Abstract
目的
分析伴侣动物中抗生素抗性基因(antibiotic resistance genes, ARGs)的分布特征,为防控ARGs在伴侣动物与人之间的传播积累科学数据。
方法
于2023年12月–2024年10月期间收集成都市某大型宠物医院中伴侣动物的粪便或肛拭子样本,采用PCR及qPCR检测9大类20种ARGs,获得ARGs检出数量、检出率与相对丰度数据,分析其在不同类型、性别、年龄、健康状况、可移动遗传元件intΙ1携带情况的伴侣动物中的分布特征。
结果
共收集获得136份样本,ARGs检出数量的中位数(四分位间距)为6.5(5,9),四环素类和氨基糖苷类抗性基因的检出率较高,其中检出率最高的是tetQ和tetW(均为90.44%),相对丰度>10-2,其次是tetM(67.65%)和aph3(58.82%),相对丰度>10-3。雌性动物的sul2、strB和cmlA相对丰度高于雄性;0~2月龄动物的sul1检出率高于12月及以上龄动物(P=0.007),3~5月龄动物的大环内酯类抗性基因和ermB检出率高于12月及以上龄动物(P<0.001);携带intΙ1动物的ARGs检出数量、多种ARGs检出率及相对丰度高于未携带intΙ1的动物,差异均具有统计学意义。
结论
伴侣动物普遍携带ARGs,四环素类和氨基糖苷类抗性基因检出率高,年龄及携带intΙ1是影响伴侣动物中ARGs分布的重要因素。
Keywords: 伴侣动物, 抗生素抗性基因, 分布特征, 可移动遗传元件
Abstract
Objective
To explore the distribution characteristics of antimicrobial resistance genes (ARGs) in companion animals and accumulate scientific data for preventing the transmission of ARGs between companion animals and humans.
Methods
From December 2023 to October 2024, fecal or rectal swab samples were collected from companion animals at a large pet hospital in Chengdu. PCR and qPCR were used to detect 9 ARGs types (including 20 ARGs). The distribution characteristics of ARGs in companion animals with different species, gender, age, clinical status and the mobile genetic element intΙ1 carrying status were analyzed based on the data of ARGs quantities, detection rates and relative abundance.
Results
A total of 136 samples were collected. The median (interquartile range) of ARGs detected per animal was 6.5 (5, 9). Tetracycline and aminoglycoside resistance genes had higher detection rates, with tetQ and tetW having the highest detection rates (both at 90.44%), and relative abundance > 10-2. This was followed by tetM (67.65%) and aph3 (58.82%), with relative abundance > 10-3. Female animals had higher relative abundance of sul2, strB, and cmlA than male animals. The detection rate of sul1 in animals aged 0-2 months was higher than that in animals aged 12 months and above (P = 0.007). The detection rates of macrolide resistance genes and ermB in animals aged 3-5 months were higher than those in animals aged 12 months and above (P < 0.001). Animals carrying intΙ1 had higher ARGs detection quantities, detection rates and relative abundance of multiple ARGs than those not carrying intΙ1, with all differences being statistically significant.
Conclusion
Companion animals commonly carry ARGs, with tetracycline and aminoglycoside resistance genes having high detection rates. Age and the carriage of intΙ1 are important factors influencing the distribution of ARGs in companion animals.
Keywords: Companion animals, Antimicrobial resistance genes, Distribution characteristics, Mobile genetic element
抗生素耐药性(antimicrobial resistance, AMR)已成为全球公共卫生面临的重大挑战之一。抗生素抗性基因(antibiotic resistance genes, ARGs)是细菌具有AMR的基础,可通过细菌的垂直基因传递(vertical gene transfer, VGT)或水平基因转移(horizontal gene transfer, HGT)在“环境-动物-人群”间传播[1],对人类健康与公共卫生安全构成潜在威胁。伴侣动物是人类居住环境中分布最广泛、与人类互动最频繁的动物群体,其携带的ARGs存在影响人类健康风险。
研究发现,随着抗生素在动物疾病治疗中广泛应用,伴侣动物源细菌的耐药形势已十分严峻,其对头孢噻呋、氨苄西林、庆大霉素、恩诺沙星等常见抗生素普遍呈现耐药性,且菌株中存在sul1、aph3、qnrS等多种抗生素抗性基因[2-3],此外,伴侣动物在环境中的广泛活动、动物之间的频繁互动,也是其体内ARGs的重要来源。SCHMITT等[4]发现了携带blaCTX-M的大肠杆菌和肺炎克雷伯菌在伴侣动物、环境、人之间的传播链,ZHAO等[5]和NAZIRI等[6]也发现宠物狗与其主人粪便中的ARGs呈现显著的相似性,提示伴侣动物中的ARGs可传播至人,威胁人群健康,影响公共卫生安全。尽管ARGs在伴侣动物与人之间的传播风险已有研究,但有关伴侣动物中ARGs的分布特征及影响因素的研究鲜有报道。
因此,本研究收集成都市某大型宠物医院中伴侣动物的样本,对9大类、20种ARGs进行检测,探讨其在伴侣动物中的流行状况、分布特征及影响因素,为进一步防控ARGs在伴侣动物与人之间的传播提供科学依据。
1. 材料与方法
1.1. 材料
1.1.1. 伴侣动物样本
2023年12月–2024年10月期间,在成都市某大型宠物医院收集体检及就诊伴侣动物的粪便或肛拭子样本,共计136份。被采样的伴侣动物均来自养宠家庭,包括猫和犬两种类型,涵盖不同性别、年龄与健康状况。
1.1.2. 主要试剂及设备
QIAamp PowerFecal Pro DNA Kit购于德国Qiagen公司;PCR试剂盒和DNA Marker Ⅰ购于天根生物科技有限公司;高纯度低电渗琼脂糖、TBE速溶颗粒和GelRed核酸凝胶染料购于擎科生物科技有限公司;qPCR试剂盒购于艾科瑞生物科技有限公司;引物由生工生物工程股份有限公司合成;Nanodrop 2000分光光度计、PCR仪、iBright 1500凝胶成像仪,美国Thermo公司;qPCR仪,美国BIO-RAD公司。
1.2. 方法
1.2.1. 样本DNA提取
按照制造商的说明,使用QIAamp PowerFecal Pro DNA Kit提取样本的DNA,使用Nanodrop 2000分光光度计测定浓度和纯度,符合检测要求的DNA储存于-20 ℃保存备用。
1.2.2. 抗生素抗性基因的检测
对所有样本采用PCR定性检测9大类20种典型ARGs,类型包括四环素类(tetracycline, TE)、大环内酯类(macrolide, MAC)、磺胺类(sulphonamide, SUL)、喹诺酮类(quinolone, QL)、β-内酰胺类(β-lactam, β-LA)、氨基糖苷类(aminoglycoside, AG)、氯霉素类(chloramphenicol, CHL)、多黏菌素类(polymyxin, PM)以及可移动遗传元件(mobile genetic element,MGE)。PCR反应体系为:0.25 μL Golden DNA Polymerase, 1 μL Primer-F, 1 μL Primer-R, 12 μL 2×Reaction Mix,8.75 μL ddH2O,2 μL DNA模板。反应程序为:94 ℃ 5 min; 94 ℃ 30 s, 退火 30 s, 72 ℃ 1 min, 30个循环; 72 ℃ 5 min, 4 ℃保存。PCR产物用1.5%~2%琼脂糖凝胶电泳检测,出现明显条带的PCR产物送至上海生工公司进行测序,并利用NCBI和CARD数据库(https://card.mcmaster.ca/)进行分析确认。
对符合质量要求且信息完整的33份样本采用qPCR(SYBR Green染料法)定量检测9大类20种ARGs,反应体系和程序参考文献[7],基因的相对丰度=
,ΔCT=(CT
ARGs-CT
16S rRNA gene)。PCR和qPCR的引物均参考文献[8-9],信息见网络资源附件附表1。
1.2.3. 统计学方法
使用SPSS 25.0进行统计分析。正态分布的计量资料采用t检验或方差分析;非正态分布的计量资料采用非参数检验;率的比较采用卡方检验;在对ARGs相对丰度的统计分析中,对相对丰度进行对数转换,使数据服从正态分布以便进一步分析[10];相关性分析采用Spearman相关性分析。P<0.05表示差异有统计学意义,多组的两两比较时采用了bonferroni法控制检验水准。
2. 结果
2.1. 伴侣动物ARGs检测情况
2.1.1. ARGs检出数量与检出率
ARGs的检出数量分布如图1A所示,检出数量的中位数(四分位间距)为6.5(5,9),检出6种ARGs的伴侣动物最多,占17.65%(24/136),其次是检出5种ARGs的伴侣动物,占16.18%(22/136)。每只动物均被检出至少1种ARGs,检出数量最多的动物携带了14种ARGs,占1.47%(2/136)。
图 1.
The number and detection rate of ARGs in companion animal samples
伴侣动物样本中ARGs检出数量与检出率
A, Quantities of ARGs. B, Detection rates of 9 ARGs types. C, Detection rates of 20 ARGs.
9大类20种ARGs的检出率如图1B、1C所示,检出率最高的ARGs类型的是四环素类抗性基因(99.26%,135/136),其次是氨基糖苷类(72.06%,98/136)、磺胺类(69.12%,94/136)和大环内酯类抗性基因(51.47%,70/136)。在20种ARGs中,除cfr未检出外,其余均有检出。检出率最高的是tetQ和tetW(均为90.44%,123/136),其次是tetM(67.65%,92/136)和aph3(58.82%,80/136),并检出了β-内酰胺类抗性基因blaNDM(2.21%,3/136)和多黏菌素类抗性基因mcr-1(4.41%,6/136)。
2.1.2. ARGs相对丰度
开展ARGs定量检测的33份样本中,tetQ的tetW的相对丰度较高,中位数达10-2以上,其次是tetM、ermB、blaCTX-M和aph3,中位数达10-3以上;而oqxA和cfr的相对丰度较低,中位数低于10-5。详见图2。
图 2.
Relative abundance of ARGs
ARGs相对丰度
2.2. 不同生物学特征的伴侣动物中ARGs的分布
2.2.1. 不同类型和不同性别伴侣动物中ARGs的分布
猫和犬的ARGs检出数量、检出率和相对丰度的差异均无统计学意义。雌性动物有3种ARGs相对丰度高于雄性,分别为sul2(t=2.71,P=0.011)、strB(t=2.247,P=0.032)和cmlA(t=2.551,P=0.016)。
2.2.2. 不同年龄伴侣动物中ARGs的分布
将伴侣动物根据年龄分为了0~2月、3~5月、6~11月和12月进行分组分析。发现4个年龄组的ARGs检出数量不全相等(F=3.79,P=0.012),3~5月组的检出数量高于12月及以上组(P=0.002)。在ARGs检出率方面,0~2月组的sul1检出率高于12月及以上组(χ2=7.226,P=0.007),3~5月组的大环内酯类抗性基因和ermB检出率均高于12月及以上组(χ2=14.366,P<0.001)。详见表1。
表 1. Differences in ARGs detection rate among companion animals with different biological characteristics and clinical status.
不同生物学特征和健康状态的伴侣动物中ARGs检出率的差异
| Characteristic | Group | Macrolides resistance genes | Chloramphenicol resistance genes | ermB | sul1 | |||||||||||
| Case (%) | χ 2 | P | Case (%) | χ 2 | P | Case (%) | χ 2 | P | Case (%) | χ 2 | P | |||||
| * P < 0.05, ** P < 0.01. The infectious diseases group includes animals with bacterial, viral, and parasitic infections, and the gastrointestinal diseases group includes animals with gastroenteritis, diarrhea, and vomiting. | ||||||||||||||||
| Species | Cat (n = 85) | 44 (51.76) | 0.008 | 0.929 | 30 (35.29) | 0.211 | 0.646 | 44 (51.76) | 0.096 | 0.757 | 31 (36.47) | 0.596 | 0.440 | |||
| Dog (n = 51) | 26 (50.98) | 20 (39.22) | 25 (49.02) | 22 (43.14) | ||||||||||||
| Gender | Female (n = 67) | 31 (46.27) | 1.431 | 0.232 | 26 (38.81) | 0.237 | 0.627 | 31 (46.27) | 1.054 | 0.305 | 25 (37.31) | 0.152 | 0.696 | |||
| Male (n = 69) | 39 (56.52) | 24 (34.78) | 38 (55.07) | 28 (40.58) | ||||||||||||
| Age/month | 0-2 (n = 37) | 18 (48.65) | 14.264 | 0.003** | 18 (48.65) | 4.920 | 0.178 | 18 (48.65) | 14.940 | 0.002** | 21 (56.76) | 8.541 | 0.036* | |||
| 3-5 (n = 20) | 18 (90.00) | 9 (45.00) | 18 (90.00) | 9 (45.00) | ||||||||||||
| 6-11 (n = 12) | 5 (41.67) | 4 (33.33) | 5 (41.67) | 3 (25.00) | ||||||||||||
| ≥12 (n = 67) | 29 (43.28) | 19 (28.36) | 28 (41.79) | 20 (29.85) | ||||||||||||
| Clinical status | Healthy (n = 42) | 22 (52.38) | 1.335 | 0.721 | 13 (30.95) | 9.015 | 0.029* | 22 (52.38) | 1.879 | 0.598 | 19 (45.24) | 4.308 | 0.230 | |||
| Infectious diseases (n = 34) |
20 (58.82) | 17 (50.00) | 20 (58.82) | 16 (47.06) | ||||||||||||
| Gastrointestinal diseases (n = 35) |
16 (45.71) | 16 (45.71) | 15 (42.86) | 12 (34.26) | ||||||||||||
| Other diseases (n = 25) | 12 (48.00) | 4 (16.00) | 12 (48.00) | 6 (24.00) | ||||||||||||
将进行了定量检测的伴侣动物根据年龄分为0~2月(n=11)和6月及以上(n=22)两组进行相对丰度的比较,发现0~2月组的tetM的相对丰度高于6月及以上组(t=3.035,P=0.005)。
2.2.3. 不同健康状况伴侣动物中ARGs的分布
将伴侣动物根据不同的健康状况分为了健康、患感染性疾病、患胃肠道疾病和患其他疾病四组进行分析。四个不同健康状况组的ARGs检出数量不全相等(H=8.84,P=0.031),患感染性疾病组的检出数量高于患其他疾病组(P=0.02)。四个不同健康状况组ARGs检出率的差异如表1所示,患感染性疾病组的氯霉素类抗性基因检出率高于患其他疾病组(χ2=7.265,P=0.007)。
2.3. 不同intΙ1携带情况的伴侣动物中ARGs的分布
携带intΙ1组的ARGs检出数量高于未携带intΙ1组(U=1254,P=0.002)。两组ARGs检出率的差异如表2所示,携带intΙ1组的四大类以及六种ARGs的检出率均高于未携带intΙ1组。在ARGs相对丰度方面,携带intΙ1组的两种ARGs的相对丰度高于未携带intΙ1组,分别为qnrS(t=-2.453,P=0.020)和strB(t=-3.113,P=0.004)。进一步对intΙ1的相对丰度与各ARGs的相对丰度进行了相关性分析,intΙ1的相对丰度与两种ARGs的相对丰度均呈正相关,分别为blaCTX-M(P<0.001,rs=0.579)和cmlA(P<0.001,rs=0.612)。
表 2. Differences in ARGs detection rate among companion animals with different intΙ1 carrying status.
不同intΙ1携带情况的伴侣动物中ARGs检出率的差异
| ARGs | intΙ1 carrying status/case (%) | χ 2 | P | |
| Carry intΙ1 (n = 39) | Not carry intΙ1 (n = 97) | |||
| sul1 | 21 (53.85) | 32 (33.00) | 5.087 | 0.024 |
| β-lactam resistance genes | 16 (41.03) | 16 (16.49) | 9.303 | 0.002 |
| bla CTX-M | 16 (41.03) | 14 (14.43) | 11.441 | 0.001 |
| Quinolone resistance genes | 22 (56.41) | 23 (23.71) | 13.433 | < 0.001 |
| qnrS | 20 (51.28) | 19 (19.56) | 13.662 | < 0.001 |
| Chloramphenicol resistance genes | 20 (51.28) | 30 (30.92) | 4.957 | 0.026 |
| cmlA | 18 (46.15) | 17 (17.53) | 11.928 | 0.001 |
| Aminoglycoside resistance genes | 33 (84.62) | 65 (67.01) | 4.282 | 0.039 |
| strB | 25 (64.10) | 41 (42.27) | 5.309 | 0.021 |
| ahp3 | 30 (76.92) | 50 (51.55) | 7.395 | 0.007 |
3. 讨论
本研究通过对伴侣动物中20种典型ARGs的检测,探讨了伴侣动物中ARGs的流行状况、分布特征及影响因素。
值得注意的是,本研究首次发现了幼年伴侣动物的ARGs检出数量、部分ARGs检出率及相对丰度更高。类似的研究结果也在农业养殖动物中被观察到,例如,CAREY等[11]的研究发现,部分ARGs的检出率在新生组、断乳组的奶牛中更高;GAIRE等[12]也发现猪粪便中的ARGs数量与丰度随着年龄增长而降低。推测可能因为动物在成年前,免疫系统尚未发育完全,且在环境中频繁活动,更易受到环境中携带ARGs的细菌的感染,提示在伴侣动物ARGs的防控中可能应该重点关注幼年动物。此外,本研究也观察到雌性伴侣动物的部分ARGs丰度高于雄性,与THAMES等[13]的结果类似,这种差异可能是由不同性别伴侣动物肠道菌群结构的差异所致[14]。本研究还发现患感染性疾病的伴侣动物的ARGs检出数量与部分ARGs检出率更高,这可能因为在其治疗过程中抗生素被频繁使用,导致产生了ARGs,提示在伴侣动物的临床治疗中需谨慎使用抗生素。另外,肠道微环境状态可能影响ARGs的表达与传播[15],因此本研究对患胃肠道疾病的伴侣动物进行了单独分组,但未发现ARGs分布上的差异,可能因为研究的区域相对集中以及定量检测的样本量较少所致。
在本研究中,四环素类抗性基因的检出率最高,尤其是tetQ和tetW,检出率高达90.44%。CHEKABAB等[16]的研究也观察到类似现象,推测这些ARGs可能不仅是抗生素诱导的结果,还可能因为其可长期存在于环境中并易发生水平基因转移[17],从而传播给了动物。本研究还发现氨基糖苷类和大环内酯类抗性基因也是伴侣动物中检出率和相对丰度较高的ARGs,与YANG等[18]的研究结果类似。此外,YANG等的研究进一步观察到宠物猫的氨基糖苷类抗性基因丰度显著高于人类,且此类抗性基因在养猫人士中的丰度显著高于非养猫人士,ROKEN等[19]对养宠物犬家庭的研究也发现了类似结果;而在人群中,四环素类、大环内酯类和喹诺酮类抗性基因的检出率达95%以上,氨基糖苷类抗性基因的检出率却低于30%[20-21],提示伴侣动物中的氨基糖苷类抗性基因较丰富,可能构成ARGs由伴侣动物向人类传播的风险,应重点关注。需要注意的是,本研究在伴侣动物中检测到了blaNDM和mcr-1,这两种基因可对β-内酰胺类和多黏菌素类抗生素(最后一道抗生素防线)产生耐药性,目前主要在禽畜中被检测到,而在人类中的检出率相对较低[22]。De MENDIETA等[23]和WANG等[24]也曾在伴侣动物源细菌中检测到blaNDM和mcr-1,并观察到其通过质粒的介导发生水平基因转移,提示伴侣动物携带对人类健康构成严重威胁且具有传播风险的ARGs,防控伴侣动物携带的这些ARGs向人类的传播至关重要。
intΙ1是细菌中最常见的MGE之一,能够捕获和整合ARGs,并在不同基因组之间移动,介导ARGs的水平基因转移,加剧耐药扩散[25]。LIU等[26]和YANG等[27]的研究分别发现了马驹粪便和猪粪便中的intΙ1与氨基糖苷类、β-内酰胺类、喹诺酮类等抗性基因的丰度呈正相关,本研究在伴侣动物中也发现了类似结果,携带intΙ1的伴侣动物其ARGs检出数量、部分ARGs检出率及相对丰度更高,且intΙ1的相对丰度与blaCTX-M和cmlA的相对丰度呈正相关,在一定程度上提示intΙ1是驱动伴侣动物中的ARGs流行与传播的潜在机制之一。
伴侣动物作为现代社会中人类的重要伙伴,长期与人类共同生活、密切接触,其携带的ARGs传播给人的风险不容忽视。本研究通过对伴侣动物中ARGs的检测与分布特征研究,发现伴侣动物普遍携带ARGs,尤其四环素类和氨基糖苷类抗性基因的检出率较高,年龄及携带可移动遗传元件intΙ1是影响伴侣动物中ARGs分布的重要因素,为明确未来需重点关注的ARGs类型与伴侣动物群体积累了科学数据,对促进动物-人协同耐药防控、守护公众健康具有积极意义。但本研究采样时间与区域相对有限,后续还需增加时间跨度、加大样本量,进一步探讨伴侣动物中ARGs的动态变化趋势以及更多因素(如地域、饮食、肠道菌群等)对伴侣动物中ARGs分布的影响,为抗生素耐药性防控提供更全面的数据支持。
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作者贡献声明
王瑞雪负责正式分析、调查研究和初稿写作,安龙懿负责调查研究、研究方法和可视化,裴晓方负责论文构思、研究项目管理和审读与编辑写作,陈嘉熠负责论文构思、经费获取和审读与编辑写作。所有作者已经同意将文章提交给本刊,且对将要发表的版本进行最终定稿,并同意对工作的所有方面负责。
利益冲突
所有作者均声明不存在利益冲突
Author Contribution
WANG Ruixue is responsible for formal analysis, investigation, and writing--original draft. AN Longyi is responsible for investigation, methodology, and visualization. PEI Xiaofang is responsible for conceptualization, project administration, and writing--review and editing. CHEN Jiayi is responsible for conceptualization, funding acquisition, writing--review and editing. All authors consented to the submission of the article to the Journal. All authors approved the final version to be published and agreed to take responsibility for all aspects of the work.
Declaration of Conflicting Interests
All authors declare no competing interests.
Funding Statement
国家自然科学基金(No. 82373646)和四川省科技厅四川省自然科学基金项目(No. 2023NSFSC1737)资助
Contributor Information
瑞雪 王 (Ruixue WANG), Email: wangruixue@stu.scu.edu.cn.
嘉熠 陈 (Jiayi CHEN), Email: cjy.210@163.com.
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