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快生长分枝杆菌(rapidly growing mycobacteria,RGM)是非结核分枝杆菌(nontuberculous mycobacteria,NTM)的一种,生长速度较快,在罗氏或7H10培养基上一周内可见菌落生长。近年来,全球RGM感染的发病率在不断增加[1,2],但是根据临床症状或影像学特征,通常很难区分RGM感染和结核分枝杆菌感染[3];脓肿分枝杆菌、龟分枝杆菌和偶发分枝杆菌是最常见的致病RGM。脓肿分枝杆菌是最常见的引起NTM肺病的菌种,约50%的NTM肺病由其引起,临床症状与肺结核相似,难以鉴别。此外,脓肿分枝杆菌也常引起皮肤软组织和骨关节感染。偶发分枝杆菌与脓肿分枝杆菌类似,既可引起肺病,也可引起皮肤软组织和骨关节感染[4]。而龟分枝杆菌主要引起人类的皮肤、骨骼和软组织感染,极少引起肺部感染。近年来,龟分枝杆菌感染也成为外科手术和医学美容手术常见的伴发感染,如器官移植、整容手术、美容注射吸脂手术及纹身等[5,6]。
材料和方法
1.菌株选择:收集首都医科大学附属北京胸科医院2015年1月至2019年12月从临床样本中分离出的98株RGM,包括25株偶发分枝杆菌、70株脓肿分枝杆菌和3株龟分枝杆菌。这些临床分离株的患者来源于中国的北方,包括13个省份及北京和天津两个直辖市。选取在对硝基苯甲酸(500μg/ml)生长试验阳性的菌株,进行hsp65、rpoB和16S rRNA、16-23S rRNA基因的测序,将所有临床分离株鉴定至菌种水平。龟分枝杆菌(ATCC14472)、偶发分枝杆菌(ATCC6481)和脓肿分枝杆菌(ATCC19977)标准株购自美国标准菌株库(American Type Culture Collection,ATCC)。
2.试剂仪器:药敏试验使用Sensititre RAPMYCO 药敏板(美国赛默飞公司)进行检测,其可在不需要仪器设备的情况下定量评估临床分离株对13种抗生素的药物敏感性[7]。该板是含有冻干抗生素的微孔板,用于检测RGM菌株的最低抑菌浓度(minimal inhibitory concentration,MIC)[9]。该板含有以下浓度的13种抗生素:阿米卡星,1~64μg/ml;妥布霉素,1~16μg/ml;阿莫西林-克拉维酸,2/1μg/ml~64/32μg/ml;头孢西丁,4~128μg/ml;亚胺培南,2~64μg/ml;环丙沙星,0.12~4μg/ml;莫西沙星,0.25~8μg/ml;克拉霉素,0.03~16μg/ml;利奈唑胺,1~32μg/ml;米诺环素,1~8μg/ml;多西霉素,2~64μg/ml;替加环素,0.015~4μg/ml;甲氧苄啶-磺胺甲噁唑,0.25/4.75μg/ml~8/152μg/ml。
3.MIC测定:在无菌生理盐水中制备菌悬液,将其比浊至0.5 麦氏浓度。将50μl的菌悬液加入10ml的Mueller Hinton(MH)肉汤培养基,最终接种液细菌载量为1×105~5×105CFU/ml。颠倒混匀后向每孔加入100μl菌悬液。将100μl菌悬液转移到含有13种抗生素的平板的各个孔中。用黏性密封纸密封接种板,并在28~30℃培养箱中孵育72h。如果在对照孔中有菌生长,表现为浑浊或孔底部有沉积物,表示菌量已足够,记录各含药孔的细菌生长情况,第一个抑制细菌生长的药物浓度,即为MIC值。否则,继续培养48h再判读结果。
4.统计学处理:对实验菌株的耐药情况进行描述性分析。
结果
1.标准菌株的耐药情况:龟分枝杆菌(ATCC14472)、偶发分枝杆菌(ATCC6481)和脓肿分枝杆菌(ATCC19977)标准株在不同批次实验中MIC值显示出高度的可重复性,见表1。
表1 13种抗生素对3种快生长分枝杆菌标准菌株的最小抑菌浓度(μg/ml)
| 药品 | 龟分枝杆菌 (ATCC14472) | 偶发分枝杆菌 (ATCC6481) | 脓肿分枝杆菌 (ATCC19977) |
|---|---|---|---|
| 阿米卡星 | 32 | 1 | 8 |
| 妥布霉素 | >8 | 1 | >8 |
| 阿莫西林- 克拉维酸 | >64/32 | >4/32 | >64/32 |
| 头孢西丁 | 128 | 32 | >128 |
| 亚胺培南 | >64 | 16 | >64 |
| 环丙沙星 | >4 | 0.5 | >4 |
| 莫西沙星 | 8 | 0.25 | >8 |
| 克拉霉素 | 16 | 4 | 0.12 |
| 利奈唑胺 | 32 | >32 | 32 |
| 米诺环素 | >8 | 1 | >8 |
| 多西霉素 | >16 | 0.5 | >16 |
| 替加环素 | 1 | 0.12 | 1 |
| 甲氧苄啶- 磺胺甲噁唑 | >8/152 | 8/152 | >8/152 |
2.龟分枝杆菌临床分离株的耐药情况:除有1株龟分枝杆菌临床分离株的MIC>64μg/ml表现为对阿米卡星耐药外,阿米卡星对其余2株龟分枝杆菌的抑菌活性均较好。对于纳入的3株龟分枝杆菌临床分离株,替加环素对其MIC分别为0.12、1、2μg/ml。但莫西沙星、环丙沙星和利奈唑胺对龟分枝杆菌临床分离株几乎没有抑菌活性。
3.偶发分枝杆菌临床分离株的耐药情况:阿米卡星对偶发分枝杆菌的抑菌活性最佳,敏感率为100.0%(25/25)。替加环素也对其表现出优越的抗菌活性,敏感率可达100.0%(25/25)。对于氟喹诺酮类药物,莫西沙星和环丙沙星均表现出对偶发分枝杆菌临床分离株高度的体外抑菌活性。但只有4.0%(1/25)纳入的偶发分枝杆菌临床分离株对克拉霉素敏感(表2)。
表2 偶发分枝杆菌临床分离株的药物敏感性试验结果
| 药品 | MIC 临界浓度(μg/ml)a | 偶发分枝杆菌(25株) | |||||
|---|---|---|---|---|---|---|---|
| 敏感 | 临界浓度 | 耐药 | MIC范围 (μg/ml) | MIC50 (μg/ml) | MIC90 (μg/ml) | 敏感菌株[株 (敏感率,%)] | |
| 阿米卡星 | ≤16 | 32 | ≥64 | 1~2 | 1 | 1 | 25(100.0) |
| 妥布霉素 | ≤2 | 4 | ≥8 | 4~>16 | 16 | >16 | 0(0.0) |
| 阿莫西林-克拉维酸 | - | - | - | 64/32 | 64/32 | 64/32 | - |
| 头孢西丁 | ≤16 | 32~64 | ≥128 | 64~>128 | 64 | 128 | 0(0.0) |
| 亚胺培南 | ≤4 | 8~16 | ≥32 | 32~>64 | >64 | >64 | 0(0.0) |
| 环丙沙星 | ≤1 | 2 | ≥4 | 0.12~4 | 0.12 | 0.25 | 23(92.0) |
| 莫西沙星 | ≤1 | 2 | ≥4 | 0.25~0.5 | 0.25 | 0.25 | 25(100.0) |
| 克拉霉素 | ≤2 | 4 | ≥8 | 1~>16 | >16 | >16 | 1(4.0) |
| 利奈唑胺 | ≤8 | 16 | ≥32 | 8~>32 | 32 | >32 | 1(4.0) |
| 米诺环素 | ≤1 | 2~4 | ≥8 | 1~>8 | >8 | >8 | 2(8.0) |
| 多西霉素 | ≤1 | 2~4 | ≥8 | 0.25~>16 | >16 | >16 | 3(12.0) |
| 替加环素b | - | - | - | 0.12~1 | 0.25 | 0.25 | 25(100.0) |
| 甲氧苄啶-磺胺甲噁唑 | ≤2/38 | - | ≥4/76 | 1/19-4/76 | 4/76 | 4/76 | 5(20.0) |
注 a:每种药品的浓度界线均符合实验室标准化研究所的建议;b:替加环素的临界浓度参考米诺环素和多西霉素;MIC:最低抑菌浓度;MIC50:半数最低抑菌浓度;MIC90:90%最低抑菌浓度
4.脓肿分枝杆菌临床分离株的耐药情况:阿米卡星对脓肿分枝杆菌的抑菌活性最佳,敏感率为90.0%(63/70);替加环素也对其表现出优越的抑菌活性,敏感率可达到72.9%(51/70)。此外,大多数脓肿分枝杆菌对克拉霉素敏感或中介敏感。但莫西沙星和环丙沙星对脓肿分枝杆菌临床分离株几乎没有抑菌活性。几乎所有纳入的脓肿分枝杆菌临床分离株均对利奈唑胺耐药(表3)。
表3 脓肿分枝杆菌临床分离株的药物敏感性试验结果
| 药品 | MIC 临界浓度(μg/ml)a | 脓肿分枝杆菌(70株) | |||||
|---|---|---|---|---|---|---|---|
| 敏感 | 临界浓度 | 耐药 | MIC范围 (μg/ml) | MIC50 (μg/ml) | MIC90 (μg/ml) | 敏感菌株[株 (敏感率,%)] | |
| 阿米卡星 | ≤16 | 32 | ≥64 | 1~32 | 16 | 16 | 63(90.0) |
| 妥布霉素 | ≤2 | 4 | ≥8 | 2~>16 | 16 | >16 | 1(1.4) |
| 阿莫西林-克拉维酸 | - | - | - | 64/32~>64/32 | 64/32 | >64/32 | - |
| 头孢西丁 | ≤16 | 32~64 | ≥128 | 16~>128 | 128 | >128 | 1(1.4) |
| 亚胺培南 | ≤4 | 8~16 | ≥32 | 8~>64 | >64 | >64 | 0(0.0) |
| 环丙沙星 | ≤1 | 2 | ≥4 | 0.12~>4 | >4 | >4 | 3(4.3) |
| 莫西沙星 | ≤1 | 2 | ≥4 | 2~>8 | >8 | >8 | 3(4.3) |
| 克拉霉素 | ≤2 | 4 | ≥8 | 0.06~>16 | 1 | 16 | 43(61.4) |
| 利奈唑胺 | ≤8 | 16 | ≥32 | 2~>32 | 32 | >32 | 2(2.9) |
| 米诺环素 | ≤1 | 2~4 | ≥8 | 1~>8 | >8 | >8 | 2(2.9) |
| 多西霉素 | ≤1 | 2~4 | ≥8 | 0.12~>16 | >16 | >16 | 2(2.9) |
| 替加环素b | - | - | - | 0.06~>4 | 1 | 4 | 51(72.9) |
| 甲氧苄啶-磺胺甲噁唑 | ≤2/38 | - | ≥4/76 | 0.25/4.75~8/152 | >8/152 | >8/152 | 3(4.3) |
注 a:每种药品的浓度界线均符合实验室标准化研究所的建议;b:替加环素的临界浓度参考米诺环素和多西霉素;MIC:最低抑菌浓度;MIC50:半数最低抑菌浓度;MIC90:90%最低抑菌浓度
5.不同药品对3种RGM耐药情况比较:阿米卡星对3种RGM均具有很好的体外抑菌效果,对偶发分枝杆菌和脓肿分枝杆菌的敏感率分别为100.0%(25/25)和90.0%(63/70);3株龟分枝杆菌临床分离株中,有2株对阿米卡星敏感。替加环素对RGM的体外抑菌效果更好,对偶发分枝杆菌和脓肿分枝杆菌的敏感率分别为100.0%(25/25)和72.9%(51/70)。莫西沙星和环丙沙星对偶发分枝杆菌临床分离株表现出较强的抑菌活性,敏感率分别为100.0%(25/25)和92.0%(23/25),而对脓肿分枝杆菌临床分离株几乎没有体外抑菌活性,耐药率均为95.7%(67/70)。几乎所有检测的RGM临床分离株均对头孢西丁、多西环素、利奈唑胺、亚胺培南和甲氧嘧啶-磺胺甲噁唑耐药。
讨论
美国胸科协会对于RGM(龟分枝杆菌、脓肿分枝杆菌和偶发分枝杆菌)的推荐治疗方案是依据体外药敏试验的结果选择药物。对于偶发分枝杆菌引起的肺病,推荐使用至少2种以上体外敏感的药物进行治疗,直至痰菌阴转12个月;对于脓肿分枝杆菌肺病,推荐使用含克拉霉素的多药物治疗方案[4]。实验室标准化研究所推荐对RGM(包括脓肿分枝杆菌、偶发分枝杆菌、龟分枝杆菌和Fuerth分枝杆菌)应进行以下药品的药敏试验,包括阿米卡星、头孢西丁、克拉环素、阿奇霉素、亚胺培南、莫西沙星、左氧氟沙星、环丙沙星、多西环素、美罗培南等[10]。本研究所用的RAPMYCO MIC 药敏板涵盖了实验室标准化研究所推荐对RGM药敏试验的全部抗生素,其药敏试验结果可为RGM感染的治疗提供重要依据。
研究显示,阿米卡星对脓肿分枝杆菌、龟分枝杆菌和偶发分枝杆菌的抑菌活性强,而妥布霉素仅对龟分枝杆菌有效,这与其他研究结果一致[7,11]。与多西霉素/米诺环素相比,替加环素在先前的研究及本研究中均对RGM表现出较低的MIC,目前被认为是一种有前景的药品[11]。笔者研究数据显示,几乎所有纳入的临床分离株均对头孢西丁、多西环素和甲氧嘧啶-磺胺甲噁唑具有抗性,这与Cavusoglu等[9]研究一致。对于亚胺培南,所有纳入的RGM分离株均对其耐药,这也与先前的研究一致,即偶发分枝杆菌对亚胺培南的敏感率每年都以9.5%的速度下降[12]。本研究中,纳入的脓肿分枝杆菌对利奈唑胺的敏感率仅为2.9%,与Hatakeyama等[13]的研究结果不一致,其报告了脓肿分枝杆菌对利奈唑胺的敏感率为76.9%(10/13)。陈品儒等[14]发现广东地区分离的脓肿分枝杆菌耐药率呈现逐年增加的趋势,2010年的敏感率为87.1%(27/31),而2011年则降为68%(17/25)。随着利奈唑胺在我国临床上的广泛应用,势必导致耐药菌株的数量不断增加。
本研究采用商品化的RAPMYCO MIC 药敏板检测RGM的药物敏感性,该方法已经获得国家食品药品监督管理局批准可用于临床诊断。与传统的罗氏药敏试验相比,RAPMYCO MIC药敏板无需手工制备不同药物浓度的培养基,操作简单;与MGIT 960液体药敏试验相比,RAPMYCO MIC药敏板法成本低、检测药物种类更多且可以报告MIC,可以更好地指导临床用药。因此,RAPMYCO MIC药敏板是一种快速、简单且经济的药敏试验检测方法。
综上所述,阿米卡星、替加环素、环丙沙星、莫西沙星和克拉霉素对RGM表现出良好的抑菌活性,此结果可为RGM感染的治疗提供参考。目前,RGM感染治疗可供选择的药物仍非常有限,需要对RGM感染治疗方案的优化提供更多的数据。RAPMYCO MIC药敏板操作简单,可以方便、快速地检测出RGM对13种抗生素的敏感性,适合在临床上常规开展使用。
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Rapidly growing mycobacteria in Singapore, 2006—2011
Antimicrobial susceptibility testing of rapidly growing mycobacteria isolated in Japan
Background: Difficult-to-treat infections caused by rapidly growing mycobacteria (RGM) are increasingly observed in clinical settings. However, studies on antimicrobial susceptibilities and effective treatments against RGM in Japan are limited.Methods: We conducted susceptibility testing of potential antimicrobial agents, including tigecycline and tebipenem, against RGM. Clinical RGM isolates were collected from a university hospital in Japan between December 2010 and August 2013. They were identified using matrix-assisted laser desorption/ionization time-offlight mass spectrometry and the sequencing of 16S rRNA, rpoB, and hsp65 genes. The samples were utilized for susceptibility testing using 16 antimicrobials, with frozen broth microdilution panels.Results: Forty-two isolates were obtained: 13, Mycobacterium abscessus complex; 12, Mycobacterium chelonae; 9, Mycobacterium fortuitum; and 8, M. fortuitum group species other than M. fortuitum. Different antimicrobial susceptibility patterns were observed between RGM species. Clarithromycin-susceptible strain rates were determined to be 0, 62, and 100% for M. fortuitum, M. abscessus complex, and M. chelonae, respectively. M. abscessus complex (100%) and > 80% M. chelonae isolates were non-susceptible, while 100% M. fortuitum group isolates were susceptible to moxifloxacin. Linezolid showed good activity against 77% M. abscessus complex, 89% M. fortuitum, and 100% M. chelonae isolates. Regardless of species, all tested isolates were inhibited by tigecycline at very low minimal inhibitory concentrations (MICs) of = 0.5 mu g/mL. MICs of tebipenem, an oral carbapenem, were <= 4 mu g/mL against all M. fortuitum group isolates.Conclusions: Our study demonstrates the importance of correct identification and antimicrobial susceptibility testing, including the testing of potential new agents, in the management of RGM infections.
致病性速生型非结核分枝杆菌 MIC 法检测及药敏结果分析
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