中华预防医学杂志    2018年04期 空气介质中耐药细菌和耐药基因的研究进展    PDF     文章点击量:72    
中华预防医学杂志2018年04期
中华医学会主办。
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文章信息

李菁 要茂盛
LiJing,YaoMaosheng
空气介质中耐药细菌和耐药基因的研究进展
State-of-the-art status on airborne antibiotic resistant bacteria and antibiotic resistance genes
中华预防医学杂志, 2018,52(4)
http://dx.doi.org/10.3760/cma.j.issn.0253-9624.2018.04.021

文章历史

投稿日期: 2017-11-30
上一篇:2016年新疆农村环境卫生现状调查与分析
下一篇:细菌耐药机制:内源活性氧分子的角色
空气介质中耐药细菌和耐药基因的研究进展
李菁 要茂盛     
李菁 100871 北京大学环境科学与工程学院
要茂盛 100871 北京大学环境科学与工程学院
摘要: 全球正在面临着由于耐药细菌感染和抗菌药物短缺而导致的更多死亡,而其重要的空气传播途径未能受到足够的关注与重视。本文结合最新文献,综述了耐药细菌和基因在不同环境空气介质中的丰度、分布、传播等方面的研究进展,并讨论监测防控的相关措施。耐药细菌和基因在职业暴露场所及其周边空气中的丰度普遍高于普通室内与室外的空气环境。与其他环境介质相比,空气介质蕴含着种类更加丰富的耐药基因,且可以通过附着在空气颗粒物上进行扩散。可通过加强监测、研究其传播机制和生物毒性以及研发病原体快速甄别技术和新型绿色抗菌药物等,有效防控细菌耐药性对人体健康及生态环境的危害。
关键词 :抗菌药;颗粒物;耐药基因;空气传播
State-of-the-art status on airborne antibiotic resistant bacteria and antibiotic resistance genes
LiJing,YaoMaosheng     
State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China
Corresponding author: Yao Maosheng, Email: yao@pku.edu.cn
Abstract:The world is facing more deaths due to increasing antibiotic-resistant bacterial infections and the shortage of new highly effective antibiotics, however the air media as its important transmission route has not been adequately studied. Based on the latest literature acquired in this work, we have discussed the state-of-the-art research progress of the concentration, distribution and spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in different environmental air media, and also analyzed some future prevention and control measures. The large use of antibiotics in the medical settings and animal husbandry places has resulted in higher abundances of ARB and ARGs in the relevant and surrounding atmosphere than in urban and general indoor air environments. ARGs can be spread by adhering to airborne particles, and researchers have also found that air media contain more abundant ARGs than other environmental media such as soil, water and sediment. It was suggested in this review that strengthening the monitoring, study on spreading factors and biological toxicity, and also research and development on pathogen accurate diagnosis and new green antibiotic are expected to help effectively monitor, prevent and control of the impacts of airborne resistant bacteria and resistance genes on both human and ecologies.
Key words :Antibiotics;Particulate matter;Antibiotic resistance genes;Aerial transport
全文

抗生素的长期滥用导致了细菌耐药性的增强、超级细菌的出现和日益增多,这些已经或正在严重危及人类健康[1,2,3,4,5]。据统计,目前耐药性的细菌感染每年在全球范围内造成约70万人死亡,预计到2050年将达到1 000万[6,7]。以耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)为例,早在2005年,美国与MRSA感染有关的死亡人数已超过同年因艾滋病和病毒性肝炎死亡的人数之和[8]。而在WHO 2017年发布的耐药细菌优先性(紧急、高、中)列表中,MRSA的级别为高[9]。列为紧急等级的耐药细菌为对碳青霉烯类药物耐药的鲍曼不动杆菌、铜绿假单胞菌和其他肠杆菌科等,由IND、GES、IMP、OXA等β内酰胺酶基因表达耐药性[9,10]。耐药基因被认为是新型的环境污染物,它的频繁出现和快速传播严重地威胁着抗生素治疗的有效性和人类健康[11]。耐药基因不受宿主细菌细胞活性的限制,因此在传播过程中比耐药细菌更持久,即便在宿主耐药细菌细胞死亡或没有抗生素选择压力时,耐药基因也可以在一定条件下在不同的细菌如致病菌与非致病菌之间,或是革兰阳性菌和阴性菌之间进行扩散传播[12,13,14,15,16]。总而言之,全球正在面临或经历着由于耐药细菌感染和新型高效抗生素短缺而导致的更多死亡的危险。
        为了评估抗生素滥用对生态平衡和人类健康带来的严重后果,已有学者针对各种地表环境中的耐药细菌和基因传播蓄积等进行了大量研究,如污水处理厂、河流、底泥、养殖场、农田等[10,17,18,19,20,21]。耐药细菌还可以通过附着在公共交通工具内座椅等表面进行远距离传播[22,23]。还有研究指出,野生动物如候鸟等,以及空气也是耐药细菌或耐药基因传播的重要媒介[24,25];耐药细菌除了可以通过室内空气直接传播外[25,26,27,28],也可以由(奶)牛、猪等养殖场的土壤中受到扰动后进入空气,进而在空气中进行传播[4,5,29,30]。然而关于耐药细菌和耐药基因(尤其是后者)在空气中的分布特性以及传播机制的研究相对于其他介质尚不多见,这不仅会影响人们对于耐药细菌及基因在空气中传播机制和环境归宿的准确理解,也可能产生细菌耐药性空气传播途径的研究盲区。2016年9月21日,联合国召开抗耐药细菌的高层会议,讨论了耐药问题的严重性,以及应对耐药细菌的多部门合作的方案[31]。虽然耐药细菌及耐药基因目前得到了足够重视,但是对其空气传播的危害和机制的研究还没有提上日程。本文结合最新文献,综述了耐药细菌和耐药基因在空气环境中的浓度、分布、传播等方面的研究进展,为今后应对细菌耐药性的空气传播途径提供研究重点和行动方向上的参考。

一、耐药细菌在空气中的来源、浓度和分布  除了不同的地表环境介质,包括土壤、水体界面如湖泊、海洋、河流等自然源,部分人为活动如污水处理、畜牧养殖、堆肥发酵等也是空气中细菌的重要来源[32,33]。同样,这些环境介质也是空气中耐药细菌和主要来源,如图1所示。地面环境介质中抗生素的使用和细菌气溶胶化的程度会影响空气介质中耐药细菌和基因的丰度与分布。

图1空气介质中耐药菌及耐药基因排放源、传播及影响
动物养殖场是抗生素使用的一个重要场所。研究认为,与动物养殖有关的农业活动是环境中细菌获得耐药性过程的主要贡献者[1,34]。根据Zhang等[35]的研究,2013年中国抗生素的总消耗量为162 000吨,动物养殖产业的使用量占比52%,美国动物养殖产业抗生素使用量则高达82%[36,37]。因此,已从养殖场及周边空气中广泛检测到耐药细菌[38,39,40,41,42,43,44,45]。以MRSA为例,Friese等[42]研究发现德国27家养猪场内的空气样品中MRSA检出率高达55.6%~85.2%,其浓度受到采样方法的影响,如撞击式采样器采集的MRSA平均浓度为257 CFU/m3,而膜采样器采集的MRSA平均浓度为802 CFU/m3。除了采样器,颗粒物粒径也是空气中耐药细菌检出浓度的影响因素。Ferguson等[45]发现养猪场内空气中的MRSA多检出于粒径大于5 μm的颗粒物上,而养猪场周边空气中的MRSA检出于粒径小于5 μm的颗粒物上。对空气中耐药细菌浓度和分布的研究大多是基于培养的方法,而评估耐药基因在空气中的丰度和分布采用的是实时荧光定量PCR技术。Ling等[29]在美国科罗拉多州的猪养殖场和奶牛养殖场的空气中均检测到了四环素类耐药基因tetX(100~200拷贝/m3)和tetW(100~400拷贝/m3)。Gao等[46]在蛋鸡养殖场和肉鸡养殖场的空气中分别检测到了四环素类耐药基因tetW(10~106拷贝/m3)和tetL(102~106拷贝/m3),虽然两种耐药基因都随着颗粒物粒径的增大丰度增高,但粒径较小的颗粒物(4.32~5.33 μm)中tetL的丰度较大,粒径较大的颗粒物(5.34~6.38 μm)中tetW的丰度较大。
        在其他职业环境中,如市政污水处理厂(urban wastewater treatment plant, UWTP)每日人均当量的出水中约含109~1012 CFU,其中至少有107~109 CFU的细菌属于获得性耐药[47]。因此污水处理厂(wastewater treatment plant, WWTP)的空气环境中耐药细菌和基因的检出近年来也逐渐被报道[48,49,50]。例如,Li等[49]在北京的一座污水处理厂内格栅间和曝气池区域的空气样本中检测到磺胺类耐药基因sul2和可以帮助其进行水平转移的整合酶基因intI1,但没有进行定量。高新磊等[50]分析了深圳市罗湖区罗芳污水处理厂内的21份空气样本中的四环素类耐药基因(tetCtetGtetOtetX)、磺胺类耐药基因(sul1sul2)和大环内酯类耐药基因(ermBermC),发现不同工艺单元区域空气样本中耐药基因的丰度不同,如曝气池和污泥脱水车间的空气样品中sul1sul2tetGtetX的丰度较高,范围在102~105拷贝/ng DNA之间。
        此外,医院作为人类疾病相关抗生素的主要消耗场所,其空气环境被认为是各种耐药细菌、甚至是多重耐药(multi-drug resistant, MDR)细菌的储藏库[26,51,52,53,54]。邱玉玉等[53]在山东省5所大中型医院的250份室内空气样品中分离出219株金葡菌,其中MRSA 88株,这些MRSA均对青霉素类及头孢曲松耐药,对庆大霉素、红霉素和四环素的耐药率超过90%。Solomon等[54]发现在埃塞俄比亚中南部的Wolaita Sodo大学教学医院的室内空气中73.6%~84.6%的常见耐药致病菌(凝固酶阴性葡萄球菌、金黄色葡萄球菌、粪肠球菌和屎肠球菌、不动杆菌属、大肠杆菌和铜绿假单胞菌)表现为多重耐药。足以见得,医院环境已经成为一个滋生耐药细菌及耐药基因的重要温床。
        职业暴露场所空气中的耐药细菌和基因会对场所内部和周边的人群健康造成呼吸暴露风险。以MRSA为例,医院获得性MRSA(hospital-acquired MRSA, HA-MRSA)的传播媒介除了医护人员本身(如医护服、医用手套等)及其所使用的医疗用具外[27,55],气源传播也被认为是发生MRSA呼吸道感染及创面感染的重要途径[27,56,57]。Zheng等[28]利用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)从呼吸道感染患者的呼出气中检测到了MRSA,证明MRSA可以通过患者的呼出气进行传播。在畜牧业相关的职业暴露场所中,MRSA可以大量存在于感染畜群产生的灰尘中,导致工作人员吸入携带MRSA的灰尘颗粒物而被感染[42,58,59,60,61,62,63]。Angen等[63]发现在养猪场内工作的志愿者鼻腔内的MRSA浓度与养猪场内空气中的MRSA浓度显著正相关。此外,Laube等[43]在德国肉鸡养殖场的肉鸡体内、养殖场土壤、养殖场空气和养殖场下风向50米处的空气中均检测到了基因型完全相同的产ESBL/AmpC酶大肠杆菌菌株。Navajas-Benito等[44]在西班牙奶牛养殖场和其周边的空气样本中均检测到了基因型相同的多重耐药大肠杆菌菌株ESTE50。此外,McEachran等[4]设计实验发现耐药基因在牛养殖场下风向颗粒物样本中的丰度均显著高出上风向样本约100~4 000倍(P值<0.002),证明耐药基因可以附着在颗粒物上进行传播。越来越多的研究证实耐药细菌和基因可以通过空气进行扩散并能够在一定时间内保持活性。

二、普通室内与室外空气中耐药细菌和基因的丰度、分布和传播  细菌耐药性问题的棘手之处在于即使在普通室内和室外的空气介质中,耐药细菌和基因也已经逐渐被检测到。Gandolfi等[64]使用纸片扩散法分析了米兰市区夏季和冬季PM10颗粒物中分别分离出来的281和288株细菌,结果显示耐药细菌在不同季节的分布不同,如肠球菌属耐药细菌多出现在夏季,而不动杆菌属耐药细菌多出现在冬季;假单胞菌科和肠杆菌科细菌始终表现出对竹桃霉素和万古霉素的较高耐药率(80%~100%);而葡萄球菌属细菌则表现出了最广泛的耐药性。Zhou和Wang[65]研究了上海地铁内空气样本中分离得到的葡萄球菌菌株,发现它们平均对2.64种抗生素耐药,58%的菌株至少对3种抗生素耐药;这些菌株中MRSA相关耐药基因mecA和耐消毒剂基因qac的携带率分别为28%和40%,甚至高于医院病房的室内空气(19.23%和35.62%),并显著高于公园的空气环境(均为5.56%)。Echeverriapalencia等[66]在美国加利福尼亚州Fresno、Bakersfield、Los Angeles和San Diego四个城市公园的空气样品中检测到了β内酰胺类耐药基因bla SHA和磺胺类耐药基因sul1,浓度范围分别为10-1~102和10-2~103拷贝/m3
        可以看出,目前对于空气环境中耐药基因的研究多集中于四环素类或有限的几种基因片段,造成这种情况的主要原因是受限于传统的荧光定量PCR技术。而高通量荧光定量PCR技术可以实现同时对多达上百种抗性基因或多个样品进行定量分析[67]。例如,Zhu等[19]采用这种技术定量检测了我国3个大型养猪场及周边地区的土壤样品中的244种耐药基因,共检测到149种耐药基因。然而目前高通量荧光定量PCR技术在空气颗粒物样本中应用的研究尚不多见。宏基因组测序近几年来也得到大力发展,成为可用于定量分析不同环境介质中耐药基因丰度的有力工具。Pal等[10]和Hu等[68]重新分析了Cao等[69]关于北京2014年1月8—14日重污染天气时颗粒物样本宏基因组测序的数据。Pal等[10]分析发现与其他环境介质如人体(皮肤、呼吸道、口腔、泌尿生殖道和肠道)、河水/河流沉积物、污水/污泥、制药废水、土壤等相比,北京空气颗粒物中蕴藏着丰富度最高的耐药基因,平均为64.4种。同时,北京空气颗粒物中耐药基因的丰度也不低,与人体肠道和污水/污泥中耐药基因的相对丰度水平相当,均为平均0.3拷贝/16S rRNA[10]。值得注意的是,北京空气颗粒物中检测到了几种碳青霉烯类耐药基因,包括IND、GES、IMP、OXA-50、OXA-51和OXA-58型β内酰胺酶基因。碳青霉烯类抗生素被称为最后的抗生素[10,70],其耐药基因在空气样本中的检出,无疑为人类滥用抗生素造成的健康风险和生态危害敲响了警钟。
        关于空气介质中耐药基因的影响因素,Hu等[68]分析发现空气细菌微生态、颗粒物理化指标和气象因素都会影响耐药基因的丰度分布,但也只能解释约54%的分布特性;此外,灰霾天空气颗粒物中耐药基因的丰度显著大于清洁天。虽然Hu等[68]的数据分析研究表明空气中耐药基因的丰度分布和土壤相似,但根据Pal等[10]的研究,北京大气颗粒物中耐药基因的组成与其他任何环境介质的相似度均不高。这些研究均证实了气源传播是特定环境中的耐药细菌和耐药基因传播到外部环境的重要途径,同时也是更复杂、更分散的过程。通过空气传播的耐药基因的环境摄取将使细菌的耐药性进一步蔓延、扩大。通过空气传播,使得耐药菌或耐药基因可以到达诸多遥远的,甚至人类从未踏足的地区。人类驱使下的抗生素的大量使用不仅影响耐药细菌、耐药基因的增加,而且也在广泛影响着细菌种群的结构变化[71]。如图1所示,通过呼吸暴露,不论是耐药细菌还是耐药基因,对人类的免疫系统、生命健康以及生态环境均构成潜在的危险。

三、研究展望  经过多方努力,我国在解决大气污染问题上已经取得了一定进展;但空气中的耐药基因作为新型空气污染物的出现没能得到足够的重视,特别是我国是抗生素使用大国。耐药细菌和耐药基因均能够形成生物气溶胶,不仅能在一定的条件下引起气源感染,对人类的生命健康造成威胁,而且可以通过沉降影响微生态平衡。与其他环境介质相比,空气介质蕴含着种类更加丰富的耐药基因;然而关于其来源、分布和传播机制等的文献报道仍旧匮乏,使得空气生物污染问题被严重低估。综合现有的状况,除了从国家和政府层面上继续加强管理和控制抗生素在畜牧养殖业和医院临床治疗中的使用以外,在未来应对耐药细菌以及耐药基因的相关研究及行动中,特别是在空气介质中的,作者认为如下几点可能需要特别关注,从而可以为有效防控细菌耐药性的扩散、更好地保护人群健康。

1.开展环境中耐药细菌监测:  大力开展对职业环境、室内环境和大气环境空气中耐药细菌和基因丰度和分布的监测工作,获得空气中常见耐药细菌和基因(包括基因转移元件)的清单,同时为建立抗生素使用和空气中耐药细菌和基因的丰度之间的数学模型提供数据基础;此外,目前对于空气中耐药基因的研究,大多没有区分其究竟是来自有活性的宿主细胞,还是裸露游离的DNA分子,这影响了我们对耐药基因在不同环境介质中的扩散机制的理解,在未来的研究中应注意区分。

2.研究细菌耐药和基因传播机制:  利用实验室研究和模拟研究分析耐药细菌和基因的传播机制,如传播距离,以及经过远距离传输后是否还具有生物活性等,是否仍然具有危害性,分析环境因子如气象条件、大气辐射等对耐药细菌和基因传播效率的影响。

3.开展空气中耐药细菌和基因毒理学研究:  利用动物模型等对空气中耐药细菌和基因进行毒理学研究,并深入了解耐药细菌和基因对呼吸道微生态的影响,为建立空气中耐药细菌和基因的风险评估体系和空气耐药微生物浓度标准体系提供数据支持。

4.发展病原体快速、精准的甄别技术:  该技术的发展也是遏制细菌耐药性发展的关键。例如,过去研究人员通过利用生物传感器可以在几分钟内检测到临床患者呼出气中H3N2病毒和H1N1病毒[72,73],从而可以帮助实现呼吸系统感染病原体的快速分型,减少抗生素的误用和滥用。未来研发及时现场护理(point-of-care,POC)便携式病原体快速分型在临床上能有效阻断抗生素的滥用,减少耐药菌的滋生,延长抗生素的有效性。

5.加大开展新型抗菌药物的研发工作:  Ling等[74]在缅因州的土壤内发现了一种名为Teixobactin的抗生素,这种抗生素主要通过结合细菌的细胞壁中的脂肪酸而不是蛋白质来消灭细菌,病原体因此很难对其产生抗药性。最新研究建议老药新用,考虑采用甲氧西林联合他汀类药物治疗MRSA导致的感染,同时因为他汀类药物没有给MRSA造成生存压力,所以不会促进细菌耐药性的产生[75]。这些代表性的工作都为寻找新型绿色抗生素的(不产生耐药性)开辟了新的思路。

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