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Antibacterial effect of N-acetylcysteine on common canine otitis externa isolates N-乙酰半胱氨酸对犬外耳炎常见分离株的抗菌作用
作者：Elizabeth R. May, Katherine A. Conklin and David A. Bemis
翻译：许晓鸥
Background – N-Acetylcysteine (NAC) has the potential to be a useful therapeutic agent for the treatment of otitis externa due to its antimicrobial and mucolytic properties, as well as its ability to disrupt bacterial biofilm. Hypothesis/Objectives – To determine the antibacterial activity of NAC against common bacterial isolates associated with canine otitis externa. Animals – Twenty two isolates from canine clinical cases of otitis externa were identified and tested, including five Staphylococcus pseudintermedius, six Pseudomonas aeruginosa, five Corynebacterium spp. and six β-haemolytic Streptococcus spp. isolates. Methods – Each isolate was grown on blood agar for 24 h and transferred to Mueller Hinton Broth (MHB) to achieve a final concentration of 5*105 CFU/mL. NAC was diluted in MHB to a starting concentration of 160 mg/mL and serial two-fold microdilution assays were performed in triplicate with negative controls for all isolates tested. Concentrations of NAC tested ranged from 0.125 to 80 mg/mL. A 50 µL volume of bacterial suspension was used to inoculate each well. Results – The minimum inhibitory concentration (MIC) of NAC for all isolates tested ranged from 5 to 20 mg/mL. Conclusions and Clinical Relevance – N-Acetylcysteine inhibits clinically relevant and drug resistant bacteria in vitro, and has potential for use as a novel agent for treatment of otitis externa.
背景-N-乙酰半胱氨酸 (NAC) 因为具有抗菌和粘液溶解特性，以及破坏细菌生物膜的能力，有可能成为治疗外耳炎的有效治疗药物。 假设/目的-确定 NAC 对犬外耳炎相关的常见细菌分离株的抗菌活性。 动物-犬外耳炎临床病例分离的22株分离株进行了鉴定和检测，包括5株假中间型葡萄球菌、6株铜绿假单胞菌、5株棒状杆菌属和6株β-溶血链球菌分离株。 方法-将每种分离株在血琼脂上生长24h，并转移至Mueller Hinton液体培养基(MHB) 中，使最终浓度为 5*105 cfu/mL。在MHB中将NAC稀释至起始浓度160mg/mL，并使用阴性对照对所有供试分离株进行3次重复的2倍微量稀释试验。试验NAC浓度范围为0.125-80mg/mL。每孔接种50µL的细菌悬液。 结果-NAC对所有供试分离株的最小抑菌浓度 (MIC) 范围为 5-20 mg/mL。 结论和临床相关性- N-乙酰半胱氨酸在体外可抑制临床相关的耐药菌，有可能作为治疗外耳炎的新型药物。
Introduction 介绍 Canine otitis externa is often a chronic and recurrent problem with limited options for treatment. It is estimated to occur frequently in the population that seeks veterinary care, being listed as the second most common condition for US canine health insurance claims.Underlying allergic conditions, such as atopic dermatitis or cutaneous adverse reactions to food, contribute to the development of primary inflammation, with secondary bacterial otitis as a complicating, perpetuating factor. Bacterial pathogens frequently associated with canine otitis externa include Staphylococcus pseudintermedius, Pseudomonas aeruginosa, β-haemolytic Streptococcus spp. and Proteus spp. Corynebacterium spp. has also been recovered and is typically cultured in association with other bacterial species. 犬外耳炎通常是一种慢性和复发性问题，治疗选择有限。据估计，其就诊率较高中，被列为美国犬健康保险索赔的第二常见疾病。潜在的过敏性疾病，如特应性皮炎或食物不良反应，是导致原发性炎症原因，继发细菌性耳炎使其复杂和持久的因素。犬外耳炎常见细菌包括假中间型葡萄球菌、铜绿假单胞菌、β-溶血性链球菌属和变形杆菌属。棒状杆菌属也有，常与其他细菌一起培养出来。
Commercially available products with documented efficacy against commonly encountered otitis pathogens are available; however, they contain antibacterials from a limited number of drug classes. In addition, these topical treatments often contain potentially ototoxic agents, such as aminoglycosides, that can cause temporary or even permanent hearing loss in veterinary patients. With limited choices available, it is desirable to identify novel, safe treatment options that are effective against organisms associated with canine otitis externa. 有对常见耳炎有效的商品化产品，但是，所含抗生素药物种类有限。此外，这些外部治疗通常含有潜在的耳毒性药物，如氨基糖苷类，可导致兽医病例出现暂时性甚至永久性听力丧失。由于可用的选择有限，因此需要对犬外耳炎相关微生物有效的新型、安全的治疗方案。
N-Acetylcysteine (NAC) is a readily available and commonly used mucolytic agent with both antibacterial and antioxidant capabilities. It is utilized therapeutically in veterinary medicine for the treatment of acetaminophen toxicity, corneal ulcers and is nebulized for the adjunctive treatment of lower respiratory tract disease. In addition, it has been reported to be otoprotective and can prevent chemotherapy associated hearing loss. Because of the established safety profile and other benefits described for this compound, NAC could potentially be of great benefit when applied topically for the treatment of canine otitis externa. The purpose of this study was to evaluate the antibacterial effect of NAC on common canine otitis externa bacterial isolates in vitro. N-乙酰半胱氨酸 (NAC) 是一种易得到的、常用粘液溶解剂，具有抗菌和抗氧化能力。 在兽医学中用于治疗对乙酰氨基酚中毒、角膜溃疡，雾化吸入用于下呼吸道疾病的辅助治疗。此外，据报道，它具有耳保护作用，可以预防化疗相关的听力丧失。由于该化合物的安全性特征和其他好处已确定，当外用NAC治疗犬外耳炎时，可能会很有效果。本研究的目的是评价NAC对犬外耳炎常见细菌分离株的体外抗菌作用。
Materials and methods 材料与方法 Twenty two isolates from canine clinical cases of otitis externa evaluated by the Dermatology service and submitted to the Department of Biomedical and Diagnostic Sciences, University of Tennessee, for culture and susceptibility testing during the month of June 2014 were included. Speciation was by standard methods employed in the laboratory and antimicrobial susceptibility tests followed general guidelines for microbroth dilution testing established by the Clinical and Laboratory Standards Institute. Five Staphylococcus pseudintermedius, six Pseudomonas aeruginosa, five Corynebacterium spp. and six β-haemolytic Streptococcus spp. isolates were evaluated. 纳入了2014年6月期间由皮肤科评估并提交至田纳西大学生物医学和诊断科学系进行培养和药敏试验的22株外耳炎犬临床病例分离株。通过实验室采用的标准方法进行菌种鉴定，抗菌剂敏感性试验遵循临床和实验室标准协会制定的微量肉汤稀释试验的一般指导原则。对5株假中间型葡萄球菌、6株铜绿假单胞菌、5株棒状杆菌属和6株β-溶血性链球菌分离株进行了评估。
Preliminary testing was performed on reference isolates which were also included as controls when testing clinical isolates. American Type Culture Collection (ATCC) control strain S. aureus 29213 and ATCC control strain P. aeruginosa 27853 served as the control strains and were included. Each isolate was inoculated onto Columbia Agar (BBL, BD Diagnostic Systems; Sparks, MD, USA) with 5% sheep blood (Hemostat Laboratories; Dixon, CA, USA) and incubated for 24 hours at 35°C. Isolates were transferred to Mueller Hinton Broth (MHB) for a final inoculum concentration of 5 X 105 CFU/mL. 对相关分离株进行了初步试验，在检测临床分离株时，相关分离株也作为对照纳入。美国典型培养物保藏中心(ATCC)对照菌株金黄色葡萄球29213和ATCC对照菌株铜绿假单胞菌27853作为对照菌株，并将其纳入。将各分离株接种至含有5% 羊血的哥伦比亚琼脂上，并在35℃下孵育24小时。将分离株转移至 Mueller Hinton液体培养基(MHB)中，使最终接种物浓度为5X105cfu/mL。
A commercially available 20% NAC solution was obtained from APP Pharmaceuticals (Schaumburg, IL, USA) and diluted in MHB to a starting concentration of 160 mg/mL. For comparison, pharmaceutical grade NAC was obtained from Sigma-Aldrich (St. Louis, MO, USA) and was diluted in MHB to a starting concentration of 160 mg/mL. Both NAC formulations underwent serial two-fold microdilution assays, with concentrations of NAC tested ranging from 0.125 to 80 mg/mL. A 50 µL volume of bacterial suspension was added to each well of a Polystyrene 96 well plate (Fisher Scientific, 12-565-500; Hanover Park, IL, USA) and each assay was performed in triplicate for each bacterial isolate. Concentrations tested were based upon previous work demonstrating that NAC, at a concentration of 80 mg/mL, was consistently bactericidal against meticillin-sensitive and -resistant Staphylococcus aureus, S. epidermidis, vancomycin-resistant Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter cloacae and Klebsiella pneumoniae. Growth controls containing untreated bacteria were included for all isolates tested, including the control strains. The minimum inhibitory concentration (MIC) was defined as the lowest concentration with complete growth inhibition, as determined visually. The highest MIC value for each triplicate run was reported as the representative MIC for each organism tested, in order to reflect a concentration predicted to consistently inhibit growth. 从 APP Pharmaceuticals获得市售20%NAC溶液，并在MHB中稀释至起始浓度160mg/mL。为进行比较，从Sigma-Aldrich获得药用级NAC，并在MHB中稀释至起始浓度160mg/mL。两种 NAC制剂均进行了连续两倍微量稀释测定法，检测的NAC浓度范围为0.125-80mg/mL。向聚苯乙烯96孔板的每个孔中加入50µL的细菌悬液，每种细菌分离株的每种试验重复进行三次。检测浓度是基于先前的工作，先前的工作证明，浓度为80 mg/mL的NAC对甲氧西林敏感和耐甲氧西林金黄色葡萄球菌、表皮葡萄球菌、耐万古霉素粪肠球菌、铜绿假单胞菌、阴沟肠杆菌和肺炎克雷伯菌具有一致的杀菌作用。所有供试分离株（包括对照组菌株）均包括含未处理细菌的生长对照。最小抑菌浓度(MIC)定义为眼观具有完全生长抑制的最低浓度。将每次一式三份运行的最高MIC值作为每种供试微生物的代表性MIC，以反映预测持续抑制生长的浓度。
Results 结果 Consistent bacterial growth was evident in all control samples, as expected. The NAC minimum inhibitory concentration for all isolates tested ranged from 5 to 20 mg/ mL (Table 1). Seven of the 22 isolates tested (31.8%) had NAC MICs of 5 mg/mL or less, 95.5% of the isolates tested had NAC MICs of 10 mg/mL or less, and all isolates had NAC MICs of 20 mg/mL or less. (Table 2). 正如预期，所有对照组样本中，细菌明显生长一致。所有供试分离株的NAC最低抑菌浓度范围为5-20mg/mL（表 1）。检测的22株分离株中有7种(31.8%)的NAC MIC≤5 mg/mL，检测的分离株中有95.5%的NAC MIC≤10 mg/mL，所有分离株的NAC MIC≤20 mg/mL。（表 2）。
Discussion 讨论 The findings of this in vitro study indicate that NAC has inhibitory activity against common pathogens associated with canine otitis externa. This is in agreement with previous studies demonstrating inhibition of both Grampositive and Gram-negative bacteria, with the growth of P. aeruginosa inhibited at low concentrations. We found that inhibitory activity occurred most frequently at 10 mg/ mL of NAC, a concentration that is approximately 5% of the concentration of the commercially available product already in clinical use (Mucomyst, 20%, 200 mg/mL NAC, Bristol-Myers Squibb; Princeton, NJ, USA). This concentration would be expected to be safe and well tolerated clinically. Middle ear mucosal inflammation has been reported at concentrations greater than 20 mg/mL, and external auditory canal and middle ear inflammation, resulting in conductive hearing loss, have been reported at a concentration of 40 mg/mL. Concentrations of 20 mg/mL were not associated with an inflammatory response;therefore, the results reported in our study are expected to be inhibitory without being inflammatory. 体外研究结果表明，NAC对犬外耳炎的常见病原微生物具有抑制作用。这与先前研究一致，证明对革兰氏阳性菌和革兰氏阴性菌均有抑制作用，在低浓度下铜绿假单胞菌的生长受到抑制。我们发现，10mg/mLNAC的抑制活性最常见，该浓度是已在临床使用的商品化产品(Mucomyst，20%，200 mg/mL NAC，Bristol-Myers Squibb；Princeton，NJ，USA) 浓度的5%。该浓度在临床上安全且耐受性良好。有报道，浓度大于20 mg/mL时出现中耳黏膜炎症，有报道浓度为40 mg/mL时出现外耳炎和中耳炎，导致传导性听力下降。20 mg/mL 浓度与炎症反应无关；因此，我们研究中报告的结果预期为抑制作用，无炎症反应。
None of the isolates tested in our study were resistant to inhibition by NAC at the concentrations tested. The inhibitory activity of NAC against bacterial pathogens is known to be both inoculum size and dose dependent; however, the precise mechanism of action is unknown. There are several proposed mechanisms for the antibacterial properties of NAC, focusing predominantly on the inhibition of amino acid utilization in bacterial cells, such as competing with cysteine, or possibly interaction between proteins normally found in the bacterial cell and the sulfhydryl group of NAC. 在我们的研究中，检测的分离株在检测浓度下均对NAC不耐药。NAC对细菌性病原的抑制作用，与接种量和使用剂量相关; 然而，确切的作用机制尚不清楚。NAC的抗菌特性有几种可能的机制，主要集中在抑制细菌细胞中的氨基酸利用，如与半胱氨酸竞争，或可能在细菌细胞中常见的蛋白质与NAC的巯基之间相互作用。
Minimum inhibitory concentration values varied slightly among the bacterial species as well as among the individual isolates examined, which was not unexpected. With the exception of the control reference strains, that were shown to be within acceptable QC range, antimicrobial susceptibility profiles of the organisms included were not evaluated prior to inclusion in the study, thus different strains may possess traits that require increased concentration of NAC to inhibit growth. Despite the slight MIC concentration variability, growth inhibition was either similar or within one dilution for all bacterial isolates tested. 不同细菌种属以及检测的单个分离株之间的最小抑菌浓度值略有不同，这并不意外。除对照组相关菌株外，在可接受QC范围内，纳入研究前未评价纳入微生物的抗菌敏感性进行评估，因此不同菌株可能具有需要增加 NAC浓度以抑制生长的特征。尽管MIC浓度存在轻微差异，但所有测试细菌分离株的生长抑制要么相似，要么在一个稀释度内。
Biofilm formation produced by P. aeruginosa has been associated with respiratory tract infections that are chronic and difficult to treat in humans. N-Acetylcysteine has been shown to contribute to the detachment of biofilms associated with P. aeruginosa and is used clinically as a mucolytic agent. Although it is currently unknown whether or not biofilm formation contributes to treatment failure in cases of canine otitis, the formation of biofilm has been documented in association with isolates of P. aeruginosa from dogs with otitis externa and was reported in 40% of the isolates tested in one study. If biofilm formation is an obstacle to treatment, NAC could potentially be utilized clinically to either prevent or detach biofilms, allowing for a more successful treatment outcome via access to the bacteria. This would be expected to result in reduction of the MIC value of the pathogenic organism. 铜绿假单胞菌产生的生物膜形成与人类慢性和难以治疗的呼吸道感染有关。N-乙酰半胱氨酸已被证明有助于分离与铜绿假单胞菌相关的生物膜，并在临床上用作粘液溶解剂。尽管目前尚不清楚生物膜的形成是否会导致犬耳炎病例的治疗失败，但已证实生物膜的形成与外耳炎患犬的铜绿假单胞菌分离株相关，并且在一项研究中检测的分离株中有 40%报告了生物膜的形成。如果生物膜形成是治疗的障碍，NAC可用于临床预防或分离生物膜，从而通过进入细菌获得更成功的治疗效果。这预期会导致病原微生物的MIC值降低。
As a mucolytic and antioxidant agent with the ability to disrupt the formation of bacterial biofilm, the use of NAC is of interest in veterinary medicine. The results reported here provide information that could translate into potential new treatment options for canine otitis externa. N-Acetylcysteine actively inhibits clinically relevant and drug-resistant bacteria in vitro, and has potential for use as a novel agent for treatment of otitis externa. NAC作为一种能够破坏细菌生物膜形成的粘液溶解剂和抗氧化剂，在兽医学中具有重要应用价值。本文报告的结果可能成为犬外耳炎潜在治疗新选择。N-乙酰半胱氨酸在体外对临床相关的耐药菌的有效抑制作用，有可能作为治疗外耳炎的新型药物。
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