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Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes
Published on: March 3, 2023
Escherichia coli from Human Wounds: Analysis of Resistance to β-Lactams and Expression of RND Efflux Pumps
Martin Rihacek1, Michaela Kuthanova1, Zbynek Splichal1
1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic.
Purpose:
Resistance of pathogenic strains of Escherichia coli to β-lactams, particularly to ampicillin, is on the rise and it is attributed to intrinsic and acquired mechanisms. One important factor contributing to resistance, together with primarily resistance mechanisms, is a mutation and/or an over-expression of the intrinsic efflux pumps in the resistance-nodulation-division (RND) superfamily. Among these efflux pumps, AcrA, AcrB, TolC, and AcrD play an important role in antimicrobial co-resistance, including resistance to β-lactams.
Materials And Methods:
Twelve E. coli isolates obtained from patients' wounds and the control strain of E. coli ATCC 25922 were analyzed. The phenotypic resistance of these isolates to selected β-lactams was assessed by determination of the minimal inhibitory concentration. Additionally, the prevalence of β-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaAmpC) was screened by PCR. Real-time qPCR was used to determine the expression of the selected efflux pumps acrA, acrB, tolC, and acrD and the repressor acrR after the exposure of E. coli to ampicillin.
Results:
Phenotypic resistance to β-lactams was detected in seven isolates, mainly to ampicillin and piperacillin. This was corroborated by the presence of at least one acquired bla gene in each of these isolates. Although E. coli strains varied in the expression of RND-family efflux pumps after the ampicillin exposure, their gene expression indicated that these pumps did not play a major role in the phenotypic resistance to ampicillin.
Conclusion:
Each E. coli isolate displayed unique characteristics, differing in minimum inhibitory concentration (MIC) values, prevalence of acquired blaTEM and blaCTX-M genes, and expression of the RND-family pumps. This together demonstrates that these clinical isolates employed distinct intrinsic or acquired resistance pathways for their defense against ampicillin. The prevalence and spread of ampicillin resistant E. coli has to be monitored and the search for ampicillin alternatives is needed.
Insights
Ampicillin-resistant Escherichia coli strains exhibit diverse resistance mechanisms, with efflux pumps not being the primary driver of resistance. Monitoring resistant strains and exploring ampicillin alternatives are crucial.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Rising resistance of pathogenic Escherichia coli to β-lactams, especially ampicillin, is a growing public health concern.
- Intrinsic and acquired resistance mechanisms, including mutations and overexpression of resistance-nodulation-division (RND) superfamily efflux pumps (AcrA, AcrB, TolC, AcrD), contribute to antimicrobial co-resistance.
Purpose of the Study:
- To analyze the phenotypic resistance and underlying genetic mechanisms of E. coli isolates from patient wounds.
- To investigate the role of β-lactamase genes and RND-family efflux pump expression in ampicillin resistance.
Main Methods:
- Phenotypic resistance to β-lactams was assessed using minimal inhibitory concentration (MIC) determination.
- PCR was employed to screen for β-lactamase genes (blaTEM, blaCTX-M, blaSHV, blaAmpC).
- Real-time qPCR quantified the expression of efflux pump genes (acrA, acrB, tolC, acrD) and the repressor acrR following ampicillin exposure.
Main Results:
- Seven out of twelve E. coli isolates showed phenotypic resistance to β-lactams, primarily ampicillin and piperacillin, correlating with the presence of acquired bla genes.
- While E. coli strains showed varied expression of RND-family efflux pumps post-ampicillin exposure, these pumps did not appear to be the major contributors to phenotypic ampicillin resistance.
- Each isolate presented unique resistance profiles, including varying MIC values, gene prevalence, and efflux pump expression levels.
Conclusions:
- E. coli clinical isolates utilize distinct intrinsic or acquired resistance pathways against ampicillin.
- The study highlights the need for continuous monitoring of ampicillin-resistant E. coli prevalence and the urgent search for alternative antibiotics.
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