Impact of biocide exposure on bacterial biofilm formation and efflux systems

: (Alternative Format Thesis)

Abstract

Proteus mirabilis and Klebsiella pneumoniae are common pathogens of the catheterised urinary tract. Indwelling urinary catheters are susceptible to biofilm formation and in the case of P. mirabilis, these biofilms can become crystalline resulting in serious complications. Control of catheter associated urinary tract infections largely relies on strategies to prevent infection, in which biocides play an important role. However, there are reports of reduced susceptibility to biocides in some clinically relevant bacteria. Bacterial efflux has been implicated in both reduced biocide susceptibility and biofilm formation and it was therefore hypothesised that adaptation to biocides may also alter biofilm formation.
Here it is shown that the SmvAR efflux system is important for modulating biocide susceptibility in P. mirabilis. Mutations in smvR or its associated promoter were identified in RS47, a clinical isolate with reduced biocide susceptibility, and P. mirabilis isolates following adaptation to the biocides chlorhexidine (CHD), octenidine (OCT) and benzalkonium chloride (BZK). These mutations derepressed the SmvAR efflux system, reducing susceptibility to biocides. As has previously been reported, following CHD adaptation, mutations in smvR were also found in K. pneumoniae isolates.
In addition to the SmvAR efflux system, mutations were also identified in genes that are related to the cell envelope. In P. mirabilis, mutations were found in mipA, encoding an MltA-interacting protein, in both CHD adapted isolates and RS47. Whilst the exact role of MipA remains unresolved, it was capable of modulating CHD susceptibility whilst having no effect on the MIC of OCT or BZK. P. mirabilis is intrinsically resistant to polymyxins and although RS50a was resistant to the polymyxins tested, it had significantly lower MICs compared to other P. mirabilis isolates tested (>64-fold difference). This was predicted to be due to a glycine to aspartic acid change at position 211 in rppA, encoding a response regulator transcription factor. Following adaptation to biocides, this residue was changed to asparagine and the polymyxin MICs were restored to levels seen in other isolates. Similarly, as has been reported before, cross-resistance to polymyxins in CHD adapted K. pneumoniae was identified and predicted to be due to mutations in phoQ. Both rppA and phoQ have roles in the modification of lipid A phosphates with 4-amino-4deoxy-L-arabinose which is known to reduce polymyxin susceptibility.
Adaptation to BZK and deletions in genes encoding a major facilitator superfamily efflux pump and transcriptional regulator EmrR was only identified in P. mirabilis RS1 isolates.
The same deletion also occurred in wild type (WT) and no biocide control populations albeit at lower frequencies.
Overall, there were no significant changes in biofilm formation in biocide adapted P. mirabilis or K. pneumoniae isolates. In the case of P. mirabilis, adapted isolates were capable of persisting, elevating the pH of residual urine and blocking the catheters to the same extent as WT isolates in in vitro bladder models. A mutation in bamE was observed in a single CHD adapted K. pneumoniae and predicted to effect biofilm formation in a 96-well crystal violet assay. However, in the in vitro bladder model there was no significant difference in biofilm formation when compared to its parental WT.
Collectively, the data produced in this study show that the SmvAR efflux system is important for CHD, OCT and BZK susceptibility in both clinical and laboratory adapted P. mirabilis isolates. Mutations in genes associated with the cell envelope, such as phoQ, mipA and rppA also occur following P. mirabilis and K. pneumoniae biocide adaptation. Increases in polymyxin resistance can occur in adapted K. pneumoniae and P. mirabilis isolates raising concern that the use of biocides may promote resistance to this critical antibiotic.

Date of Award	22 Jun 2022
Original language	English
Sponsors	Medical Research Council
Supervisor	Brian Jones (Supervisor), Mark Sutton (Supervisor), Lucy Bock (Supervisor), Wendy Macfarlane (Supervisor) & Emma Denham (Supervisor)