Background 

Biofilms protect bacteria from the host immune system and many antibiotics, making the treatment of orthopaedic infections difficult. Halicin, a recently discovered antibiotic, has potent activity against nonorthopaedic infections in mice and the planktonic, free-living forms of many bacterial species, including Staphylococcus aureus, a common cause of orthopaedic infections. Importantly, halicin did not induce resistance in vitro and was effective against drug-resistant bacteria and proliferating and quiescent bacteria. Quiescence is an important cause of antibiotic tolerance in biofilms. However, whether halicin acts on biofilms has not been tested.

Questions/purposes 

(1) Does halicin reduce the viability of S. aureus in less mature and more mature biofilms as it does in planktonic cultures? (2) How do the relative effects of halicin on S. aureus biofilms and planktonic cultures compare with those of conventional antibiotics (tobramycin, cefazolin, vancomycin, or rifampicin) that are commonly used in clinical orthopaedic infections?

Methods 

To measure minimal biofilm eradication concentrations (MBECs) with less mature 3-day and more mature 7-day biofilms, we used 96-well peg plates that provided high throughput and excellent reproducibility. After S. aureus-Xen36 biofilm formation, planktonic bacteria were removed from the cultures, and the biofilms were exposed to various concentrations of halicin, tobramycin, cefazolin, vancomycin, or rifampicin for 20 hours. Biofilm viability was determined by measuring resazurin reduction or by counting colony-forming units after sonication. To determine effects of halicin and the conventional antibiotics on biofilm viability, we defined MBEC₇₅ as the lowest concentration that decreased viability by 75% or more. To determine effects on bacterial viability in planktonic cultures, minimum inhibitory concentrations (MICs) were determined with the broth dilution method. Each result was measured in four to 10 independent experiments.

Results 

We found no differences between halicin’s effectiveness against planktonic S. aureus and 3-day biofilms (MIC and MBEC₇₅ for 3-day biofilms was 25 μM [interquartile range 25 to 25 and 25 to 25, respectively]; p > 0.99). Halicin was eightfold less effective against more mature 7-day biofilms (MBEC₇₅ = 200 μM [100 to 200]; p < 0.001). Similarly, tobramycin was equally effective against planktonic culture and 3-day biofilms (MIC and MBEC₇₅ for 3-day biofilms was 20 μM [20 to 20 and 10 to 20, respectively]; p > 0.99). Tobramycin’s MBEC₇₅ against more mature 7-day biofilms was 320 μM (320 to 480), which is 16-fold greater than its planktonic MIC (p = 0.03). In contrast, the MBEC₇₅ for cefazolin, vancomycin, and rifampicin against more mature 7-day biofilms were more than 1000-fold (> 1000; p < 0.001), 500-fold (500 to 875; p < 0.001), and 3125-fold (3125 to 5469; p = 0.004) greater than their planktonic MICs, respectively, consistent with those antibiotics’ relative inactivity against biofilms.

Conclusion 

Halicin was as effective against S. aureus in less mature 3-day biofilms as those in planktonic cultures, but eightfold higher concentrations were needed for more mature 7-day biofilms. Tobramycin, an antibiotic whose effectiveness depends on biofilm maturity, was also as effective against S. aureus in less mature 3-day biofilms as those in planktonic cultures, but 16-fold higher concentrations were needed for more mature 7-day biofilms. In contrast, cefazolin, vancomycin, and rifampicin were substantially less active against both less and more mature biofilms than against planktonic cultures.

Clinical Relevance 

Halicin is a promising antibiotic that may be effective against S. aureus osteomyelitis and infections on orthopaedic implants. Future studies should assess the translational value of halicin by testing its effects in animal models of orthopaedic infections; on the biofilms of other bacterial species, including multidrug-resistant bacteria; and in combination therapy with conventional antibiotics.

中文翻译：

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Halicin 在体外有效对抗金黄色葡萄球菌生物膜

背景 

生物膜保护细菌免受宿主免疫系统和许多抗生素的侵害，使得骨科感染的治疗变得困难。Halicin 是一种最近发现的抗生素，对小鼠非骨科感染和许多细菌种类的浮游、自由生活形式具有有效活性，包括金黄色葡萄球菌（骨科感染的常见原因）。重要的是，halicin 在体外不会诱导耐药性，并且对耐药细菌以及增殖和休眠细菌有效。静止是生物膜中抗生素耐受的重要原因。然而，halicin是否作用于生物膜尚未得到测试。

问题/目的 

(1) halicin 是否会像浮游培养物中那样降低不太成熟和较成熟生物膜中金黄色葡萄球菌的活力？(2)与临床骨科感染常用的传统抗生素（妥布霉素、头孢唑林、万古霉素或利福平）相比， halicin 对金黄色葡萄球菌生物膜和浮游培养物的相对影响如何？

方法 

为了测量不太成熟的 3 天生物膜和较成熟的 7 天生物膜的最低生物膜根除浓度 (MBEC)，我们使用了 96 孔板，该板可提供高通量和出色的重现性。金黄色葡萄球菌-Xen36生物膜形成后，从培养物中除去浮游细菌，并将生物膜暴露于不同浓度的哈利星、妥布霉素、头孢唑林、万古霉素或利福平20小时。通过测量刃天青的减少或通过超声处理后计数菌落形成单位来确定生物膜活力。为了确定 halicin 和常规抗生素对生物膜活力的影响，我们将 MBEC ₇₅定义为活力降低 75% 或更多的最低浓度。为了确定对浮游培养物中细菌活力的影响，使用肉汤稀释法确定了最低抑菌浓度 (MIC)。每个结果均通过四到十次独立实验进行测量。

结果 

我们发现 halicin 对浮游金黄色葡萄球菌和 3 天生物膜的有效性没有差异（3 天生物膜的 MIC 和 MBEC ₇₅为 25 μM [四分位数范围分别为 25 至 25 和 25 至 25]；p > 0.99）。Halicin 针对更成熟的 7 天生物膜的效果要低八倍（MBEC ₇₅ = 200 μM [100 至 200]；p < 0.001）。同样，妥布霉素对浮游培养物和 3 天生物膜同样有效（3 天生物膜的 MIC 和 MBEC ₇₅为 20 μM [分别为 20 至 20 和 10 至 20]；p > 0.99）。妥布霉素针对更成熟的 7 天生物膜的MBEC _{75为 320 μM（320 至 480），比其浮游 MIC 高 16 倍（p = 0.03）。}相比之下，头孢唑林、万古霉素和利福平针对更成熟的 7 天生物膜的 MBEC ₇₅分别为 1000 倍以上 (> 1000；p < 0.001)、500 倍(500 至 875；p < 0.001) 和 3125 倍。分别比其浮游 MIC 大 3125 至 5469 倍（3125 至 5469；p = 0.004），这与这些抗生素对生物膜的相对不活性一致。

结论 

Halicin在不太成熟的 3 天生物膜中与浮游培养物中的金黄色葡萄球菌一样有效，但对于更成熟的 7 天生物膜，需要高八倍的浓度。妥布霉素是一种抗生素，其有效性取决于生物膜的成熟度，在不太成熟的 3 天生物膜中对金黄色葡萄球菌也与浮游培养物中的金黄色葡萄球菌一样有效，但对于更成熟的 7 天生物膜，需要高 16 倍的浓度。相比之下，头孢唑啉、万古霉素和利福平对较不成熟和较成熟的生物膜的活性明显低于对浮游培养物的活性。

临床相关性 

Halicin 是一种很有前途的抗生素，可能对金黄色葡萄球菌骨髓炎和骨科植入物感染有效。未来的研究应通过测试其在骨科感染动物模型中的作用来评估 halicin 的转化价值；其他细菌物种的生物膜，包括多重耐药细菌；以及与常规抗生素的联合治疗。