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Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli.

ABSTRACT: A genetic approach was undertaken to identify normal bacterial genes whose products function to limit the effective concentration of antibiotics. In this approach, a multicopy plasmid library containing cloned Escherichia coli chromosomal sequences was screened for transformants that showed increased resistance to a number of unrelated antibiotics. Three such plasmids were identified, and all contained sequences originating from the mar locus. DNA sequence analysis of the minimal complementation unit revealed that the resistance phenotype was associated with the presence of the marA gene on the plasmids. The putative marA gene product is predicted to contain a helix-turn-helix DNA binding domain that is very similar to analogous domains found in three other E. coli proteins. One such similarity was to the SoxS gene product, the elevated expression of which has previously been associated with the multiple antibiotic resistance (Mar) phenotype. Constitutive expression of marA conferred antibiotic resistance even in cells carrying a deletion of the chromosomal mar locus. We have also found that transformants bearing marA plasmids show a significant reduction in ompF translation but not transcription, similar to previously described mar mutants. However, this reduction in ompF expression plays only a minor role in the resistance mechanism, suggesting that functions encoded by genes unlinked to mar must be affected by marA. These results suggest that activation of marA is the ultimate event that occurs at the mar locus during the process that results in multiple antibiotic resistance.

SUBMITTER: Gambino L

PROVIDER: S-EPMC204606 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

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Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli.

Gambino L L Gracheck S J SJ Miller P F PF

Journal of bacteriology 19930501 10

A genetic approach was undertaken to identify normal bacterial genes whose products function to limit the effective concentration of antibiotics. In this approach, a multicopy plasmid library containing cloned Escherichia coli chromosomal sequences was screened for transformants that showed increased resistance to a number of unrelated antibiotics. Three such plasmids were identified, and all contained sequences originating from the mar locus. DNA sequence analysis of the minimal complementation ...[more]

PMID: 8491710

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Project description:Atypical antipsychotic (AAP) medication is a critical tool for treating symptoms of psychiatric disorders. While AAPs primarily target dopamine (D2) and serotonin (5HT2A and 5HT1A) receptors, they also exhibit intrinsic antimicrobial activity as an off-target effect. Because AAPs are often prescribed to patients for many years, a potential risk associated with long-term AAP use is the unintended emergence of bacteria with antimicrobial resistance (AMR). Here, we show that exposure to the AAP quetiapine at estimated gut concentrations promotes AMR in Escherichia coli after 6 weeks. Quetiapine-exposed isolates exhibited an increase in MICs for ampicillin, tetracycline, ceftriaxone, and levofloxacin. By whole-genome sequencing analysis, we identified mutations in genes that confer AMR, including the repressor for the multiple antibiotic resistance mar operon (marR), and real-time reverse transcription-quantitative PCR (RT-qPCR) analysis showed increased levels of marA, acrA, and tolC mRNAs and reduced levels of ompF mRNA in the isolates carrying marR mutations. To determine the contribution of each marR mutation to AMR, we constructed isogenic strains carrying individual mutant marR alleles in the parent background and reevaluated their resistance phenotypes using MIC and RT-qPCR assays. While marR mutations induced robust activity of the mar operon, they resulted in only modest increases in MICs. Interestingly, although these marR mutations did not fully recapitulate the AMR phenotype of the quetiapine-exposed isolates, we show that marR mutations promote growth fitness in the presence of quetiapine. Our findings revealed an important link between the use of AAPs and AMR development in E. coli. IMPORTANCE AAP medication is a cornerstone in the treatment of serious psychiatric disease. The AAPs are known to exhibit antimicrobial activity; therefore, a potential unintended risk of long-term AAP use may be the emergence of AMR, although such risk has received little attention. In this study, we describe the development of multidrug antibiotic resistance in Escherichia coli after 6 weeks of exposure to the AAP quetiapine. Investigation of mutations in the marR gene, which encodes a repressor for the multiple antibiotic resistance (mar) operon, reveals a potential mechanism that increases the fitness of E. coli in the presence of quetiapine. Our findings establish a link between the use of AAPs and AMR development in bacteria.

Project description:A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced. Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites. To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids. To the right (region II) is a transcriptional unit containing three putative ORFs (ORF125/144, ORF129, and ORF72). Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (ORF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion. One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or ORF144 (a 1-bp change). All six mutations led to increased transcription of the region II transcript. High-copy-number plasmids containing marO and the adjacent ORF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells. High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain. The nature of the Mar mutations and the results of the complementation studies suggest that ORF125/144 encodes a repressor (designated MarR) which acts at marO. The second ORF (ORF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators. A Tn5 insertion in marA inactivated the multiresistance phenotype of Mar mutants. The function of ORF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.

Project description:Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 microg/ml), kanamycin (25 microg/ml), and ofloxacin (10 microg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.

Dataset Information

Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli.

Publications

Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli.

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