RESUMEN
The antibacterial oxidative response, which relies on the production of hydrogen peroxide (H2O2) and hypothiocyanite (OSCN-), is a major line of defense protecting the human airway epithelium (HAE) from lesions when infected. The in vitro studies of the oxidative responses are performed mainly by one-shot H2O2 exposure that does not recapitulate the complex H2O2/LPO/SCN- system releasing the reactive oxygen species in airway secretions. A cell-free in vitro assay mimicking this system has been described but was not fully characterized. Here, we comprehensively characterized the hourly H2O2/OSCN- concentrations produced within this in vitro assay and assessed the resistance of Pseudomonas aeruginosa and Staphylococcus aureus clinical strains to the HAE oxidative response. We found that H2O2/OSCN- were steadily produced from 7h and up to 25h, but OSCN- was detoxified in 15 minutes by bacteria upon exposure. Preliminary tests on PA14 showed survival rates at 1-hour post-exposure (hpe) to H2O2 of roughly 50% for 105 and 107 colony-forming unit (CFU)/mL inocula, while 102 and 104 CFU/mL inocula were cleared after one hpe. Thirteen clinical strains were then exposed, highlighting that conversely to P. aeruginosa, S. aureus showed resistance to oxidative stress independently of its antibiotic resistance phenotype. Our results demonstrated how this in vitro assay can be helpful in assessing whether pathogens can resist the antibacterial oxidative HAE response. We anticipate these findings as a starting point for more sophisticated in vitro models that could serve as high-throughput screening for molecules targeting the bacterial antioxidant response.
Asunto(s)
Peróxido de Hidrógeno , Estrés Oxidativo , Pseudomonas aeruginosa , Staphylococcus aureus , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/metabolismo , Oxidación-Reducción , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/metabolismo , TiocianatosAsunto(s)
Neumonía Bacteriana , Humanos , Neumonía Bacteriana/diagnóstico , Neumonía Bacteriana/microbiología , Infecciones por Klebsiella/diagnóstico , Infecciones por Klebsiella/microbiología , Enterobacter aerogenes/aislamiento & purificación , Enterobacter aerogenes/genética , Masculino , Anciano , Técnicas de Diagnóstico Molecular/métodos , Femenino , Persona de Mediana EdadRESUMEN
An increase of carbapenemase-producing Bacteroides fragilis infections is observed. To detect such a resistance in B. fragilis, several tests exist that are expensive or show poor sensitivity and specificity. Therefore, we upgraded the Anaerobic Carbapenem Inactivation Method (Ana-CIM) to easily screen for carbapenemase-producing B. fragilis. The presence of carbapenemase cfiA gene was identified in 50 B. fragilis isolates by PCR. We modified the Ana-CIM by (1) increasing the bacterial inoculum, and (2) measuring the differences in diameter between the negative control and the testing disc. We correctly classified the cfiA-negative and positive isolates and could define a cut-off of positivity at 2 mm. Our modified Ana-CIM allowed to correctly discriminate the 31 cfiA-positive with meropenem MICs ranging from 1 to > 32 µg/mL. We anticipate that our modified Ana-CIM could be used in most clinical laboratories to easily screen for carbapenemase-producing B. fragilis, even at low levels.