Next generation decontamination: Challenging Bacillus subtilis biofilms with cold plasmas

Abstract

Introduction: Biofilms are highly resistant microbial communities that exhibit advanced protection mechanisms against extreme environmental conditions and aggressive disinfectants. These surface-associated consortia are embedded in extracellular polymeric substances (EPS), a self-built protecting matrix made up of different biopolymers. Due to their high resistances and difficulty of removal, biofilms provoke serious problems in almost all man-made settings including the international space station. Therefore, there is an urgent need for reliable sterilization approaches. Cold plasmas demonstrated already in previous studies a high efficiency in biofilm disruption but nonetheless complex mechanisms leading to inactivation are still not fully understood. This project aims to improve the decontamination processes of microbial biofilms by plasmas. Therefore, it is crucial to uncover underlying protection mechanisms and understand interactions between individual plasma elements and biofilm structures. Methods: Since biofilms are highly heterogeneous communities developing over time, various maturation states were treated with UV-C (254nm). Here, UV-C represents one of the major biocidal components formed during plasma gas discharge. To determine detrimental effects, colony forming units (CFU) of B. subtilis were calculated. In addition, the impact of matrix components in UV-C and plasma resistance were investigated by using a biofilm-forming deficient species (lacking epsA-O). Furthermore, the contribution of spores in UV-C and plasma resilience was tested by treating sporulation deficient biofilms (ΔsigG). The same biological setup was applied for treatments with double inductively coupled plasma (DICP) under synthetic air for maximum of 15 minutes. Results: UV-C radiation is a potent DNA damaging agent resulting in the inactivation of microorganisms. This study shows that it is highly biocidal to spores as the spore forming wildtype shows instant and tremendous decrease after the first treatment dose of 100 J m2 at the 0h biofilm growth state. However, since consortia of biofilms are formed there is no decrease in CFU regardless maturity. On the other hand, multicomponent sterilization by plasma is highly effective as biofilm inactivation approach, whereas within EPS deficient biofilms inactivation is the highest. Conclusion: UV-C seems to be a suitable decontamination method against monolayered spores. Since consortia of microbes as in biofilms are formed, UV-C radiation has no inactivation effect. Nevertheless, UV-C is one of the major active components emitted in plasma. Since plasma is a multicomponent system, the lethal effects are most likely achieved by synergistic procedures.