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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
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RESUMO: A resistência aos antibióticos é uma ameaça cada vez maior na era da medicina moderna, na qual a
resistência aos β-lactâmicos se tornou um tema premente uma vez que é a classe de antibióticos mais usada
na clínica. Escherichia coli neutraliza os β-lactâmicos através da produção de β-lactamases, no entanto, a
actividade de efluxo e redução da expressão de porinas também contribuem para este fenótipo de
resistência. Neste trabalho, pretendemos optimizar um método colorimétrico económico para a detecção
rápida de β-lactamases AmpC, β-lactamases de largo espectro (ESBLs), metallo-β-lactamases (MBLs), e
carbapenemases, bem como a detecção simultânea de actividade de efluxo em E. coli para uso de rotina.
As 27 estirpes clínicas de E. coli estudadas foram genotipadas por ERIC-PCR. A susceptibilidade aos
antibióticos foi avaliada por Kirby-Bauer. As concentrações mínimas inibitórias (CMIs) dos β-lactâmicos foram
determinadas na presença e ausência dos seguintes inibidores específicos de β-lactamases: cloxacillina para
AmpC; clavulanato para ESBLs; EDTA para MBLs e ácido borónico para carbapenemases. As CMIs dos βlactâmicos foram também realizadas na presença e ausência dos inibidores de efluxo clorpromazina, “phearg-β-naphthylamide” (PAβN) e “carbonyl cyanide m-chlorophenyl hydrazone” (CCCP) em combinação com
os inibidores de β-lactamases. As CMIs foram efectuadas usando o método de microdiluição em placa de 96
poços e MTT para detecção colorimétrica. Os resultados foram validados usando os métodos fenotípicos
padrão para detecção de β-lactamases por difusão em disco e a detecção de genes que codificam βlactamases.
A produção de β-lactamasesfoi detectada em todas as estirpes clínicas e incluiu a presença de AmpC,
TEM com OXA-1, SHV, ou CTX-M ou sobrexpressão de AmpC. Foi possível reduzir as CMIs dos β-lactâmicos
na presença dos inibidores de efluxo em combinação com os inibidores de β-lactamases demonstrando que,
apesar da presença de β-lactamases, as CMIs dos β-lactâmicos podem ser reduzidas na presença de
inibidores de efluxo.
Neste trabalho, optimizámos um método para a detecção simultânea de β-lactamases AmpC, ESBLs,
MBLs, carbapenemases e actividade de efluxo. Este método apresenta potencialidade para ser usado em
países em desenvolvimento uma vez que é um método de fácil execução e não necessita de reagentes
dispendiosos nem de equipamento especializado. Quando associado ao uso de um composto colorimétrico
para a detecção, como o MTT, a interpretação dos resultados é directa uma vez que não está dependente do
operador, evitando deste modo resultados falso-negativos bem como falsos-positivos.
Baseado nos resultados obtidos, o efeito sinérgico observado entre inibidores de β-lactamases e
inibidores de efluxo sugere um aumento da concentração de β-lactâmicos no interior da célula, aumentando
assim a sua acção bactericida.
ABSTRACT: 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.
ABSTRACT: 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.
Descrição
Palavras-chave
Escherichia coli β-lactâmicos β-lactamases Bombas de efluxo Inibidores de efluxo MTT Escherichia coli β-lactams β-lactamases Efflux pumps Efflux inhibitors MTT