Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing

Biofilms protect bacteria from stress or stimuli by providing a thick layer of extracellular proteins [1]. Biofilm in bacterial communities is formed through initial and irreversible attachment, microcolony formation, biofilm maturation, and biofilm dispersion [2]. Biofilm matrix comprises polysaccharides, proteins, and nucleic acids that can be dissolved by enzymatic degradation [3]. Proteinaceous components, such as the non-ribosomally generated peptide aureusimine (phevalin) in Staphylococcus aureus [4] and three exopolysaccharides (Psl, Pel, and alginate) in Pseudomonas aeruginosa [2, 3], play important roles in biofilm structural maintenance and are highly resistant to antibiotics and disinfectants. The biofilm-forming bacteria S. aureus and P. aeruginosa are major opportunistic pathogens implicated in hospital-acquired infections; these bacteria are additionally known to promote the spread of chronic diseases, including osteomyelitis and endocarditis, contaminate medical device implants, and persist in lung cystic fibrosis [2, 4]. Specifically, endoscopy-related infections caused by biofilm adhesion within the narrow luminal cavities of endoscope channels are attributed to inadequate cleaning routines and ineffective disinfection procedures [5, 6] despite adherence to guideline recommendations [7, 8]. These are often reported in conjunction with pseudo-outbreaks of biofilm-producing bacteria [9‐11]. Additionally, biofilms are recalcitrant to endoscope reprocessing, resulting in infection by P. aeruginosa and Mycobacterium chelonae isolates, such as those with high resistance to glutaraldehyde (GA) [12, 13].

In order to effectively remove biofilms from endoscope channels, automated endoscope reprocessors (AERs) have become widespread in Japan, particularly in combination with novel high-level disinfectants (HLDs) such as peracetic acid (PAA) and ortho-phthalaldehyde (OPA); these broad-spectrum disinfectants have replaced GA [14‐16]. The exposure time required for bactericidal effectiveness of HLDs, according to current Japanese multisociety guides on reprocessing flexible GI endoscopes, is routinely recommended as 5 min for 0.3% PAA, 10 min for 0.55% OPA, and 10 min for 2.0% GA. Other guidelines state that this duration is dependent on the concentration of the specific HLDs [17‐19]. In addition, the ability of OPA to fix stains (bacterial proteins or biofilms) onto materials [20, 21] and involvement of efflux as a GA resistance mechanism in P. fluorescens and P. aeruginosa biofilms [22] have been reported; however, the mechanism associated with PAA activity, specifically its ability to fix or remove biofilms from materials, is not completely understood. Furthermore, to date, comparison of the exposure times for bactericidal effectiveness of PAA and OPA against biofilms, under the same conditions, has not been attempted.

Here, we investigated the exposure time for bactericidal effectiveness of three HLDs used in Japan, using in vitro biofilm models. The study aimed to examine AMR outcomes associated with biofilms, including in drug-resistant strains, and to visualize the bacterial inner-cell and outer-surface morphological structures by electron microscopy. The findings offer novel insights into PAA activity against biofilms.

Our study is the first to utilize TEM and SEM to document biofilm formation and investigate the bactericidal effects of PAA. Our in vitro biofilm models exhibited mature biofilm development inside a narrow catheter lumen, as reported previously, thus mimicking biofilm formation inside the endoscope channel [23]. Notably, different HLDs presented varying bactericidal effects on S. aureus and P. aeruginosa biofilms in our in vitro biofilm models. Accordingly, PAA had the most rapid bactericidal activity (< 1 min), in line with a report by Tote et al. [24]. In contrast, OPA required >10 min and 30 min to completely eradicate S. aureus and P. aeruginosa biofilms, respectively. These results indicate that P. aeruginosa shows reduced susceptibility to OPA due to overproduction of biofilm matrix components [25]. The P. aeruginosa biofilm is comprised of bacteria embedded in a matrix of extracellular polymeric substances, which functions as both a structural scaffold and/or as a protective barrier against harsh environments. Alginate or other polysaccharides have been identified as the main matrix ingredients in P. aeruginosa biofilms, with important roles in structural maintenance and AMR [2]. Additional compounds affecting biofilm matrix and tolerance of P. aeruginosa biofilms to OPA are currently being investigated. Polysaccharide intercellular adhesin has also been described as a major proteinaceous component of the S. aureus biofilm matrix, which is also rich in teichoic acids [4]. Other cellular components are likely to be present, and await further investigation. To address a study limitation, our future work will investigate the influence of HLDs on several known biofilm matrices from other bacterial strains. However, as disinfection exposure time recommended by guidelines for endoscope reprocessing may differ by biofilm matrix, as indicated by testing OPA in this study, it would be necessary to select an HLD with stronger and fast-acting bactericidal effects, such PAA, in practical use.

Although mature S. aureus and P. aeruginosa biofilms are characterized by production of matrix components that hinder biofilm killing, PAA appears to be an effective disinfectant. TEM and SEM observations enabled a comparative analysis of the mechanism of action of PAA, revealing changes in shape after 5 min and structural damage after 30 min. In the current report, mature P. aeruginosa biofilms aged 96 h were eradicated at 3000 ppm (0.3%) of PAA after 5-min exposure [26]. Our findings indicate that the exposure time for effective bactericidal activity is 5 min under high concentrations of HLD, e.g., 0.3% PAA.

These alterations might be caused by a sharp decrease in bacterial inner pressure resulting from high permeability to PAA for a short period. Additionally, PAA is thought to act as an oxidizing agent by releasing hydroxyl radicals, which subsequently attack essential biofilm matrix components [27‐29]. To date, no PAA-resistance mechanisms have been reported for either cell suspensions or biofilms. Here, spectroscopic measurements of bacterial survival correlated well with TEM and SEM observations of biofilm structures and surfaces. This in vitro biofilm model associated with electron microscopy represents an effective tool to investigate the mechanistic action of disinfectants such as PAA. Future work on biofilm formation should help elucidate the nature of interactions under changing environmental conditions (e.g., pH) or in the presence of plasma for various durations of exposure.

In summary, we found that PAA is a useful HLD for endoscope channel reprocessing, even in the presence of strongly adhering bacterial biofilms. The current developments should allow for shorter exposure time for effective bactericidal activity during endoscope reprocessing in healthcare settings. This is expected to enable the prevention of endoscopy-related infections resulting from potential contamination by S. aureus and P. aeruginosa biofilms.

Morphological observations of in vitro biofilm models by electron microscopy showed that PAA exhibited more rapid bactericidal effects than OPA or GA against S. aureus and P. aeruginosa biofilms, which are associated with endoscopy-related infections. Therefore, these findings suggest that PAA has fast-acting effects against S. aureus and P. aeruginosa biofilms.