Data_Sheet_1_The Latest Time Point of Retreatment (LTPR) as a Novel Method to Determine Antibacterial Effects for Binary Use of Cold Atmospheric Plasma and Conventional Agents.docx

Multi-resistant microorganisms are a long-standing problem for public healthcare, as inactivating those resistant pathogens with conventional antibiotics or antiseptics often no longer achieves the expected clinical success. The aim of this in vitro study was to investigate the antibacterial efficacy of binary combinations of conventional antibacterial agents with cold atmospheric plasma (CAP), when both are applied in non-lethal concentrations. In this study, Enterococcus faecalis biofilms were treated with CAP in binary combinations with benzalkonium chloride (BAC), chlorhexidine (CHX), or ciprofloxacin (CIP), respectively, which were applied in different sequences. In order to evaluate effects of binary use of two different antibacterial approaches, the so-called latest time point of retreatment (LTPR) was defined. For this purpose, regrowth curves of the bacteria were measured following the respective treatment combinations. LTPR is defined as the time component of the inflection point of a normalized regrowth curve and allows the rating and interpretation of single or binary treatments with different agents or approaches. Furthermore, LTPR designates the latest time point where a retreatment appears to be appropriate for preventing regrowth of the bacteria in case the first treatment was not lethal. Here in our study, the binary combination of 10 min CAP with BAC, CHX, or CIP leads to higher LTPRs as compared to single treatments for both sequences of application. Overall, the combination of two antimicrobial approaches is an effective alternative for inactivating bacteria in biofilms instead of a single treatment. Thus, LTPR provides a novel promising way to determine antibacterial effects for single or binary use of given antimicrobial approaches.

多重耐药微生物长期以来都是公共医疗卫生领域的棘手难题，常规抗生素或消毒剂往往无法再有效灭活这类耐药病原菌，难以达成预期的临床疗效。本体外（in vitro）研究旨在探究：当常规抗菌剂与冷大气等离子体（cold atmospheric plasma, CAP）均以非致死浓度应用时，二者联合使用的体外抗菌效果。本研究中，研究人员以不同给药顺序，分别将粪肠球菌（Enterococcus faecalis）生物膜与苯扎氯铵（benzalkonium chloride, BAC）、氯己定（chlorhexidine, CHX）或环丙沙星（ciprofloxacin, CIP）联合冷大气等离子体进行处理。为评估两种不同抗菌策略联合应用的效应，研究定义了“最晚再治疗时间点（latest time point of retreatment, LTPR）”这一核心指标。具体而言，通过测定各处理组合后的细菌再生长曲线来测算该参数。LTPR被定义为归一化再生长曲线拐点对应的时间分量，可用于评级与解读单一或联合使用不同抗菌剂或策略的抗菌效果。此外，当首次治疗未达到致死效果时，LTPR指的是可进行再治疗以防止细菌再生的最晚时间节点。本研究结果显示，无论采用哪种给药顺序，10分钟冷大气等离子体与BAC、CHX或CIP的联合处理，其LTPR均高于单一处理组。总体而言，相较于单一治疗手段，两种抗菌策略联合使用是灭活生物膜内细菌的有效替代方案。因此，LTPR为评估单一或联合使用指定抗菌策略的抗菌效果提供了一种新颖且颇具应用前景的方法。