抗生素最初是用来治疗动物疾病,后来逐渐作为饲料添加剂、预防剂和生长促进剂,使用量和使用频次逐年上升[1-2]。据报道全球73%的水产养殖国家使用土霉素、磺胺嘧啶和氟苯尼考,55%的国家在水产养殖中使用磺胺二甲氧嘧啶、红霉素、阿莫西林和恩诺沙星[3]。抗生素的使用会直接影响鱼体肠道菌群的稳定。鱼类肠道菌群在营养合成和代谢、肠道上皮发育以及免疫系统调节等方面发挥重要作用[4],肠道菌群紊乱则会进一步影响鱼体的生理功能。
此外,抗生素的生物利用率仅为10%～30%,致使大量抗生素以母体及其代谢物形式进入养殖水环境中[5]。同时,抗生素持续不断地输入造成在水环境中形成一种假持久性现象[6]。一些抗生素在亚致死剂量下能显著促进质粒介导的抗生素抗性基因(antibiotic resistance genes, ARGs)水平转移,比如常见的氟诺喹酮类药物可通过接合转移促进ARGs在不同细菌之间进行传播[7-8]。一旦细菌获得ARGs,即使在选择压力消失后,它们也可以在环境中长期存在[9]。ARGs在生态环境中的传播、迁移和扩散已成为影响全球公共卫生安全的重要问题[10]。2021年9月,Nature发表的综述文章指出在水产养殖业中超过50%的水产养殖环境和养殖生物存在耐药性问题[11]。研究发现,已有90%以上的水生细菌能够抵抗超过1种抗生素,大约20%的细菌能抵抗5种甚至更多抗生素[12]。根据世界卫生组织(2019)[13]的报告,如果不采取行动,到2050年抗生素耐药性每年将导致1 000万人死亡,并使全球经济陷入危机。然而,抗生素的使用和ARGs的产生和传播之间的关联机制仍未明确。因此,本文总结归纳了养殖水环境中的抗生素残留现状,着重以抗生素对鱼类肠道菌群结构、功能及抗性组的影响为主线进行总结讨论,以期为环境中抗生素对鱼类肠道健康和ARGs传播的影响研究提供参考,也为在水产养殖中合理使用抗生素提供借鉴。
1 抗生素在养殖水环境中的残留现状(Status of antibiotic residues in aquaculture water environment)
1.1 养殖水环境中抗生素残留的原因、特点及变化趋势(Causes, characteristics and trend of antibiotic residues in aquaculture water)
水产养殖中抗生素的污染来源主要有:(1)水产养殖中直接使用的抗生素;(2)家禽粪/牛粪等水产饲料中的抗生素残留;(3)受污染的养殖用水中的抗生素。其中直接使用的抗生素是最重要的来源之一[14-15]。鱼类是全球贸易量最大的粮食商品之一,为人类的粮食安全和充足营养供应做出了重大贡献,目前野生鱼类捕捞量已趋平稳,能够持续大幅增长的用于人类消费的鱼类供应主要来源于水产养殖[16]。当前,世界范围内的水产养殖模式已从传统养殖系统向集约化养殖系统转变,导致高密度养殖现象,显著增加了感染细菌性疾病发生和传播的风险[17]。为了预防和治疗这些疾病,抗生素在水产养殖中得到了广泛的应用。此外,抗生素,特别是土霉素和氟苯尼考也被用作水产养殖生物的生长促进剂[18]。世界范围内水产养殖中抗生素的使用量显著增加,抗生素的施用方式主要有混合饲料饲喂法、沐浴法和池洒法,其中混合饲料饲喂是抗生素的常见施用途径,这些饲喂方式都容易导致抗生素在水产养殖环境中残留,严重污染养殖环境和养殖生物,增加养殖鱼类的抗生素暴露风险[19-20]。
近年来,在水产养殖水环境中频繁检出抗生素,主要包括磺胺类、四环素类以及氟喹诺酮类药物。表1列举了国内外部分养殖水体中检测频率和浓度较高的抗生素种类及其残留量。由数据可知,养殖水体中抗生素的残留量较高,大多在ng·L-1～μg·L-1水平。其中检测到磺胺类残留量最高达7.42 μg·L-1,氟喹诺酮类最高达958 μg·L-1,四环素类最高达15.163 μg·L-1。
表1 养殖水体中抗生素浓度
Table 1 Concentration of antibiotics in aquaculture water
抗生素种类Antibiotic type养殖水体采样位置Sampling location of aquaculture water浓度/(ng·L-1)Concentration/(ng·L-1)参考文献Reference磺胺嘧啶Sulfadiazine中国茅尾海Maowei Sea, ChinaND^28.60[21]中国北部湾Beibu Gulf, ChinaND^24.42[22]中国杭州湾Hangzhou Bay, China0.86^2.67[23]中国北江Beijiang River, ChinaND^7 418.00[24]中国洪湖Lake Honghu, China8.00^261.10[25]中国桂林Guilin, China32.30^333.71[26]韩国Republic of KoreaND^5.69[27]孟加拉国BangladeshND^17.97[28]磺胺甲恶唑Sulfamethoxazole中国北部湾Beibu Gulf, ChinaND^3.30[22]中国杭州湾Hangzhou Bay, China2.25^15.17[23]中国北江Beijiang River, ChinaND^29.60[24]中国洪湖Lake Honghu, China6.00^260.50[25]中国桂林Guilin, China116.76^652.69[26]中国黄海Yellow Sea, ChinaND^3.43[29]中国莱州湾Laizhou Bay, China0.60^1 273.70[30]中国太湖Tai Lake, ChinaND^7 189.10[31]恩诺沙星Enrofloxacin中国北江Beijiang River, ChinaND^20.40[24]中国桂林Guilin, China181.49^678.48[26]中国黄海Yellow Sea, ChinaND^995.02[29]中国莱州湾Laizhou Bay, China0.31^82.25[30]中国太湖Tai Lake, ChinaND^183.61[31]中国广州Guangzhou, ChinaND^100.00[32]马来西亚MalaysiaND^958 000.00[33]诺氟沙星Norfloxacin中国北部湾Beibu Gulf, China4.31^97.30[22]中国杭州湾Hangzhou Bay, China15.20^115.28[23]中国桂林Guilin, ChinaND^191.07[26]中国黄海Yellow Sea, ChinaND^8.73[29]中国莱州湾Laizhou Bay, ChinaND^72.37[30]中国太湖Tai Lake, ChinaND^210.57[31]马来西亚MalaysiaND^6 670.00[33]环丙沙星Ciprofloxacin中国北部湾Beibu Gulf, China5.42^182.00[22]中国桂林Guilin, China135.57^247.64[26]中国黄海Yellow Sea, ChinaND^61.29[29]中国莱州湾Laizhou Bay, ChinaND^73.84[30]马来西亚MalaysiaND^131 000.00[33]中国海陵岛Hailing Island, ChinaND^186.00 [34]中国广东Guangdong, China15.80^61.80[35]土霉素Oxytetracycline中国杭州湾Hangzhou Bay, China12.35^38.33[23]中国北江Beijiang River, ChinaND^89.40[24]中国黄海Yellow Sea, ChinaND^41.03[29]中国莱州湾Laizhou Bay, ChinaND^175.02[30]中国太湖Tai Lake, ChinaND^198.83[31]中国海陵岛Hailing Island, ChinaND^15 163.00[34]越南Vietnam200.00^250.00[36]韩国Republic of KoreaND^9 935.00[37]
续表1抗生素种类Antibiotic type养殖水体采样位置Sampling location of aquaculture water浓度/(ng·L-1)Concentration/(ng·L-1)参考文献Reference四环素Tetracycline中国杭州湾Hangzhou Bay, China9.89^34.11[23]中国北江Beijiang River, ChinaND^303.00[24]中国洪湖Lake Honghu, China98.50^1 019.20[25]中国莱州湾Laizhou Bay, ChinaND^1.73[30]中国太湖Tai Lake, ChinaND^250.63[31]马来西亚MalaysiaND^73.00[33]中国海陵岛Hailing Island, ChinaND^2 305.00[34]越南Vietnam130.00^190.00[36]红霉素Erythromycin中国茅尾海Maowei Sea, ChinaND^1 290.00[21]中国北部湾Beibu Gulf, China0.62^45.80[22]中国北江Beijiang River, China1.00^68.80[24]中国广州Guangzhou, China80.00^1 400.00[32]氟苯尼考Florfenicol中国北部湾Beibu Gulf, ChinaND^578.00[22]中国杭州湾Hangzhou Bay, China0.48^47.97[23]中国太湖Tai Lake, ChinaND^2 708.60[31]中国广东Guangdong, ChinaND^1 282.00[35]韩国Republic of KoreaND^1 600.00[37]
养殖水体中抗生素残留水平主要与水产养殖的空间分布、养殖生物种类、生长阶段以及季节等因素有关。在我国检测到较高抗生素残留量的养殖水体主要分布于东部和南部,包括黄海、莱州湾、海陵岛和北江等地。这和我国水产养殖的空间分布有关,据报道东部和南部地区的养殖面积可达北部和西部地区的600倍,广东、山东、福建、江苏和湖北是产量最大的5个省份[19]。
不同养殖生物对抗生素的需求差异会影响养殖水体中抗生素残留水平。大菱鲆的抗病性较差,养殖中通常伴随着高剂量的抗生素使用,导致其养殖水体抗生素含量高于海参池和软体动物池[30]。大环内酯类药物仅在虾/蟹池中检测到,在鱼池中未检出,这是由于大环内酯类药物对藻类有较强毒性,可以控制高温期间在虾/蟹池经常发生的藻华[31]。王敏等[38]检测了鱼、虾、蟹养殖水体中抗生素的残留水平,抗生素检出种类情况为鱼(6种)>虾(5种)>蟹(3种),其中诺氟沙星仅在虾池中检出,磺胺二甲嘧啶、磺胺甲恶唑和甲氧苄氨嘧啶仅在鱼池和虾池中检出,氧氟沙星和磺胺嘧啶仅在鱼池和蟹池中检出。李贞金等[39]在不同生物的养殖池中检出的抗生素种类也不相同,其中鱼池10种、虾池9种、蟹池仅5种。氟喹诺酮类在鱼池中的检出浓度远高于虾池和蟹池,喹乙醇在虾池中的检出浓度远高于鱼池和蟹池,磺胺甲恶唑仅在蟹池中被检出。养殖生物的不同生长阶段对抗生素的需求也有较大差异,幼年时期的抵抗力较差,需要更多的抗生素用于疾病防治和促进生长。而在成年时期会减少或停止抗生素的使用,以最大限度地减少水产品中抗生素的残留[21-22, 34]。抗生素在养殖水体中的残留量也受季节因素影响,夏季比冬季检测到的抗生素浓度更高,种类更多。夏季是水产养殖的主要季节,高温会导致水生生物疾病频发,抗生素用量增加[21, 25, 29-30, 40]。
1.2 养殖水环境中抗生素残留引发生态环境问题(Antibiotic residues in aquaculture water may cause ecological and environmental problems)
水产养殖系统和周围水域之间存在交叉污染现象。养殖水体中抗生素的大量残留会对周围水域造成污染。养殖池塘中多种广泛用于水生生物的抗生素(如磺胺甲氧嘧啶、磺胺甲恶唑和环丙沙星)的检测浓度高于附近的河流[35, 40]。此外,开放水域养殖渔业的水源是从地表水或附近海水中收集的,而周边水体中由人为活动产生的污染物也会对养殖水体产生影响,包括医用抗生素(林可霉素、红霉素等)、咖啡因、布洛芬和工业化学品(防腐剂和全氟化合物)[40]。如果养殖池中含有高浓度抗生素的废水不加限制地排放到附近的水域中,将严重破坏相关水域生态系统的稳定性,而养殖池附近水域的水环境保护对养殖业公共健康也至关重要。
养殖水体中的抗生素残留最终会成为水产养殖生态系统的潜在生态风险。长期接触低浓度抗生素会影响水生生物的生长和繁殖,造成慢性中毒,导致畸形或死亡,在养殖生物体中积累的抗生素通过食物链进入人体,并可能影响人体健康[41]。此外,水生环境中ARGs的迁移和转化可能比环境中的抗生素残留物危害更大。抗生素在养殖水体中的持续残留会导致ARGs的产生和富集,进而会使抗生素失效,水产养殖生物更容易感染疾病,导致养殖产业的巨大经济损失。而食用受ARGs污染的水产品或暴露在受污染的水生环境中可能会增加人类健康风险[42]。
2 鱼类肠道菌群功能(Fish gut microbiota function)
鱼类肠道菌群参与鱼类营养物质的消化、吸收和免疫防御等,能促进宿主的生长发育维持宿主的健康。
2.1 促进生长(Promote the growth of fish)
肠道菌群可通过促进宿主的能量获取直接促进鱼体生长,研究发现,梭菌科、纤毛菌科、动球菌科和毛螺菌科的细菌可以决定鱼体的生长速度,这些细菌与宿主互利共生[43]。研究证实花斑溪鳉、大西洋鳕鱼的生长速率与肠道菌群组成密切相关[44-45]。Tan等[46]报道,从尼罗罗非鱼肠道分离出的Rummeliibacillusstabekisii菌株可以提高蛋白酶、木聚糖酶和纤维素酶的活性,这些酶可以帮助宿主消化蛋白质和糖类物质,提高营养物质利用率,促进鱼体的生长。此外,肠道菌群在脂质代谢中也发挥重要作用。Falcinelli等[47]发现,在鼠李糖乳杆菌的作用下,斑马鱼肠道中厚壁菌丰度增加,放线菌丰度降低,这些肠道菌群的变化导致胆固醇和甘油三酯代谢相关基因(如fit2、agpat4、dgat2和mgll)表达下调,最终调节宿主的脂质代谢。Semova等[48]的研究显示,厚壁菌可以刺激肠上皮细胞中脂肪酸的摄取,导致肠细胞中脂滴的积累和膳食中脂肪酸在肠外组织中的积累。因此,鱼类肠道菌群可通过调节宿主蛋白质吸收、糖类吸收和脂质代谢在调节鱼体营养吸收方面发挥重要作用。
2.2 免疫功能(The immune function)
鱼类有复杂的免疫系统。第一道防线由皮肤和黏膜组成,为宿主提供屏障,并具有杀菌活性。第二道防线是先天免疫系统,第三条防线是适应性免疫系统。鱼类的肠道菌群是免疫系统的重要组成部分。
肠道菌群可作为鱼类免疫的第一道防线——直接屏障[49]。微生物生物膜在宿主的发育和功能中发挥着重要作用,并保护宿主免受病原体的侵害。Burtseva等[50]从海洋鱼类肠道中分离出的生物发光细菌菌株可以在体外形成生物膜,这表明鱼类肠道菌群中也存在能够抵抗外来病原体的微生物生物膜系统。此外,肠道内的共生细菌可通过竞争或改变生态位和可用营养素等抑制肠道内机会性病原体的定植和增殖。例如,益生菌双歧杆菌通过肠道环境酸化(种间屏障效应)防止致病性大肠杆菌入侵[51]。肠道共生菌还可以产生细菌素和蛋白毒素,专门抑制相同或相似细菌物种的成员(种内屏障效应)生长或定植。
肠道菌群与鱼类的先天免疫和适应性免疫密切相关。鱼类肠道菌群可以通过调节肠道中免疫相关细胞的功能发挥免疫调节作用。研究表明,鱼类肠道菌群在肠道上皮细胞更新和成熟过程中发挥关键作用[52],肠道上皮细胞的更新和成熟是免疫反应过程的重要部分。与无菌斑马鱼相比,传统饲养的斑马鱼与上皮细胞增殖和先天免疫反应相关的基因表达量更高[53]。成熟的肠道黏膜可通过模式识别受体(PRRs,如TOLL样受体、RIG-I样受体、NOD样受体和AIM2样受体)区分病原体和共生菌,PRRs能检测到细菌抗原并激活信号级联反应以调节免疫反应。例如,TOLL样受体家族是PRRs的代表成员,能识别病原体中的保守结构,可以招募和调节免疫和炎症细胞,从而启动和介导全身免疫反应[54]。研究表明患有肠炎的石斑鱼肠道中弧菌、链球菌等病原体增加,激活TOLL样受体、RIG-I样受体和NOD样受体信号通路[55]。在斑马鱼中,肠道共生细菌是中性粒细胞募集的主要来源,共生细菌通过TLR/MyD88/NF-kB信号通路诱导促炎细胞因子和抗病毒介质的表达[56]。从成年黑鲈肠道菌群中分离得到的Lactobacillus delbrueckiis sp.,被喂食给黑鲈幼鱼以研究其对肠道免疫系统发育和分化的影响,观察到处理鱼的T细胞和嗜酸粒细胞数量显著高于对照鱼[57]。有研究表明在没有肠腔细菌的情况下,B细胞和T细胞从肠道固有层迁移,同时免疫球蛋白A也不再分泌[54]。
2.3 增强鱼体抵抗环境污染物的能力(Enhance the ability of fish body to resist environmental pollutants)
外源性污染物的毒性可在肠道细菌代谢后降低。研究表明,菌群可将环境污染物生物催化转化,这些转化过程包括还原、水解、官能团去除、N-氧化物裂解、蛋白质水解和反硝化等[58]。研究证明,肠道细菌的生物转化能力可以有效降解多种污染物,如药品[59]、金属[60]、农药[61]以及多环芳烃[62]。
2.4 肠道菌群与疾病(Gut microbiota and disease)
正常情况下,鱼的肠道菌群始终保持动态平衡,从而维持正常的生理功能。然而,环境的变化,如水温、食物类型、污染物接触、病毒和细菌感染等均可破坏肠道菌群的平衡和稳态,由此将可能导致病原菌的异常生长,与鱼类相关疾病的发生密切相关。
患红盖病的鲫鱼肠道菌群多样性和稳定性改变,条件致病菌包括弧菌、气单胞菌和希瓦氏菌丰度增加[63]。鳗鲡弧菌感染的香鱼肠道菌群多样性和均匀度下降,梭菌目增加,该目包括病原体艰难梭菌[64]。患疖病的圆口铜鱼肠道菌群多样性下降,病原体杀鲑气单胞菌显著增加[65]。患有肠炎的草鱼肠道中Dechloromonas和Methylocaldum等致病菌种类增加,进一步导致氨基酸代谢、外源性物质生物降解和代谢以及碳水化合物代谢增强,这可能会加剧肠炎的发展[66]。Parshukov等[67]对患病虹鳟(皮肤表面溃疡、鱼鳍坏死、贫血和肠道炎症)的肠黏膜进行测序,发现患病虹鳟肠道中的主要细菌门是变形菌、放线菌、厚壁菌和软壁菌,而健康虹鳟鱼肠道中主要为拟杆菌和梭杆菌。患病罗非鱼(皮肤出血性败血症、眼球突出)肠道菌群多样性下降,变形菌门取代梭菌门成为最主要的门,弧菌属取代头孢菌属成为最主要的属[68]。Ye等[69]发现,在高脂肪饮食下,斑马鱼肠道中不动杆菌、假单胞菌和气单胞菌的丰度显著增加。此外,不动杆菌的增加导致肠内分泌细胞的形态变化和营养不敏感状态,这导致代谢性疾病(包括肥胖和胰岛素抵抗)的发病率和严重程度更高。
3 抗生素对鱼类肠道菌群结构和功能的影响(Effects of antibiotics on intestinal structure and function of fish)
3.1 氟喹诺酮类抗生素(Fluoroquinolone antibiotics)
关于氟喹诺酮类抗生素对鱼类肠道菌群影响的研究主要集中在氟喹诺酮类抗生素对淡水鱼的短期暴露实验研究(表2)。环境水平的恩诺沙星暴露会导致斑马鱼肠道中立克次氏体和假单胞菌减少,黄杆菌比例增加,此外,肠道菌群紊乱会进一步影响斑马鱼的免疫功能[70]。虹鳟给药恩诺沙星(25 mg·kg-1)5d后,其肠道中拟杆菌属和支原体减少,梭菌属和鲸杆菌属增加[71]。高浓度环丙沙星(1 mg·L-1)会导致香鱼肠道中变形菌门、厚壁菌门和疣微菌门增加,放线菌门、拟杆菌门和软壁菌门减少,进一步导致肠道杯状细胞减少,免疫失调,肠上皮细胞坏死[72]。短期暴露于环境水平的诺氟沙星会导致大黄鱼肠道菌群多样性显著降低,变形菌门的相对丰度降低,但软壁菌门增加。从菌群功能的角度来看,诺氟沙星抑制细菌的代谢、细胞防御机制和信息转导过程,最终会抑制鱼体生长[73]。
表2 氟喹诺酮类抗生素对鱼类肠道菌群结构和功能的影响
Table 2 Effects of fluoroquinolone antibiotics on the structure and function of fish intestinal microbiota
抗生素种类Antibiotic type抗生素浓度及暴露时间Antibiotic concentration and exposure time物种Species肠道菌群结构变化Structural changes of intestinal microbiota肠道菌群相关功能变化Functional changes of intestinal microbiota参考文献Reference恩诺沙星Enrofloxacin0.01、0.1、1、10、100 μg·L-1; 10 d斑马鱼Zebrafish黄杆菌目Flavobacteriales↑、立克次氏体目Rickettsiales↓、假单胞菌目Pseudomonadales↓诱导免疫抑制Inducing immune suppression[70]25 mg·kg-1;5 d虹鳟Oncorhynchus mykiss梭菌属Clostridium↑、鲸杆菌属Cetobacterium↑、拟杆菌属Bacteroides↓细菌衍生脂肪酸减少Bacteria-derived fatty acids decrease[71]环丙沙星Ciprofloxacin1 000 μg·L-1;7 d香鱼Plecoglossus altivelis变形菌门Proteobacteria↑、厚壁菌门Firmicutes↑、疣微菌门Verrucomicrobia↑、放线菌门Actinobacteria↓、拟杆菌门Bacteroidetes↓、软壁菌门Tenericutes↓;弯曲杆菌属Ancylobacter↑、链球菌属Streptococcus↑、普雷沃菌属Prevotella↓杯状细胞减少,免疫失调,肠上皮细胞坏死The number of goblet cells decreased, im-mune dysregulation, intestinal epithelial cell necrosis[72]诺氟沙星Norfloxacin 0.1、10、100、1 000 μg·L-1; 14 d大黄鱼Large yellow croaker软壁菌门Tenericutes↑、变形菌门Proteobacteria↓代谢和免疫功能失调,抑制鱼的生长Metabolic and immune dysfunction, fish growth is inhibited[73]
3.2 四环素类抗生素(Tetracycline antibiotics)
四环素类抗生素对鱼类肠道菌群的研究比较充分,其中在环境中残留浓度较高的土霉素对鱼类肠道菌群的研究最集中。另外有研究报道四环素和强力霉素在不同的暴露方式下对肠道菌群结构和功能的影响(表3)。斑马鱼暴露于环境水平的四环素(1 μg·L-1和100 μg·L-1)后,肠道菌群多样性增加,变形菌门、拟杆菌门以及厚壁菌门显著失调,拟杆菌门和厚壁菌门的比例增加,进而导致斑马鱼体质量增加[74]。四环素暴露金鱼21 d,鱼肠道耐药菌比例增加,梭杆菌门取代厚壁菌门成为绝对优势菌群,鲸杆菌属增加,伟荣球菌属减少,菌群紊乱会进一步导致能量代谢失调,菌群耐药性增强[75]。无论是暴露还是给药方式的土霉素均能显著改变肠道菌群组成[76-83],导致致病菌增加,免疫和代谢功能失调。强力霉素暴露导致斑马鱼肠道菌群多样性减少,梭杆菌门减少,具有潜在毒性的丛毛单胞菌属增加[84]。
表3 四环素类抗生素对鱼类肠道菌群结构和功能的影响
Table 3 Effects of tetracycline antibiotics on the structure and function of fish intestinal microbiota
抗生素种类Antibiotic type抗生素浓度及暴露时间Antibiotic concentration and exposure time物种Species肠道菌群结构变化Structural changes of intestinal microbiota肠道菌群相关功能变化Functional changes of intestinal microbiota参考文献Reference四环素Tetracycline1、100 μg·L-1; 30 d斑马鱼Zebrafish变形菌门Proteobacteria↑、拟杆菌门Bacteroidetes↓、厚壁菌门Firmicutes↓脂质代谢失调,体质量增加Dysregula-tion of lipid metabolism and weight gain[74]0.285、2.85 μg·L-1; 21 d金鱼Goldfish梭杆菌门Fusobacteria↑、厚壁菌门Firmicutes↓;鲸杆菌属Cetobacterium↑、伟荣球菌属Erysipellotriceae↓菌群耐药性增强,能量代谢失调Bacteri-al resistance increased, energy metabolism disorders[75]
续表3
抗生素种类Antibiotic type抗生素浓度及暴露时间Antibiotic concentration and exposure time物种Species肠道菌群结构变化Structural changes of intestinal microbiota肠道菌群相关功能变化Functional changes of intestinal microbiota参考文献Reference土霉素Oxytetracycline1、5 μg·L-1; 90 d斑马鱼Zebrafish黄杆菌属Flavobacterium↑病原菌定植,菌群耐药性增强,代谢紊乱Pathogen colonization, bacterial resist-ance increased, metabolic disorders[76]0.42 μg·L-1; 42 d斑马鱼Zebrafish梭杆菌门Fusobacteria↑、CKC4门↑、变形菌门Pro-teobacteria↓、浮霉菌门Planctomycetes↓免疫功能受损,引发肠道炎症反应Im-mune function is impaired, causing intesti-nal inflammation[77]10、10 000 μg·L-1; 60 d斑马鱼Zebrafishα-变形菌纲Alphaproteobacteria↑、放线菌纲Actinobacte-ria↑、γ-变形菌纲Gammaproteobacteria↓代谢紊乱Metabolic disorders[78]10 000 μg·L-1; 5 d斑马鱼Zebrafish假单胞菌属Pseudomonas↑、鲸杆菌属Cetobacterium↓、芽孢杆菌属Bacillus↓能量代谢失调Dysregulation of energy metabolism[79]80 mg·kg-1; 42 d斑马鱼ZebrafishCKC4门↑;肠球菌属Enterococcus↑、气单胞菌属Aero-monas↑免疫和能量代谢失调,引发肠道炎症Immune and energy metabolism are dis-rupted, and intestinal inflammation is trig-gered[80]100 mg·kg-1; 8 d尼罗罗非鱼Nile tilapia梭杆菌门Fusobacteria↑、变形菌门Proteobacteria↓、放线菌门Actinobacteria↓;邻单胞菌属Plesiomonas↑病原菌定植,菌群耐药性增强Pathogen colonization, bacterial resistance increased[81]80 mg·kg-1; 35 d尼罗罗非鱼Nile tilapia微杆菌属Microbacterium↑、Chlamydiae↑、邻单胞菌属Plesiomonas↑、Bosea属↑、不动杆菌属Acinetobacter↑、军团菌属Legionella↑、芽孢杆菌属Bacillus↑免疫系统受损,能量代谢失调,生长性能降低,存活率降低The immune system are impaired, energy metabolism is malad-justed, growth performance is reduced, and survival rate is reduced[82]100 mg·kg-1; 5 d虹鳟Rainbow trout软壁菌门Tenericutes↑、厚壁菌门Firmicutes↑、梭杆菌门Fusobacteria↓、变形菌门Proteobacteria↓代谢紊乱Metabolic disorders[83]强力霉素Doxycycline 10、30、100 μg·L-1; 21 d斑马鱼Zebrafish梭杆菌门Fusobacteria↓;丛毛单胞菌属Comamonas↑病原菌定植Colonization of pathogenic bacteria[84]
3.3 磺胺类抗生素(Sulfonamide antibiotics)
磺胺类抗生素的研究主要集中在磺胺甲恶唑和磺胺间甲氧嘧啶(表4)。长期暴露于环境浓度磺胺甲恶唑(1 μg·L-1和5 μg·L-1)导致致病性黄杆菌的积累,此外,一些代谢途径,包括叶酸生物合成、氧化磷酸化和生物素代谢途径在暴露样品中显著富集[76]。磺胺甲恶唑(5、90和450 μg·L-1)暴露成年斑马鱼3周,会导致其肠道中变形菌门、拟杆菌门丰度增加,厚壁菌门减少,一些机会致病菌包括军团菌属和Clostridium sensustricto 13出现[85]。以不同剂量磺胺甲恶唑给药均能显著影响鱼体肠道菌群组成[80, 86-87]。此外,这些研究结果显示的肠道菌群结构变化并不一致,这可能受给药剂量、给药时间以及实验生物多方面因素的影响。磺胺间甲氧嘧啶(200 mg·kg-1和300 mg·kg-1)喂养尼罗罗非鱼4周,其肠道菌群丰富度显著下降,放线菌减少,厚壁菌门(芽孢杆菌科)增加,并显著增强了肠道菌群的抗生素抗性[88]。
表4 磺胺类抗生素对鱼类肠道菌群结构和功能的影响
Table 4 Effects of sulfonamides antibiotics on the structure and function of fish intestinal microbiota
抗生素种类Antibiotic type抗生素浓度及暴露时间Antibiotic concentration and exposure time物种Species肠道菌群结构变化Structural changes of intestinal microbiota肠道菌群相关功能变化Functional changes of intestinal microbiota参考文献Reference磺胺甲恶唑Sulfamethoxazole1、5 μg·L-1;90 d斑马鱼Zebrafish黄杆菌属Flavobacterium↑病原菌定植,菌群耐药性增强,代谢紊乱Pathogen colonization, bacterial resist-ance increased, metabolic disorders[76]100 mg·kg-1; 42 d斑马鱼ZebrafishCKC4门↓;芽孢杆菌科Bacillaceae↑、梭菌科Clostridi-aceae↑抗氧化系统受损,能量代谢失调,鱼体质量增加The antioxidant system is dam-aged, the energy metabolism is maladjust-ed, and the fish gain weight[80]5、90、450 μg·L-1; 21 d斑马鱼Zebrafish变形菌门Proteobacteria↑、拟杆菌门Bacteroidetes↑、厚壁菌门Firmicutes↓;军团菌属Legionella↑、Clostridium sensustricto 13↑、Hydrobacter↑、葡萄球菌属Staphylococ-cus↓、气单胞菌属Aeromonas↓脂质代谢失调,杯状细胞数量减少,肠上皮细胞脱落溶解Lipid metabolism is maladjusted, the number of goblet cells is reduced, and intestinal epithelial cells are exfoliated and dissolved[85]3 000 mg·kg-1; 56 d加州鲈Micropterus salmoides梭杆菌门Fusobacteria↑、螺旋体门Spirochaete↑、蓝藻菌门Cyanobacteria↓、栖热菌门Thermophilic↓;鲸杆菌属Cetobacterium↑、螺旋体属Spirochaeta↑、邻单胞菌属Plesiomonas↓、Mangrovibacter↓、希瓦氏菌属Shewanella↓、肠球菌属Enterococcus↓、双歧杆菌属Bifidobacterium↓诱导氧化应激、炎症和细胞凋亡,器官指数和体质量增加Induce oxidative stress, inflammation and apoptosis, organ index and body weight gain[86]20、200、1 000 mg·kg-1; 28 d尼罗罗非鱼Nile tilapia梭杆菌门Fusobacteria↑、变形菌门Proteobacteria↓脂质代谢失调,影响生长性能Lipid me-tabolism is maladjusted and growth per-formance is affected[87]磺胺间甲氧嘧啶Sulfamonomethoxine200、300 mg·kg-1; 28 d尼罗罗非鱼Nile tilapia厚壁菌门Firmicutes↑、放线菌门Actinobacteria↓破坏肠道菌群稳定性,菌群耐药性增强Damage the stability of intestinal microbi-ota, bacterial resistance increased[88]
3.4 其他种类抗生素(Other classes of antibiotics)
其他种类抗生素对鱼体肠道菌群结构和功能的影响也有研究(表5),其中氟苯尼考[84, 89-91]研究较多。此外也有不少研究评价了抗生素混合物对鱼体肠道菌群的影响[92-93]。
表5 其他抗生素对鱼类肠道菌群结构的功能的影响
Table 5 Effects of other antibiotics on the structure and function of fish intestinal microbiota
抗生素种类Antibiotic type抗生素浓度及暴露时间Antibiotic concentration and exposure time物种Species肠道菌群结构变化Structural changes of intestinal microbiota肠道菌群相关功能变化Functional changes of intestinal microbiota参考文献Reference氟苯尼考Florfenicol10、30、100 μg·L-1; 21 d斑马鱼Zebrafish变形菌门Proteobacteria↑、梭杆菌门Fusobacteria↓;布劳特氏菌属Blautia↑、埃希氏杆菌属Escherichia-Shigella↑、多尔氏菌属Dorea↑、叶瘤杆菌属Phyllobacterium↑病原菌定植,能量代谢失调Pathogen colonization, energy metabolism disorders[84]10 mg·kg-1;10 d布氏鲳鲹Trachinotus blochii变形菌门Proteobacteria↑、广古菌门Euryarchaeota↓、厚壁菌门Firmicutes↓;弧菌属Vibrio↑、肠弧菌属En-terovibrio↑、发光菌属Photobacterium↑、假单胞菌属Pseudomonas↑、希瓦氏菌属Shewanella↑、沙雷氏菌属Serratia↓、甲烷杆菌属Methanobacterium↓、不动杆菌属Acinetobacter↓、芽孢杆菌属Bacillus↓病原菌定植,菌群耐药性增强Pathogen colonization, bacterial resistance increased[89]2 000 mg·kg-1;12 d大西洋鲑鱼Atlantic salmon变形菌门Proteobacteria↑、放线菌门Actinobacteria↑、拟杆菌门Bacteroidetes↑、软壁菌门Tenericutes↓肠道菌群稳定性遭到破坏The stability of intestinal microbiota is destroyed[90]500 mg·kg-1; 3、6、10 d斑点叉尾鮰Channel catfish邻单胞菌属Plesiomonas↑、志贺氏杆菌属Shigella↑、气单胞菌属Aeromonas↑病原菌定植Colonization of pathogenic bacteria[91]红霉素和氨苄西林Erythromycin and Ampicillin3 900μg·L-1红霉素Eryth-romycin;3 125 μg·L-1氨苄西林Ampicillin;30 d青鳉Oryzias latipes变形菌门Proteobacteria↑、梭杆菌门Fusobacteria↓;维氏气单胞菌Aeromonasveronii↑、副溶血弧菌Vibrio parah-aemolyticus↑免疫失调,诱发炎症Immune dysfunc-tion, inducing inflammation[92]甲硝唑、硫酸新霉素和万古霉素Metronidazole, neo-mycin sulfate and vancomycin4 000 mg·kg-1甲硝唑Met-ronidazole;4 000 mg·kg-1硫酸新霉素Neomycin sulfate; 2 000 mg·kg-1万古霉素Vancomy-cin;6 d杂交石斑鱼Hybrid grouper拟杆菌门Bacteroidetes↑、厚壁菌门Firmicutes↓;不动杆菌属Acinetobacter↓、芽孢杆菌属Bacillus↓、梭菌属Clostridium↓、雷尔氏菌属Ralstonia↓脂质代谢、能量代谢失调Dysregulation of lipid metabolism and energy metabo-lism[93]
抗生素主要用于杀灭病原菌,有时也用做生长促进剂,但大量的研究证实抗生素在使用时会严重破坏肠道共生菌,这些菌在肠道定植抗力(抵抗病原体的能力)、免疫调节以及代谢等方面发挥重要作用,所以会进一步导致病原菌定植,肠道菌群多样性和代谢活性改变,从而影响宿主生理功能。抗生素通过影响肠道菌群的结构和功能,会进一步导致鱼体内抗氧化系统和免疫系统失调,诱发炎症反应,代谢紊乱,生长性能受到影响等。综上所述,抗生素能通过影响肠道菌群的结构和功能影响鱼体的健康。
4 抗生素对鱼类抗生素抗性组的影响(Effect of antibiotics on antibiotic resistome of fish)
抗生素抗性组,即抗生素抗性基因组,是一组编码具有抵抗抗生素作用蛋白质的微生物基因集。抗生素在养殖水环境中的长期残留导致水产养殖环境中抗性菌的出现,鱼类病原体的抗性能力增加。据报道,从世界各地的鱼类,如斑马鱼、金鱼、鲤鱼、沙丁鱼和银牙鱼等都分离得到了抗性病原菌,有许多表现出多重耐药性[94-98]。肠道菌群一直被视为ARGs的关键储库,在一些选择压力下如抗生素暴露,会导致ARGs的急剧富集,进而导致肠道中抗生素抗性菌的富集,这反过来会导致治疗无效。此外肠道菌群成员间还可以进行ARGs的交换,从而传播抗生素抗性组[99]。
4.1 抗生素诱导抗性基因富集(Antibiotic induced enrichment of resistance genes)
恩诺沙星暴露斑马鱼10 d,通过qPCR技术检测到肠道中7种氟喹诺酮类抗性基因(qnrA、catA1、cmeA、cmx(A)、mexF、mexE和cmlA1)显著富集[70]。四环素暴露金鱼3周,检测到肠道中编码四环素外排基因(tetA、tetG、tetL和tetZ)以及磺胺类抗性基因(sul1)丰度显著增加[75]。土霉素和磺胺甲恶唑暴露斑马鱼4个月后,通过宏基因组技术检测到肠道中四环素类抗性基因(tetX6)以及磺胺类抗性基因(sul2)显著富集[76]。土霉素喂养尼罗罗非鱼8 d,导致肠道中抗性基因tetA、tetM显著富集,并与条件致病菌邻单胞菌呈正相关[81]。氟苯尼考和土霉素的大量使用导致大西洋鲑鱼肠道中氟苯尼考和土霉素抗性细菌显著富集,另外还检测到对8种抗生素显示出抗性的“超级耐药”细菌,大多数抗性菌株中检测到氯霉素类抗性基因(floR、fexA)和四环素类抗性基因(tetA、tetB、tetE、tetH、tetL、tet34和tet35)[100]。口服氟苯尼考的细鳞肥脂鲤肠道菌群中ARGs总相对丰度显著增加,属于多药类、氯霉素类、氟喹诺酮类、氨基香豆素类、四环素类和肽类的抗性基因显著富集[101]。这些研究均证实抗生素能够导致鱼体肠道中ARGs显著富集。其中,所有的研究都表明使用一种特定抗生素能够使鱼类肠道菌群产生对同类抗生素的抗性,包括恩诺沙星暴露增加鱼类肠道中氟喹诺酮类抗性基因的相对丰度,四环素和土霉素的使用显著富集四环素类抗性基因,使用氟苯尼考能够显著富集氯霉素类抗性基因。但抗生素使用和抗性基因产生之间的关系并非如此简单,一种特定抗生素不仅可以产生对同类抗生素的抗性,还可以产生对其他类抗生素的抗性,即抗生素具有潜在“协同选择”作用[102]。这主要与交叉耐药和共耐药有关。当一个抗性基因可以对多种抗生素产生抗性时,就会发生交叉耐药[103]。共耐药是指多种抗性基因可能存在于同一个可移动遗传元件(MGE)上,如阿莫西林和甲氧苄啶抗性基因[104]。因此,使用一种抗生素治疗可能对另一种抗生素产生抗性。部分关于抗生素对鱼类肠道抗性基因富集的研究也体现了抗生素对ARGs的“协同选择”作用,包括四环素和土霉素的使用能够富集磺胺类抗性基因,氟苯尼考和土霉素的大量使用导致鱼肠道中出现对8种抗生素显示出抗性的“超级耐药”细菌以及口服氟苯尼考使多药类、氟喹诺酮类和四环素类抗性基因显著富集。
4.2 抗生素诱导抗性基因转移(Antibiotic induced resistance gene transfer)
因自发基因突变产生ARGs并不会造成抗性基因的广泛传播,水平基因转移(horizontal gene transfer, HGT)是导致ARGs传播和扩散的主要因素,环境选择压力和由MGE(整合子、质粒和转座子等)介导的HGT是细菌获得外源抗性基因的主要原因[105]。HGT主要有接合、转导和自然转化3种类型[106]。接合转移最为常见,ARGs通过接合转移可在不同种属的微生物间传播[107]。HGT使更多的细菌可以获得ARGs,甚至打破了特定微生物生存环境中环境(非致病性)细菌和病原体之间的物种屏障[108]。
研究表明,抗生素是促进抗性基因水平转移的主要驱动因素之一。氟苯尼考和土霉素的大量使用导致大西洋鲑鱼肠道中多重耐药菌的富集,几乎100%耐药菌株检测到典型的MGE-1类整合子,也有研究在口服氟苯尼考的细鳞肥脂鲤肠道菌群中检测到MGE的总相对丰度显著增加,这都表明抗生素增加了MGE介导的鱼肠道ARGs水平转移的可能性,导致ARGs在水产养殖系统中的富集和传播[97-98]。此外,还有研究在大西洋鲑鱼养殖场中发现,在大量使用抗生素后鱼类肠道菌群中携带ARGs的1类整合子显著富集[109]。
目前抗生素对鱼类肠道中ARGs转移的影响研究主要集中在对MGE的定量分析,具体的转移过程以及转移机制并不明确。因此未来抗生素选择压力下的ARGs转移相关研究应与接合转移实验模型及相关技术相结合明确转移过程和转移机制。另外噬菌体已被证明更适合作为ARGs转移的载体,这也表明转导在HGT过程中发挥重要作用[110]。目前只有少数研究对抗生素与噬菌体ARGs转移之间的关系进行了探讨。研究表明抗生素治疗增强了人类和小鼠肠道中携带ARGs的噬菌体数量[111-112]。然而,关于ARGs在鱼类噬菌体基因组中的分布特征以及噬菌体介导的HGT在鱼体内的作用机制,仍存在许多不确定性,鲜有相关研究报道。
4.3 鱼类抗生素抗性问题的健康风险研究(Health risk of antibiotic resistance in fish)
由于过度使用或滥用抗生素,会导致越来越多的抗性菌和ARGs出现。肠道微生物群作为一个与环境“半隔离”但与应用的抗生素直接接触的系统,是来自不同环境(包括人类临床环境)的细菌之间发生相互作用的理想场所[87]。现已在鱼类病原菌(沙门氏气单胞菌、迟缓爱德华氏菌、鳗鲡弧菌、嗜水气单胞菌、鱼型链球菌和鱼巴氏杆菌等)中检测到ARGs的普遍存在[113-114],其中一些病原体,例如爱德华氏菌、气单胞菌和链球菌,可以感染人类并产生人畜共患感染[115-116]。ARGs的转移不受物种屏障的限制,人类可能通过受感染的鱼或鱼类生存环境直接接触到抗生素抗性菌和ARGs。另外,鱼类作为人类的食物来源之一,其体内残留的抗生素或抗生素抗性菌可能因其被人类摄入而经由食物链传播最终到人类。鱼类细菌和人类病原体之间的ARGs可能存在双向流动,导致高风险ARGs在人群中广泛传播,会导致人类健康风险。在鱼和人类致病性气单胞菌以及住院患者分离的大肠杆菌中出现了类似的IncU不相容组质粒,其中含有抗性基因(Tn1721 TetA)决定簇和1类整合子[117-118]。鱼体中水生希瓦氏菌、发光杆菌、气单胞菌和弧菌与大量人类革兰氏阴性病原菌(如大肠杆菌和克雷伯菌)共享喹诺酮类抗性基因qnrA、qnrS和qnrVC[119]。鱼类病原体(如鲁克氏菌、气单胞菌和爱德华氏菌)和人类病原体(例如鼠疫菌、沙门氏菌和霍乱弧菌)共同具有IncA/C多药耐药质粒[120-122]。这些研究均表明鱼类细菌和人类病原体之间的ARGs可能存双向传递过程。抗性基因转移最终将导致抗生素抗性(药物无法抑制细菌生长,失去用于治疗细菌感染能力的情况)问题,已成为全球范围的一个重要的公共卫生问题。
5 结论与展望(Conclusion and prospect)
正常情况下,肠道菌群处于动态平衡状态,益生菌占主导地位,与中性菌、致病菌相互制约和相互依存,保持肠道正常运转。目前大量研究证明环境水平的抗生素会严重破坏鱼体肠道生态平衡,并伴随着鱼体代谢和免疫能力的改变,但未能阐明其中的因果关系,缺乏系统的机制研究。同种抗生素导致的菌群变化结果并不一致,因此未来需要进一步建立系统的实验方案,从更深层次的分子机制探索抗生素与鱼类肠道菌群以及生理功能之间的关系,使相关研究更加标准化,增加结果的可信度和说服力。目前抗生素导致鱼类肠道ARGs富集和转移的相关研究也主要集中于对ARGs和MGE种类和相对丰度的基础调查,缺乏对ARGs富集和转移机制的进一步探索。此外,自然水环境中含有多种抗病毒药、重金属、微塑料、农药和人工甜味剂等污染物,这些污染物已被证实对肠道菌群结构和ARGs转移产生影响。当前研究多集中于单一污染物对肠道菌群以及ARGs的影响,鲜有从抗生素与其他污染物共污染角度研究肠道菌群结构和功能变化以及ARGs的传播和转移问题,尤其是污染物共选择压力的机制尚不明确。未来需要重视抗生素和其他污染物共污染对生物肠道菌群和ARGs的影响以及ARGs在水产养殖环境中传播带来的生态和健康风险,为全面评价水产养殖环境中的抗生素等污染物影响ARGs转移和扩散带来的生态健康风险提供科学依据,为医疗、农业和水产养殖业中抗生素的合理使用提供科学指导,有利于实现“绿色、健康、安全”的水产养殖业发展模式。
通信作者简介：崔鹏飞(1988—),男,博士,副教授,主要研究方向为生态毒理学。
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