on 06-Dec-2024 (Fri)

Among vegetative bacteria, mycobacteria are considered the least susceptible to biocides owing to their lipid-rich outer layer of mycolic acids surrounding the cell44

Nature Reviews Microbiology Review article Bacterial endospores provide the best evidence of biocide resist- ance derived from intrinsic cell properties. Bacterial endospores are formed through a sporulation process to facilitate survival under adverse conditions 46 . The lack of susceptibility of endospores from the two main spore-forming bacterial genera Bacillus spp. and Clostridium spp. (including Clostridioides difficile) has been well reported 47 . The mechanisms of bacterial endospore resistance to biocides have been previously described and can be divided broadly into permeability barriers and nucleic acid protection46 (Fig. 3)

In addition to the com- monly described ‘wet’ biofilms, which are associated with moist envi- ronments, biofilms can develop on environmental dry surfaces 48 . These dry-surface biofilms are widespread on surfaces within health-care environments49,50 and are highly resilient to surface disinfection

Alongside cellular debris, EPS forms a matrix that acts as a diffusion bar- rier while also quenching the activity of biocides

EPS is the main factor affecting susceptibility of Pseudomonas aeruginosa biofilms to per- acetic acid and benzalkonium chloride, and its removal through washing yields cells with comparable susceptibility to vegetative bacteria 52 .

The ability of a biocide to penetrate a biofilm does not entirely explain the differ- ences observed in antibiofilm performance55, and the EPS does not fully account for biocide resistance15, exemplifying the importance of other mechanisms

However, the impact of muta- tions on decreasing susceptibility to biocides, as measured by mini- mum inhibitory concentration (MIC), is more widely reported 58 .

For example, a recent report showed that repeated sub-MIC/MIC exposure to QACs induced mutations in regulators (acrR, marR, soxR and crp), outer membrane proteins and transporters (mipA and sbmA) and RNA polymerase (rpoB and rpoC) genes in Escherichia coli 59 .

Efflux has been widely linked to increases in biocide MIC63–65 and decreased susceptibility to some antibiotics66–70

The qac transporter, which belongs to the small multidrug resistance (SMR) family within the drug and metabolite transporter (DMT) superfamily, exports lipophilic cations such as QACs and is particularly notable in the context of biocides 71 .

General diffusion porins, such as OmpC, allow a wide range of substrates to traverse the membrane, whereas others may exhibit a higher degree of substrate specificity

For example, decreased expression of Msp-type porins in mycobacteria results in increased resistance to glutaraldehyde and ortho-phthalaldehyde and a number of antibiotics including rifampicin, vancomycin, clarithromycin and erythromycin 80 .

For example, decreased susceptibility to ionic silver can result from multiple mechanisms (such as those encoded by silA-S genes) that encompass efflux, reduced penetration and neutralization and reduc - tion of ionic silver to its inactive metallic form

The ability of bacteria to repair damage following exposure to a biocide has generally received little attention 84–86 , yet repair is essential to bacterial survival (Fig. 3). The impact of repair on bacterial survival is better considered in the food industry, in which bacterial ability to repair injuries inflicted with chemical and physical agents is important to evaluate potential food contamination post-processing87.

Emerging small colony variants (SCVs) following antibiotic 89,90 or biocide exposure91 is driven by mutations92,93. SCVs are associ- ated with several survival advantages, including intracellular persis- tence and reduced antimicrobial susceptibility, and are implicated in disease94.

The alteration of metabolic pathways is part of this global response 66,98,100–103 . Sublethal exposure to biocides may indi- rectly induce oxidative stress response regulators such as marA and soxS104–106.

Mutations in global regulators can also impact bac - terial susceptibility to biocides and promote cross-resistance to anti- biotics.

Metabolic changes following biocide exposure have sometimes been associated with a change in antibiotic susceptibility, for example, aminoglycoside resistance in Listeria monocytogenes 111 or isoniazid resistance in Mycobacterium smegmatis112, both following triclosan exposure

Although antibiotic resistance may be clearly defined by clinical breakpoints 113–115 , similar definitions for ‘biocide resistance’ are lacking and there is little consensus as to what it should be and how it should be measured 5 .

Although there are no clinical breakpoints for biocides, evaluation of biocide resistance inadequately aligns with tests designed for deter- mining antibiotic efficacy, which principally measure the MIC; this test measures bacterial growth in medium with various concentrations of a biocide and over a period of 24 h (refs. 5,19). The efficacy of biocides may be substantially affected by the growth medium composition and even the type of plastic used in the assay plate116.

Similarly, mini- mum bactericidal concentration (MBC), the minimum concentration required to inactivate bacteria, is typically ascertained following 24 h of contact. MBCs are often determined following the use of an MIC determination protocol and rarely use a neutralization step that inac- tivates the biocide.

Quenching the activity of a biocide is paramount for evaluating the efficacy of a biocide and failing to do so can result in overestimation of biocide efficacy25,117

Many studies define ‘biocide resistance’ as a change in MIC, as low as a twofold increase (Supplementary Box 2). As the concentration of biocide within disinfectant products is typically 100–1,000-fold higher than the MIC, and the goal is typically to kill microorganisms within a short contact time rather than prevent their growth, MIC-based pro- tocols have been criticized poor markers of biocide resistance: such small increases in MIC are unlikely to lead to disinfection failure 5,43