FINDING THE ACHILLES HEEL: INVESTIGATING HOW TO

Overview

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Key numbers

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Table 1.1. Selected cationic biocides and their application. … 19
Table 1.2. Examples of definitions available in the literature for biocide resistance/tolerance.
Table 2.1. List of bacterial strains used. … 33
Table 3.1. Selected hits from the volcano plots for E. faecalis ST40 under biocide stress. … 64
Table 5.1. MICs for 6 different antibiotics and 2 biocides against E. faecalis ST40 determined
Table 5.3. Summary of the SNPs in annotated and hypothetical genes with known biological
Table 6.1. Selected S. aureus (SA) and E. faecalis (EF) evolved isolates with SNPs in fakA.
Table 7.1. Log10 CFU/mL reductions for S. aureus S188 and E. faecalis ST40 at different
Table 7.2. MIC and MBC values of the tested agents against S. aureus ST 188, E. faecalis ST40
Table A.I. List of bacterial isolates used in the optimisation experiments. …….……………….. 218
Table A.IV. Primers used to create ΔfakA mutants. ……………………………….……..…………….…. 232
Table A.V. PCR programmes used in the cloning strategy (Step 1) to create ΔfakA mutants.
Table A.XIV. Summarised sequencing details for S. aureus ST188, E. faecalis ST40 and S.
2014; PHE, 2012). A wide range of factors can increase the risk of acquiring HCAIs such as:
healthcare settings have several limitations; 7 % of the patients in healthcare facilities have a
risk of acquiring at least one HCAIs in high-income countries (HICs), compared to 10 % of the
S. aureus were implicated in 11.6 % of HCAIs, Enterococcus spp. in 9.8 %, and coagulase-
(80 - 90 % clinical isolates) and E. faecium (5 - 10 % clinical isolates) are the main causative
directly attributable to resistance (Murray et al., 2022). The highest burden from AMR is
Gram-positive infections, the incidence of infections caused by S. aureus increased by 14 %,
in hospitals, increased by 3.5 % over the last 5 years (PHE, 2020).
The World Bank has estimated that by 2050 the global annual GDP loss could range from 1.1
% to 3.8 %, and the additional healthcare costs needed to combat MDROs would reach to
use of Watch and Reserve antibiotics, aiming to reach at least 60 % of antibiotic consumption
2022a), shows that only 65 % of the reporting countries have achieved the AWaRe-target so
(NIH), 80% of all bacterial infections in humans possess a biofilm component (NIH, 2002). In
associated with “wet biofilms” is around 65 % (Percival et al., 2014). In 2021, biofilms were
and biguanides (Table 1.1.) were selected.
Table 1.1. Selected cationic biocides and their application.
ranges for biocide susceptibility testing (BST) (Schug et al., 2022) should also be implemented
As seen (Table 1.2.), the variety of terms used and the criteria to interpret results in BST is
Table 1.2. Examples of definitions available in the literature for biocide resistance/tolerance.
and a range of antibiotics (and other disinfectants) can occur (Hegstad et al., 2010).
outbreaks in HCAIs had on the spread of MDROs. The model revealed a 10 % increase in AMR
rates and colonisation by 14 %. However, it also showed how COVID-19 control measures,
along with more stringent IPC protocols, helped limit the spread of MDROs (with a 28 %
faecalis (an ear swab) were selected as representatives of each species (Table 2.1.). The
Table 2.1. List of bacterial strains used.
to media to a final concentration of 50 mg/L. Antibiotics were purchased from Sigma-Aldrich,
(Sigma-Aldrich, UK) and 0.5 % (w/v) OCT (Alfa Aesar, US) were prepared in double-distilled
mg/L in accordance with EUCAST guidelines (EUCAST, 2024a). For each antibiotic, MH agar
washing time the pins were washed with 70 % ethanol for 4 minutes, and 8 minutes if there

Methods (brief)

each sample in E. faecalis isolates exposed to CHX … 237
each sample in E. faecalis isolates exposed to OCT … 238
sample in S. aureus isolates exposed to CHX … 239
each sample in S. aureus isolates exposed to OCT … 240
Figure 7.5. Congo red intensity of the WTs and the evolved samples. … 178
microbiome (Ma et al., 2022), or even by reintroducing AMPs from extinct organisms (Maasch
(ESPAUR) collected data on antimicrobial use and resistance patterns. Data for 2019-2020
collection (> 2000 strains) of commensal isolates recently collected from babies in the NICU
concentration (MIC) of the tested antimicrobials against the controls and WT samples
evolved samples using the multipoint inoculator (Denley Instruments Ltd, UK) to identify any
planktonic samples from the bead condition and planktonic samples were stored in 800 µL
of TSB and 800 µL of 20 % glycerol in a 96-deep well plates. In parallel, samples were stored
Samples from parental generation, early timepoint (2nd passage), middle timepoint (7th
for some OCT samples where the middle of the experiment corresponded to the 8th passage)
and (‘late’) were sampled to confirm no relevant changes had occurred. Three random
each sample was included. Kruskal-Wallis test was performed to assess differences in biofilm
samples (Population, C1, C2, C3).
replicates were included for each sample. Plates were incubated without shaking at 30 °C for

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Update history

The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git. When DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.

Commit	Date	Change	Description
b01ec52c	2026-08-04	major	2 sections added
d49e450f	2026-08-03	major	5 sections added; narrative text revised