Antimicrobial resistance (AMR) poses an ever-increasing threat to public health; the prevalence of resistant bacterial strains has reduced the clinical efficacy of many existing therapeutics and is therefore contributing to rising mortality rates due to difficult to treat bacterial infections. Two key approaches used to mitigate the threat of AMR are the discovery of novel therapeutics with activity against these resistant strains, and educating the wider public about the impact of AMR, and steps that can be taken to reduce the development of resistance. We are combining both approaches to enhance the impact of our public engagement activities. During a recent event at the University of Plymouth, a member of the public isolated the bacterial strain ‘36A’ from the button of a lift control panel. Simultaneous antagonistic screening identified antimicrobial activity against a range of both Gram-positive and Gram-negative bacteria. 36A was then subjected to draft genome sequence determination via the MinION platform (Oxford Nanopore). Growth media were optimised to enhance antimicrobial activity, with fermentation in LB broth and subsequent purification of the culture supernatant via multi-stage column chromatography resulting in the isolation of four putative antimicrobial compounds. Initial characterisation has shown that each compound has a peptidic component, all showing stability and potency at a relatively low concentration against MRSA, E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Structural characterisation has been carried out using mass spectrometry, with further characterisation and cell toxicity studies ongoing. The producing strain has been identified as a member of the Paenibacillus genus.
Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as “resistant” to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.
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