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1.
Sci Rep ; 12(1): 12149, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35840757

RESUMO

Bacterial resistance to the antiseptic chlorhexidine (CHX), is a growing problem, recently shown to be caused by deleterious mutations to the phospholipid transport system component (mlaA) as well as efflux pump overexpression. Comparisons of CHX resistance mechanisms, such as porin deletions (ompCF), and over-expressed efflux pumps (acrB, qacE, aceI), are lacking and may be distinguishable using antiseptic rapid fluorescent dye testing assays. Using E. coli K-12 CHX adapted isolates (CHXR1), gene deletion mutants, and over-expressed transformants the phenotypes of these CHX resistance genes were compared using antimicrobial susceptibility tests (AST), rapid fluorescent propidium iodide dye-based membrane integrity assays (RFDMIA), and scanning electron microscopy (SEM). AST findings showed CHXR1, ΔacrB, ΔompCF, and transformants pCA24N-aceI and pCA24N-mlaA conferred greater (two to fourfold) MIC changes when compared to matched controls. Examination of these mutants/transformants using CHX RFDMIA showed that porin dual-deletions (ΔompCF) and mlaA alterations (ΔmlaA; pCA24N-mlaA, CHXR1) were distinguishable from controls. Results for over-expressed (pMS119EH-aceI) and deleted (ΔacrB) efflux pump RFDMIA could not be distinguished with propidium iodide, only with ethidium bromide, suggesting propidium iodide is better suited for detecting porin and mlaA associated CHX resistance mechanisms. SEM of CHXR1 and unadapted E. coli cells exposed to increasing CHX concentrations revealed that CHX does not visibly damage cell envelope integrity at any tested concentration but did identify elongated CHXR1 cells. ΔmlaA confers similar levels of CHX resistance as efflux overexpression and porin deletions, however, only outer membrane-altering porin and mlaA deletions can be reliably distinguished using RFDMIA.


Assuntos
Anti-Infecciosos Locais , Proteínas de Escherichia coli , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Clorexidina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Corantes Fluorescentes , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fenótipo , Porinas/genética , Propídio
2.
Sci Rep ; 10(1): 20543, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239659

RESUMO

Biocides such as quaternary ammonium compounds (QACs) are potentially important contributors towards bacterial antimicrobial resistance development, however, their contributions are unclear due to a lack of internationally recognized biocide testing standards. Methods to detect QAC tolerance are limited to laborious traditional antimicrobial susceptibility testing (AST) methods. Here, we developed a rapid fluorescent dye-based membrane impermeant assay (RFDMIA) to discriminate QAC susceptibility among Gram-negative Enterobacterales and Pseudomonadales species. RFDMIA uses a membrane impermeant fluorescent dye, propidium iodide, in a 30-min 96-well fluorescent microplate-based assay where cell suspensions are exposed to increasing QAC concentrations. Our results demonstrate that RFDMIA can discriminate between QAC-susceptible and QAC-adapted Escherichia coli tolerant phenotypes and predict benzalkonium and cetrimide tolerance in all species tested except for intrinsically fluorescent Pseudomonas aeruginosa. RFDMIA identified a close association to minimum inhibitory concentration values determined by broth microdilution AST and increasing fluorescent dye emission values. RFDMIA emission values and scanning electron microscopy results also suggest that CET-adapted E. coli isolates have a CET dependence, where cells require sub-inhibitory CET concentrations to maintain bacilliform cell integrity. Overall, this study generates a new, rapid, sensitive fluorescent assay capable of detecting QAC-susceptible Gram-negative bacteria phenotypes and cell membrane perturbations.


Assuntos
Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana/métodos , Compostos de Amônio Quaternário/farmacologia , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Anti-Infecciosos Locais/farmacologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Compostos de Benzalcônio/farmacologia , Desinfetantes/farmacologia , Corantes Fluorescentes/farmacologia , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-33077665

RESUMO

Here, we characterize the fosA genes from three Escherichia coli clinical isolates recovered from Canadian patients. Each fosA sequence was individually overexpressed in E. coli BW25113, and antimicrobial susceptibility testing was performed to assess their role in fosfomycin resistance. The findings from this study identify and functionally characterize FosA3, FosA8, and novel FosA7 members and highlight the importance of phenotypic characterization of fosA genes.


Assuntos
Infecções por Escherichia coli , Fosfomicina , Antibacterianos/farmacologia , Canadá , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Infecções por Escherichia coli/tratamento farmacológico , Fosfomicina/farmacologia , Hospitais , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos , beta-Lactamases/genética
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