Sedani, Keval (2020) The action of metal cations on antimicrobial breakpoints in veterinary isolates of 'Escherichia coli' and 'Pseudomonas' spp. (MSc(R) thesis), Kingston University, .
Abstract
Antibiotic use and misuse since their introduction have led to increasing bacterial resistance which coupled with the lack of development of new antimicrobials poses a global threat to both human and veterinary treatments. As a result of antibiotic resistance, treating bacterial infections in human and agricultural populations has become more difficult. A gold standard method for testing antimicrobial susceptibility of clinical isolates was adopted within the UK in the 1950s with the current standard recommended by EUCAST. However, no set guidance is available for testing agricultural isolates with very few antibiotic breakpoints available. The overall aim of this study was to identify whether certain metal cations within testing media affects the susceptibility of clinical and agricultural Pseudomonas aeruginosa and Escherichia coli isolates towards 4 panels of antimicrobials and to determine if this could lead to over or under reporting the efficacy of antimicrobials. A representative sample of bovine gut (BG) and environmental (ENV) isolates from previous research at Kingston University were selected for use within this study, totalling 113 bacterial isolates. These had been previously identified as P. aeruginosa (14 BG and 17 ENV) and E. coli (63 BG and 19 ENV) with their antimicrobial resistance profiles confirmed. High tetracycline and β-lactam resistance was observed in the initial study, with multidrug resistance seen in many of the isolates. Following confirmational testing of antimicrobial susceptibility, A proportion of these isolates (N=40) distributed between both species was redetermined using the EUCAST method and changes in antimicrobial susceptibility profiles previously noted in 20 of the isolates selected for progression in this project identified. Increases in antimicrobial susceptibility was seen in 6 of 10 E. coli isolates (4 BG and 2 ENV) and 8 of 10 P. aeruginosa (3 BG and 5 ENV) isolates now displaying sensitivity towards at least one antimicrobial following antimicrobial susceptibility testing. An attempt to re-establish initial antimicrobial susceptibility profiles through double exposure towards an antimicrobial was achieved with β-lactam and tetracycline resistance seen to return in the environmental isolates of E. coli (n=3) and P. aeruginosa (n=3). The EUCAST gold standard antimicrobial susceptibility testing method utilises media which contains trace amounts cations to allow for homeostasis within the bacterial cultures. The concentration of cations varies from manufacturer which has been seen to affect the susceptibility of antimicrobials on isolates (Girardello et al., 2012). Cations may be seen as a contributing factor to antimicrobial resistance with their ability to alter ion gradients within and around the cell affecting cell surface charges and interactions with protein channels like efflux pumps which are present in prokaryotes as well as interacting with antimicrobials contributing to development of antimicrobial resistance. Efflux pump inhibitors are used to target efflux pumps within prokaryotes and have been studied in an attempt to combat antimicrobial resistance with several modes of actions proposed. The main focus of efflux pump inhibitors are as potential therapeutic agents to rejuvenate antimicrobials which have lost activity. The efflux pump inhibitor Phenylalanine arginine β-naphthylamide (PAβN) was selected as a well-studied inhibitor and a target for the resistant nodule division efflux pumps which have a strong prevalence in both E. coli and P. aeruginosa. A final concentration of 10 µg/ml was utilised in this study to identify changes in minimum inhibitory concentration of: Cefoxitin, Ceftazidime, Cefepime, Ciprofloxacin, Imipenem, Amoxicillin, Amoxicillin+ Clavulanic acid and Tetracycline. Some changes in the minimum inhibitory concentration of ceftazidime against P. aeruginosa bovine gut isolates with one isolate showing a decrease from 1 μg/mL on Mueller-Hinton agar to 0.5 μg/mL with the cation adjusted media and with Phenylalanine arginine β- naphthylamide. A different isolate showing a decrease with the presence of the efflux pump inhibitor and cation adjusted media from 6 μg/mL to 4 μg/mL towards cefepime. A decrease in minimum inhibitory concentration for cefepime was also observed in an E. coli environmental isolate with the combination efflux pump inhibitor and cation adjusted media with a decrease against normal Mueller-Hinton agar from 0.125 μg/mL to 0.064 μg/mL. The preliminary results indicate that changes in antimicrobial susceptibility testing conditions like increased concentration of cations and the presence of efflux pump inhibitors can affect the susceptibility of isolates towards antimicrobials and lead to changes in reporting of isolate susceptibility. However, these were initial observations and further testing is required with various concentrations of Phenylalanine arginine β-naphthylamide and cations present within the media to confirm these results on veterinary isolates.
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