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Realism-based Approaches for Evaluating Bacterial Susceptibility to Antimicrobials used in Home and Personal Care Products
[Thesis]. Manchester, UK: The University of Manchester; 2016.
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Abstract
ABSTRACT OF THESIS submitted by Nicola Louise Cowley for the Degree of Doctor of Philosophy entitled Realism-based Studies for Evaluating Bacterial Susceptibility to Antimicrobials used in Home and Personal Care Products. May 2016.Microbicides are used in consumer products worldwide to enhance their antibacterial potency in disinfection, for antisepsis or as preservatives. With the widespread use of these compounds, concerns have been expressed about their potential to select for reduced susceptibility. Whilst in vitro studies have reported the laboratory generation of bacterial insusceptibility for certain combinations of bacterium and microbicide, true microbicide resistance, which is defined as a change in susceptibility that is likely to affect the outcome of treatment, has not been frequently reported or observed in the environment. Importantly, risk assessments on the use of microbicides have been largely based on laboratory studies whereby pure cultures of bacteria are exposed to microbicides in aqueous solution. In use however, microbicides are formulated with various excipients and bacteria are exposed to them most commonly in complex biofilm communities. Work described in this doctoral thesis evaluates the effects of exposing bacteria to microbicides using exposure conditions intended to increase realism, with the ultimate aim of informing improved risk assessment methods that better reflect deployment of microbicides in the real-world, taking in to account the effects of formulation, growth in multi-species communities and potential reduced competiveness in adapted bacteria. Test bacteria (8 species, 7 genera) were repeatedly exposed to selected microbicides in aqueous solution and in various formulations reflecting their use in the domestic environment, such as general-purpose cleaners and laundry detergents. Minimum inhibitory concentrations, minimum bactericidal concentrations and minimum biofilm eradication concentrations were determined before and after 14 passages (P14) in the presence of microbicides (benzalkonium chloride (BAC), benzisothiazolinone (BIT), chlorhexidine (CHX), didecyldimethyl ammonium chloride (DDAC), Glydant (DMDM-hydantoin), polyhexamethylene biguanide (PHMB), thymol and triclosan) in aqueous solution or in formulation, using a previously validated gradient plating system. Bacteria were subsequently passaged a further 14 times in the absence of any antimicrobial agent to determine the stability of any adaptations (X14). In bacterial isolates that demonstrated marked changes in susceptibility, further phenotypic analysis was conducted to test for any induced alterations in antibiotic susceptibility, planktonic growth rate, biofilm formation, competitive fitness and relative pathogenicity. Exposure of microbial communities was carried out using a previously validated domestic drain biofilm simulator within constant depth film fermenters (CDFF). The CDFFs were exposed to increasing concentrations of BAC in aqueous solution or BAC formulation over a 32-week period. Changes in bacterial community composition and antimicrobial susceptibility distributions were assessed via replica plating onto selective and antimicrobial-containing agars as well as through the use of next generation sequencing technologies via the illumina Miseq platform and QIIME software. The formulation of microbicides significantly increased antibacterial and anti-biofilm potency and reduced the incidence and extent of the development of insusceptibility isolated bacteria (7 non-revertible bacteria in MBCs for microbicides, whilst 2 non-revertible bacteria in MBCs for formulations). In bacteria that develop marked changes in antimicrobial susceptibility after repeated exposure show changes to biofilm growth rates (10 increases and 6 decreases after microbicide exposure; 1 increase and 3 decreases after formulation exposure), as well as alterations in competitive fitness (6 decreases and 19 increases after microbicide exposure; whilst all exposed to formulation had decreased fitness) and virulence (9/13 decreased and 1/13 increased after microbicide exposure; 4/7 decreased and 2/7 increased after formulation exposure). In the multispecies microcosm system, long-term exposure to BAC or BAC formulation induced shifts in bacterial community dynamics and resulted in a decrease in BAC and various antibiotic susceptibilities (1 log10 reduction in the BAC system; 2.5 log10 reduction in the BAC formulation system of viable bacteria). Such shifts in community dynamics after antimicrobial treatment are theorised to be mainly due to clonal expansion of innately insusceptible bacteria (abundance of Achromobacter sp. increased by 39% in BAC system and 10% in BAC formulation system). Understanding the potential selectivity of microbicide-containing formulations is likely to better serve by testing formulations as well as actives in aqueous solutions. This highlights the need to conduct risk assessments of induced microbicide susceptibility changes using conditions that more accurately reflect their deployment.