Publicação
Detection and inhibition of β-lactamase activity in clinical isolates of Escherichia coli combining β-lactamase and efflux inhibitors using an MTT-based method : phenotypic and genotypic validation
| Resumo: | Antimicrobial resistance is an ever-increasing threat in the era of modern medicine, of which resistance to β-lactams has become an urgent issue as they are the most common used antibiotics. Escherichia coli counteracts β-lactams with production of β-lactamases, however efflux mechanisms and porins have been known to contribute to this resistance phenotype. In this work, we aim to establish a costeffective colorimetric method for the fast screening of β-lactamases AmpC, extended spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), carbapenemases and the simultaneous presence of increased efflux activity in E. coli for routine use. The 27 E. coli clinical strains studied were typed by ERIC-PCR. Drug susceptibility was assessed by Kirby-Bauer. Minimum inhibitory concentrations (MICs) of β-lactams were performed in presence and absence the following β-lactamase-specific inhibitors: cloxacillin for AmpC; clavulanate for ESBLs; EDTA for MBLs and boronic acid for carbapenemases. MICs of β-lactams were also performed in presence and absence the efflux inhibitors chlorpromazine, phe-arg-β-naphthylamide (PAβN) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) in combination with the β-lactamase-specific inhibitors. MICs were done using the 96-well plate microdilution method and MTT for colorimetric detection. The results were validated using the phenotypic standard methods for β-lactamases detection by disc diffusion and the screening of genes coding β-lactamases. β-lactamase production was detected in all clinical strains and included the presence of AmpC, TEM with OXA-1, SHV, or CTX-M or AmpC overexpression. We were able to reduce the MICs of β-lactams in the presence of the efflux inhibitors in combination with the β-lactamase-specific inhibitors demonstrating that despite the presence β-lactamases, the MICs of β-lactams can be reduced in the presence of efflux inhibitors. In this work we optimize a method for the simultaneous detection of β-lactamases AmpC, ESBLs, MBLs, carbapenemases and efflux activity. This method has the potential to be used in low-income countries since does not require expensive reagents nor specialized equipment. When used in association with a colorimetric reagent for the detection, as MTT, the interpretation of the results is straightforward being independent of the operator, avoiding false-negative as well false-positive results. Based on the results obtained, the synergistic effect observed between β-lactamase inhibitors and efflux pump inhibitors suggest the accumulation of β-lactams within the bacteria, thus potentiating their bactericide action. |
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| Autores principais: | Serra, Débora Carvalho |
| Assunto: | Escherichia coli β-lactâmicos β-lactamases Bombas de efluxo Inibidores de efluxo MTT Escherichia coli β-lactams β-lactamases Efflux pumps Efflux inhibitors MTT |
| Ano: | 2020 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Antimicrobial resistance is an ever-increasing threat in the era of modern medicine, of which resistance to β-lactams has become an urgent issue as they are the most common used antibiotics. Escherichia coli counteracts β-lactams with production of β-lactamases, however efflux mechanisms and porins have been known to contribute to this resistance phenotype. In this work, we aim to establish a costeffective colorimetric method for the fast screening of β-lactamases AmpC, extended spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), carbapenemases and the simultaneous presence of increased efflux activity in E. coli for routine use. The 27 E. coli clinical strains studied were typed by ERIC-PCR. Drug susceptibility was assessed by Kirby-Bauer. Minimum inhibitory concentrations (MICs) of β-lactams were performed in presence and absence the following β-lactamase-specific inhibitors: cloxacillin for AmpC; clavulanate for ESBLs; EDTA for MBLs and boronic acid for carbapenemases. MICs of β-lactams were also performed in presence and absence the efflux inhibitors chlorpromazine, phe-arg-β-naphthylamide (PAβN) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) in combination with the β-lactamase-specific inhibitors. MICs were done using the 96-well plate microdilution method and MTT for colorimetric detection. The results were validated using the phenotypic standard methods for β-lactamases detection by disc diffusion and the screening of genes coding β-lactamases. β-lactamase production was detected in all clinical strains and included the presence of AmpC, TEM with OXA-1, SHV, or CTX-M or AmpC overexpression. We were able to reduce the MICs of β-lactams in the presence of the efflux inhibitors in combination with the β-lactamase-specific inhibitors demonstrating that despite the presence β-lactamases, the MICs of β-lactams can be reduced in the presence of efflux inhibitors. In this work we optimize a method for the simultaneous detection of β-lactamases AmpC, ESBLs, MBLs, carbapenemases and efflux activity. This method has the potential to be used in low-income countries since does not require expensive reagents nor specialized equipment. When used in association with a colorimetric reagent for the detection, as MTT, the interpretation of the results is straightforward being independent of the operator, avoiding false-negative as well false-positive results. Based on the results obtained, the synergistic effect observed between β-lactamase inhibitors and efflux pump inhibitors suggest the accumulation of β-lactams within the bacteria, thus potentiating their bactericide action. |
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