Nitric oxide tolerance and antimicrobial susceptibility in Escherichia coli clinical isolates

The emergence of antibiotic resistance has been a serious concern for the last few decades. It hinders the treatment of infectious diseases, raising mortality and morbidity rates, as well as increasing the cost of healthcare. To investigate the problem of antibiotic resistant E. coli, 50 E. coli bacteraemia clinical isolates (Kent collection) were collected from East Kent Hospitals University NHS Foundation and phenotypically/genotypically characterized for antibiotic resistance, virulence factors, and the presence of putative plasmids. High levels of resistance were detected for amoxicillin and trimethoprim, and 14% of the isolates showed a multidrug-resistant phenotype. ST73 isolates exhibited the highest virulence potential while ST131 exhibited the highest levels of antibiotic resistance, although no correlation was detected between the two variables. In accordance with previous observations, co-carriage of CTX-M-15 and aac(3)-IIa, aac(6')Ib-cr, and blaOXA was observed in the collection, providing a possible explanation on why ESBL-producing isolates are often multidrug resistant.

The production of nitric oxide (NO) by the mammalian immune system and its well-known anti-bacterial properties has prompted the investigation exogenously administered NO as an alternative to antibiotics. While combinatorial treatments of NO and antibiotics have proved to be successful against bacterial biofilms, this strategy has not been investigated in planktonic bacterial cells. Moreover, recent studies have shown that the generation of reactive oxygen species (ROS) resulting from hyperactivation of the aerobic respiratory chain of E. coli occurs in response to treatment with bactericidal antibiotics. This secondary effect of antibiotics is an important part of the as part of the lethality of bactericidal antibiotics under aerobic conditions. Given that NO is a well-known respiratory inhibitor, it was hypothesised this would diminish the toxic effects of antibiotics. To test this hypothesis, the effect of NO upon the lethality of a bactericidal antibiotic (gentamicin) was tested on a multidrug resistant E. coli. Pre-exposure to the NO-donor GSNO or NOC-12 prior to gentamicin treatment was found to increase bacterial tolerance to the antibiotic in planktonic cells: the presence of NO elicits a 10-fold increase in IC50 for gentamicin lethality against an E. coli clinical isolate. Further investigation showed that cytochrome bd-I, a NO-tolerant respiratory oxidase expressed maximally under microaerobic conditions in E. coli, is largely responsible for the sensitization of E. coli to gentamicin during NO exposure.

The work herein reports that NO elicits a dramatic increase in the tolerance of E. coli to antibiotics. Hence, this work has revealed a huge void in knowledge related to

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antibiotic potency during conditions relevant to infection (i.e. in the presence of NO). Furthermore, this work reveals that the cytochrome bd-I respiratory oxidase sensitises E. coli to antibiotics in the presence of NO. These findings shed light on how NO encountered during infection could impair the function of antibiotics and will prompt future research into how controlling levels of respiratory inhibition during infection may be used to improve antibiotic efficacy.