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首页> 外文会议>World biomaterials congress >Evaluating activities of novel antimicrobial peptides against Staphylococcus aureus biofilms
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Evaluating activities of novel antimicrobial peptides against Staphylococcus aureus biofilms

机译：评价新型抗菌肽对金黄色葡萄球菌生物膜的活性

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Introduction: There is an increasing recognition that bacteria biofilm play a significant role in the morbidity and mortality associated with device related and wound infections.There is an unmet need for new antimicrobial technologies that are effective against biofilm and won't select for antibiotic resistance. The goal of this study was to evaluate and compare the efficacy of two novel antimicrobial peptides against Staphylococcus aureus (SA) in preformed biofilms. A secondary focus was to evaluate the effect of pH and the presence of serum proteins on the concentration of each peptide required to eradicate biofilm. Materials and Methods: Two proprietary cationic, amphiphilic antimicrobial peptides, ASP-1 and ASP-2 were synthesized by Bachem. Afresh overnight culture of SA Rosenbach (ATCC 10390) was diluted to ~107 bacteria in cation adjusted Muller Hinton broth (CAMHB) and used to inoculate pegs on the lid of a 96-well MBEC device (Innovotech). Biofilms were allowed to develop for 24 hrs at 37°C and 110rpm. Biofilms were challenged with serially diluted peptide solutions for 24 hrs at three pHs: acetate buffer saline (AcBS) pH6.0, phosphate buffer saline (PBS) pH7.4 and borate buffer saline (BBS) pH 8.9. The impact of biologically relevant proteins on peptide activity was also studied using buffers containing 1) Bovine serum albumin (BSA) at 30mg/ml, or 2) 50% heat-inactivated human serum with 0.05% peptone (HISP). All challenging media contained 2% CAMHB. Minimum biofilm eradication concentration (MBEC) was determined by sonicating pegs in Dey-Engley neutralizer broth followed by a 24 hr re-growth period and OD reading at 600nm. MBEC was assigned to the lowest concentration that gave OD＜0.1. Bacteria recovery was quantified by serially diluting sonicates and plating on TSB/Agar. Results and Discussion: ASP-1 and ASP-2 displayed nearly identical MBEC values ranging from 1.2 to 19 μg/ml, which are similar to previously reported minimum inhibitory concentrations for planktonic bacteria (Table 1). An increase in pH from 6.0 to 8.9 led to a 2 fold decrease in MBEC. Higher peptide concentrations were required to eradicate biofilm in the presence of proteins with an 8-fold increase observed for 50% HISP at pH 6.0 and 7.4. As shown in the Figure 1, both peptides possess strong biocidal properties over a wide pH range demonstrating more than 3 log reductions in colony forming units at low micromolar concentrations even in the presence of albumin. Conclusion: The two engineered antimicrobial peptides demonstrate potent activity against biofilm in a broad range of media simulating clinical conditions. These properties combined with high intrinsic stability and low likelihood for developing antibiotic resistance offer significant practical advantages and high potential for wide range of applications requiring infection prevention and control.
机译：简介：人们越来越认识到细菌生物膜在与设备相关的疾病和伤口感染相关的发病率和死亡率中起着重要作用。对新的有效对抗生物膜并且不会选择对抗生素产生抗性的抗菌技术的需求尚未得到满足。这项研究的目的是评估和比较两种新型抗菌肽对预先形成的生物膜中金黄色葡萄球菌（SA）的功效。第二个重点是评估pH和血清蛋白的存在对根除生物膜所需的每种肽的浓度的影响。材料和方法：Bachem合成了两种专有的阳离子，两亲性抗菌肽ASP-1和ASP-2。将SA Rosenbach（ATCC 10390）的新过夜培养物在阳离子调节的Muller Hinton肉汤（CAMHB）中稀释至约107个细菌，并用于在96孔MBEC设备（Innovotech）的盖子上接种钉子。使生物膜在37℃和110rpm下显影24小时。生物膜用连续稀释的肽溶液在以下三个pH值下攻击24小时：醋酸缓冲液（AcBS）pH6.0，磷酸盐缓冲液（PBS）pH7.4和硼酸缓冲液（BBS）pH 8.9。还使用包含1）30mg / ml牛血清白蛋白（BSA）或2）50％热灭活的人血清和0.05％ept（HISP）的缓冲液研究了生物学相关蛋白对肽活性的影响。所有具有挑战性的培养基均含有2％CAMHB。通过在Dey-Engley中和液中对钉进行超声处理，然后再进行24小时的重新生长和在600nm处的OD读数来确定最小生物膜清除浓度（MBEC）。 MBEC被指定为产生OD ＜0.1的最低浓度。通过连续稀释超声并在TSB /琼脂上平板接种来量化细菌回收率。结果与讨论：ASP-1和ASP-2的MBEC值几乎相同，范围为1.2至19μg/ ml，与先前报道的浮游细菌最低抑菌浓度相似（表1）。 pH从6.0增加到8.9导致MBEC降低2倍。在存在蛋白质的情况下，需要更高的肽浓度来根除生物膜，在pH 6.0和7.4下，对于50％HISP，观察到的肽浓度增加了8倍。如图1所示，两种肽在很宽的pH范围内都具有很强的杀菌性能，这表明即使在存在白蛋白的情况下，在低微摩尔浓度下，菌落形成单位的对数减少也超过3个对数。结论：两种工程改造的抗菌肽在模拟临床状况的各种培养基中均显示出对生物膜的有效活性。这些特性与高固有稳定性和极低的发展抗药性相结合，提供了显着的实用优势，并在需要预防和控制感染的广泛应用中具有很高的潜力。

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