Biofilms of a Bacillus subtilis Hospital Isolate Protect Staphylococcus aureus from Biocide Action

Abstract

The development of a biofilm constitutes a survival strategy by providing bacteria a protective environment safe from stresses such as microbicide action and can thus lead to important health-care problems. In this study, biofilm resistance of a Bacillus subtilis strain (called hereafter NDmedical) recently isolated from endoscope washer-disinfectors to peracetic acid was investigated and its ability to protect the pathogen Staphylococcus aureus in mixed biofilms was evaluated. Biocide action within Bacillus subtilis biofilms was visualised in real time using a non-invasive 4D confocal imaging method. The resistance of single species and mixed biofilms to peracetic acid was quantified using standard plate counting methods and their architecture was explored using confocal imaging and electronic microscopy. The results showed that the NDmedical strain demonstrates the ability to make very large amount of biofilm together with hyper-resistance to the concentration of PAA used in many formulations (3500 ppm). Evidences strongly suggest that the enhanced resistance of the NDmedical strain was related to the specific three-dimensional structure of the biofilm and the large amount of the extracellular matrix produced which can hinder the penetration of peracetic acid. When grown in mixed biofilm with Staphylococcus aureus, the NDmedical strain demonstrated the ability to protect the pathogen from PAA action, thus enabling its persistence in the environment. This work points out the ability of bacteria to adapt to an extremely hostile environment, and the necessity of considering multi-organism ecosystems instead of single species model to decipher the mechanisms of biofilm resistance to antimicrobials agents.

Is peracetic acid suitable for the cleaning step of reprocessing flexible endoscopes?

“…Under natural environmental conditions, biofilms are likely to be composed of a mixture of different species [131,132] . In the laboratory, they can be grown on various materials and devices, including polystyrene microtitre plates [133][134][135][136] , haemolysis glass tubes [137,138] , stainless steel coupons [134,139] and also in Teflon tubes [140][141][142][143] , similar to endoscope channels.…”

“…Similar results were found with an E. coli biofilm and peracetic acid/H2O2 [153] . The resistance of biofilms can often further increase when the communities are composed of more than one bacterial species [134,136,[160][161][162][163] which may include resistance against 0.35% peracetic acid, which is a concentration used in many formulations [133] . Especially "build-up" biofilms mimicking repeated endoscope reprocessing cycles exhibited a significantly higher survival rate than 'traditional' biofilms [158] .…”

“…Especially "build-up" biofilms mimicking repeated endoscope reprocessing cycles exhibited a significantly higher survival rate than 'traditional' biofilms [158] . The mechanisms underlying disinfectant-resistant phenotypes appear to be multifactorial [133,148,151,153,164] .…”

Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

“…In a previous study, we proposed a new methodology to visualize and quantify B. subtilis submerged biofilms using a microplate-based model combined with confocal laser scanning microscopy (CLSM) (27,28). This experimental system enabled the identification of a remarkable B. subtilis strain (NDmed), isolated from an endoscope washer-disinfector (29), capable of forming thick and protruding biofilms highly resistant to the biocides commonly used for endoscope disinfection in hospitals (30).…”

“…In most cases, interspecies relationships increase the fitness and the resistance to environmental stresses of the biofilm (30 -35). Indeed, we previously demonstrated the ability of B. subtilis NDmed to protect the pathogen S. aureus from biocide action in dual-species biofilms (30).…”