or to dried Sterillium® only (n = 200). The number of experiments that showed growth of an amoxicillin-resistant EUR1 isolate was evaluated in all three groups. An additional 48 experiments were performed in which a different clinical E. coli isolate (EUR2) was exposed to remnants of the pBS-E12 treated with Sterillium®. Whole-genome sequencing and phenotypic testing for AmpC beta-lactamase production was performed to investigate the mechanism behind this resistance development.ResultsIn 22 (11.0%) of 200 experiments in which the EUR1 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained, as opposed to only 2 (1.0%) of 200 experiments involving the exposure of the EUR1 to Sterillium® only (risk difference: 10.0%; 95% CI 5.4-14.6%)) and 1 (0.5%) of 200 experiments involving the exposure of the EUR1 isolate to the remnants of the phagemid-free E12 (risk difference: 10.5%; 95% CI 6.1-14.9%). In 1 (2.1%) of the 48 experiments in which the EUR2 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained. The development of resistance in all experiments was due to mutations in the promoter/attenuator region of the chromosomal AmpC beta-lactamase (cAmpC) gene leading to cAmpC hyperproduction.ConclusionExposure of an E. coli isolate to another phagemid-containing E. coli that was treated with propanol-based hand rub increased the development of amoxicillin resistance. Although phagemids are cloning vectors that are not present in clinical isolates, this finding may have implications for hand disinfection practices in healthcare facilities.

Project description:Various forms of adaptive evolution occur in clinical isolates in response to the presence of antimicrobial drugs. Among a total of 171 CTX-M-9 group/family extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli blood isolates recovered between 2016 and 2017 in six general hospitals, 50.3% of the isolates possessed the blaCTX-M-14-like gene in their chromosome rather than in a plasmid. Focusing on this unprecedented way of the blaCTX-M ESBL gene possession, molecular epidemiology of the isolates was assessed and the chromosomal location of the acquired cephalosporinase gene was dissected in an evolutionary point of view. Taking advantage of a complete collection of E. coli blood isolates from a limited period, clonal relatedness of the E. coli isolates carrying the blaCTX-M-14-like gene was clarified and the dominant clone, ST131 H30R, was identified. To control the level of resistance and the resistance spectrum to oxyimino-cephalosporin drugs, transcription level of the blaCTX-M-14-like gene was tuned finely through positioning the gene near the chromosomal initiation dnaA gene and amplifying numbers of the gene in a chromosome using either the copy-and-paste or the tandem amplification methods. Inconspicuous fitness cost by chromosomal location of the gene and free adjustment of the oxyimino-cephalosporin resistance would urge the dominancy of E. coli clinical isolates harboring the blaCTX-M ESBL gene in their chromosome. IMPORTANCE Increasing prevalence of E. coli producing CTX-M ESBL is a major concern in clinical settings because it significantly limits treatment options. Thus, it is important to keep watching current molecular mechanisms of resistance and the scheme for dissemination. Recently, chromosomal locations of the blaCTX-M genes are often documented in clinical settings and the bacterial strategies were needed to be dissected in an evolutionary point of view. Both main mechanisms of fine tuning the chromosomal gene expression, bacterial gene amplification either by copy-and-paste or by tandem amplification and positioning the gene near the chromosomal initiation dnaA gene, were demonstrated in the study, and the fitness cost by the chromosomal location was evaluated.

Dataset Information

Development of Resistance in Escherichia coli ATCC25922 under Exposure of Sub-Inhibitory Concentration of Olaquindox.

Publications

Development of Resistance in <i>Escherichia coli</i> ATCC25922 under Exposure of Sub-Inhibitory Concentration of Olaquindox.

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