摘要
目的
探究广东省部分地区宠物源肺炎克雷伯菌的耐药和毒力情况。
方法
采集犬猫粪便拭子,通过分离培养、PCR扩增16S rRNA和khe基因进行菌株鉴定。采用琼脂扩散法测定肺炎克雷伯菌分离株对17种抗菌药物的敏感性;通过PCR检测β-内酰胺类(blaSHV、blaCTX、blaTEM)、碳青霉烯类(blaKPC、blaNDM)、氨基糖苷类(rmtB)、喹诺酮类(qnrS、oqxAB)、磺胺类(Sul1、Sul2)、酰胺醇类(floR)、四环素类[tet(A)]、磷霉素(fosA3)耐药基因以及部分毒力基因(rmpA、maga、fimH、mrkD、uge、WabG、kfu、Aerobactin、ureA)。
结果
从采集的428份粪便样品中分离得到126株肺炎克雷伯菌,分离率为29.4%。琼脂扩散法结果显示,126株分离菌对阿莫西林(75.40%)、氨苄西林(73.81%)、复方新诺明(61.90%)耐药率高,对头孢他啶、阿米卡星、安普霉素和恩诺沙星较为敏感,对替加环素、黏菌素、美罗培南敏感。耐药基因检测结果显示,磺胺类耐药基因oqxAB检出率最高,为86.51%;其次为β-内酰胺酶耐药基因blaSHV (73.81%)、四环素类耐药基因tet(A) (52.68%);其他耐药基因均有不同程度检出(0.79%-46.03%),碳青霉烯类耐药基因blaKPC、黏菌素耐药基因mcr-1和氨基糖苷类耐药基因rmtB未检出。毒力基因检测结果显示,尿素酶相关基因ureA检出率为100.00%,脂多糖相关基因uge检出率为95.54%,菌毛相关基因fimH的检出率为91.07%,其他毒力基因均有不同程度检出(2.70%-8.90%),荚膜相关基因magA与菌毛相关基因mrkD未检出。
结论
广东省部分地区宠物源肺炎克雷伯菌耐药状况严重,但致病性较弱,应加强对其耐药性与致病力的监测。此外,临床上应严格管理和合理使用抗生素,避免多重耐药肺炎克雷伯菌的产生与传播。
肺炎克雷伯菌(Klebsiella pneumoniae)是一种常见的革兰阴性菌和条件致病菌,属肠杆菌科克雷伯
宠物与主人及其家庭成员之间的密切接触,为多重耐药细菌在人和宠物之间的传播创造了有利条件,可能对人类健康造成一定威胁,对公众健康构成潜在风
1 材料与方法
1.1 样品来源
2022年和2023年分别从不同区域采集到393份和35份新鲜犬猫粪便拭子,共428份粪便样品,其中广州市部分宠物医院319份、广州市某花鸟市场76份及东莞市某宠物医院33份。将样品装入样品袋中并编号,置于4 ℃保存备用。质控菌株大肠杆菌ATCC 25922由华南农业大学广东省兽药研制与安全评价重点实验室保存。
1.2 主要试剂
BHI肉汤(brain heart infusion broth)、MH肉汤(mueller hinton broth)、LB琼脂和MH琼脂 (mueller hinton agar)均购自广东环凯微生物科技有限公司;麦康凯肌醇阿东醇羧苄青霉素(MacConkey inositol adonitol carbenicillin agar, MIAC)培养基购自青岛海博生物技术有限公司;DL2000 bp DNA marker、2×Taq PCR Master Mix均购自广东东盛生物科技有限公司;革兰染色试剂盒购自北京索莱宝科技有限公司。
1.3 抗菌药物
氨苄西林(纯度98%)、头孢他啶(纯度98%)、头孢噻呋(纯度98%)、多西环素(纯度97%)、替加环素(纯度98%)、氟苯尼考(纯度95%)、阿米卡星(纯度97%)、恩诺沙星(纯度98.5%)均购自生工生物工程(上海)股份有限公司;黏菌素(6 500 IU/mg)、庆大霉素(590 IU/mg)、安普霉素(纯度99%)、环丙沙星(纯度99%)、阿莫西林(纯度99%)、头孢噻肟(纯度98%)、新霉素(纯度92%)、美罗培南(纯度98%)均购自大连美仑生物技术有限公司;磺胺甲噁唑(纯度99%)、甲氧苄啶(纯度98%)均购自广州翔博生物科技有限公司,复方新诺明由磺胺甲噁唑和甲氧苄啶按比例混合而成。
1.4 细菌的分离培养与革兰染色镜检
将粪便样品加入无菌BHI肉汤2 mL,置于37 ℃、180 r/min培养过夜后,划线接种于MIAC培养基,37 ℃培养箱培养12 h。进一步挑取大小一致、粉红色中心发白的单个疑似菌落划线于MIAC进行纯化,37 ℃培养箱培养12 h;挑取单菌落并划线和涂布于LB琼脂,37 ℃培养过夜。取单菌落按照革兰染色试剂盒使用步骤进行革兰染色与镜检观察。
1.5 16S rRNA基因和特异性基因的检测
采用水煮法提取分离菌株的DNA,以其为模板,利用PCR扩增16S rRNA基因和khe基因。16S rRNA基因使用细菌通用引
基因 Genes | 引物序列 Primer sequences (5′→3′) | 片段大小 Target fragment (bp) | 退火温度 Annealing temperature (℃) | 参考文献References |
---|---|---|---|---|
16S rRNA | F: AGAGTTTGATCCTGGCTCAG | 1 500 | 55 |
[ |
R: ACGGCTACCTTGTTACGACTT | ||||
khe | F: TGATTGCATTCGCCACTGG | 428 | 68 |
[ |
R: GGTCAACCCAACGATCCTG | ||||
mcr-1 | F: TCGCGGCATTCGTTATA | 535 | 52 |
[ |
R: GGTGGCGTTCAGCAGTC | ||||
blaCTX | F: TTAGGAARTGTGCCGCTGYA | 688 | 56 |
[ |
R: CGATATCGTTGGTGGTRCCAT | ||||
blaTEM | F: ATAAAATTCTTGAAGACGAAA | 1 076 | 55 |
[ |
R: GACAGTTACCAATGCTTAATC | ||||
blaSHV | F: TTATCTCCCTGTTAGCCACC | 795 | 55 |
[ |
R: GATTTGCTGATTTCGCTCGG | ||||
blaKPC | F: CGTCTAGTTCTGCTGTCTTG | 566 | 55 |
[ |
R: CTTGTCATCCTTGTTAGGCG | ||||
blaNDM | F: GGTTTGGCGATCTGGTTTTC | 621 | 53 |
[ |
R: CGGAATGGCTCATCACGATC | ||||
Sul1 | F: TCGGACAGGGCGTCTAAG | 925 | 56 |
[ |
R: GGGTATCGGAGCGTTTGCA | ||||
Sul2 | F: CGGCATCGTCAACATAACCT | 721 | 57 |
[ |
R: TGTGCGGATGAAGTCAGCTC | ||||
fosA3 | F: GCGTCAAGCCTGGCATTT | 282 | 56 |
[ |
R: GCCGTCAGGGTCGAGAAA | ||||
floR | F: CTGAGGGTGTCGTCATCTAC | 673 | 57 |
[ |
R: GCTCCGACAATGCTGACTAT | ||||
rmtB | F: ACATCAACGATGCCCTCAC | 725 | 52 |
[ |
R: AAGTTCTGTTCCGATGGTC | ||||
oqxAB | F: GTCCAGCGATAATCAGGC | 669 | 54 |
[ |
R: GGTCTCGGCAATCACTTT | ||||
qnrS | F: ACGACATTCGTCAACTGCAA | 417 | 54 |
[ |
R: TAAATTGGCACCCTGTAGGC | ||||
tet(A) | F: CCAATCCATCGACAATCACC | 583 | 53 |
[ |
R: CAGCCGAATACAGTGATCC |
1.6 药物敏感性试验
采用琼脂扩散法对分离菌株进行17种抗菌药物(美罗培南、阿莫西林、头孢噻肟、头孢噻呋、氨苄西林、头孢他啶、新霉素、安普霉素、庆大霉素、阿米卡星、多西环素、黏菌素、环丙沙星、恩诺沙星、氟苯尼考、复方新诺明、替加环素)最小抑菌浓度(minimum inhibitory concentration, MIC)的测定,以大肠杆菌ATCC 25922作为质控菌,参考美国临床和实验室标准协会(Clinical and Laboratory Standards Institute, CLS
1.7 耐药基因PCR扩增
对分离出的目标菌株进行部分耐药基因的扩增,分别为黏菌素耐药基因mcr-1;β-内酰胺酶类耐药基因blaSHV、blaCTX、blaTEM;碳青霉烯类耐药基因blaKPC、blaNDM;磺胺类耐药基因Sul1、Sul2;磷霉素耐药基因fosA3;酰胺醇类耐药基因floR;氨基糖苷类耐药基因rmtB;喹诺酮类耐药基因qnrS、oqxAB;四环素类耐药基因tet(A)。参照文献[
1.8 毒力基因扩增
对分离出的目标菌株,选取部分毒力基因进行PCR扩增。参照文献[
基因 Genes | 引物序列 Primer sequences (5′→3′) | 片段大小 Target fragment (bp) | 退火温度 Annealing temperature (℃) | 参考文献 References |
---|---|---|---|---|
rmpA | F: ACTGGGCTACCTCTGCTTCA | 535 | 55 |
[ |
R: CTTGCATGAGCCATCTTTCA | ||||
mrkD | F: ATGTCGCTGAGGAAATTACTAACGC | 958 | 56 |
[ |
R: TTAATCGTACGTAAGGTTAAAGATCAT | ||||
WabG | F: ACCATCGGCCATTTGATAGA | 683 | 54 |
[ |
R: CGGACTGGCAGATCCATATC | ||||
maga | F: GGTGCTCTTTACATCATTGC | 1 283 | 53 |
[ |
R: GCAATGGCCATTTGCGTTAG | ||||
uge | F: TCTTCACGCCTTCCTTCACT | 535 | 55 |
[ |
R: GATCATCCGGTCTCCCTGTA | ||||
fimH | F: TGCTGCTGGGCTGGTCGATG | 550 | 57 |
[ |
R: GGGAGGGTGACGGTGACATC | ||||
kfu | F: GAAGTGACGCTGTTTCTGGC | 520 | 57 |
[ |
R: TTTCGTGTGGCCAGTGACTC | ||||
Aerobactin | F: GCATAGGCGGATACGAACAT | 556 | 56 |
[ |
R: CACAGGGCAATTGCTTACCT | ||||
ureA | F: GCTGACTTAAGAGAACGTTATG | 337 | 54 |
[ |
R: GATCATGGCGCTACCTCA |
2 结果与分析
2.1 细菌的分离培养与革兰染色
从428份粪便样品中分离得到126株疑似肺炎克雷伯菌,分离率为29.4%,按不同来源分别命名。疑似肺炎克雷伯菌在MIAC琼脂培养基上菌落大小一致,呈粉色、中心发白,表面光滑、中间凸起,菌落湿润(

图1 MIAC培养基的肺炎克雷伯菌菌落(A)与革兰氏染色镜检结果(B,400×)
Figure 1 Klebsiella pneumoniae colonies in MIAC (A) and Gram-stained microscopic findings (B, 400×).
2.2 16S rRNA和khe基因检测
采用PCR方法对16S rRNA基因(

图2 分离菌株16S rRNA (A)和khe (B)基因PCR扩增结果。泳道1-9:部分分离菌株的PCR扩增结果;泳道M:DL2000 DNA marker。
Figure 2 The amplification of 16S rRNA (A) and khe (B) genes of isolated strains. Lanes 1-9: PCR results of some isolated strains; Lane M: DL2000 DNA marker.
2.3 药物敏感性试验结果
采用琼脂扩散法对126株肺炎克雷伯菌进行17种药物的敏感性测定。结果显示,分离株对阿莫西林、氨苄西林、复方新诺明的耐药率较高,分别为75.40%、73.81%、61.90%;其次对庆大霉素、头孢噻呋、多西环素、头孢噻肟、氟苯尼考、新霉素和环丙沙星具有不同程度的耐药,耐药率分别为44.44%、40.48%、34.13%、26.98%、24.60%、18.25%和16.67%;对头孢他啶、恩诺沙星、阿米卡星、安普霉素、较为敏感;对替加环素、黏菌素、美罗培南敏感(

图3 肺炎克雷伯菌药敏检测结果。AMP:氨苄西林;AMO:阿莫西林;CTX:头孢噻肟;CEF:头孢噻呋;CAZ:头孢他啶;AMI:阿米卡星;GEN:庆大霉素;NEO:新霉素;APR:安普霉素;TIG:替加环素;DOX:多西环素;CIP:环丙沙星;ENR:恩诺沙星;SXT:复方新诺明;FLR:氟苯尼考;CL:黏菌素;MEM:美罗培南。
Figure 3 The result of Klebsiella pneumoniae drug sensitivity test. AMP: Ampicillin; AMO: Amoxicillin; CTX: Cefotaxime; CEF: Ceftiofur; CAZ: Ceftazidime; AMI: Amikacin; GEN: Gentamicin; NEO: Neomycin; APR: Apramycin; TIG: Tigecycline; DOX: Doxycycline; CIP: Ciprofloxacin; ENR: Enrofloxacin; SXT: Cotrimoxazole; FLR: Florfenicol; CL: Colistin; MEM: Meropenem.
2.4 耐药基因检测结果
耐药基因检测结果显示,β-内酰胺酶耐药基因blaSHV检出率为73.81%,blaCTX (26.98%)、blaTEM (30.16%)也有不同程度的检出;碳青霉烯类耐药基因blaNDM检出率为0.79%;氟喹诺酮类耐药基因oqxAB检出率最高,为86.51%,qnrS检出率为27.78%;磺胺类耐药基因Sul1检出率为16.67%,Sul2 (46.03%)检出率较高;四环素类耐药基因tet(A)检出率较高,为52.68%;磷霉素耐药基因fosA3检出率为21.43%;氟苯尼考耐药基因floR检出率为24.60%;黏菌素耐药基因mcr-1、碳青霉烯酶类blaKPC和氨基糖苷类耐药基因rmtB未检出(

图4 肺炎克雷伯菌分离株的耐药基因检测结果
Figure 4 The result of drug resistance genes in Klebsiella pneumoniae isolates.
2.5 毒力基因检测结果
毒力基因检测结果显示:荚膜相关基因rmpA的检出率为6.30%,magA未检出;脂多糖相关基因wabG、uge的检出率分别为8.90%、95.54%;菌毛相关基因fimH检出率为91.07%,mrkD未检出;铁载体相关基因kfu、Aerobactin检出率分别为7.10%、2.70%;尿素酶相关基因ureA检出率最高,为100.00% (

图5 肺炎克雷伯菌分离株的毒力基因检测结果
Figure 5 The result of virulence genes in Klebsiella pneumoniae isolates.
3 讨论
近年来,中国养宠数量持续上升,肺炎克雷伯菌引起的人和动物患病的相关报道也在迅速增多。已有报道从不同患病动物中分离出高致病性、高水平多重耐药的肺炎克雷伯
目前,抗菌药物仍是临床治疗宠物细菌性疾病的首选方式。宠物临床主要用药包括:阿莫西林克拉维酸钾用于敏感菌引起的各种感染,如皮肤、软组织、呼吸道、尿道和肠道感染;新霉素常用于眼部感染,如结膜炎和角膜炎等;恩诺沙星首选用于泌尿系统疾病,如膀胱炎和猫下泌尿道综合征;当犬猫支原体和衣原体感染致呼吸道疾病时,常用多西环素联合阿莫西林克拉维酸钾。宠物抗生素使用指南是限制细菌耐药性发生与耐药细菌在人畜共患病传播的重要工
已有研究发现,分离于宠物医院的犬源肺炎克雷伯菌的耐药程度有不断攀升的趋势,从对一代头孢菌素类抗生素耐药到对二代和三代头孢菌素类、四环素类、喹诺酮类及磺胺类等抗菌药物耐
随着宠物行业的迅猛发展,动物诊疗机构中药品管理与使用问题越来越突出,人药兽用也引发了社会各界的高度关
肺炎克雷伯菌感染机体的能力与菌毛、荚膜、铁载体与脂多糖等毒力基因相关。肺炎克雷伯菌主要表达Ⅰ型、Ⅲ型菌毛。Ⅰ型菌毛由fim基因群表达(如fimH),介导菌株黏附并侵入宿主细胞,是尿路感染的关键原因;Ⅲ型菌毛由mrk基因群表达(如mrkD),主要是黏附于内皮细胞及呼吸道和泌尿生殖道上皮细
4 结论
本研究表明,广东省部分地区宠物源肺炎克雷伯菌对常用抗菌药物耐药情况严重,但致病性较弱。提示兽用临床上应检测肺炎克雷伯菌的致病力变化,加强对抗生素的管理及使用,避免多重耐药高毒力肺炎克雷伯菌的广泛传播。
作者贡献声明
吴素娟:方案设计、数据处理与分析、数据管理、数据可视化、文稿写作及编辑;林昌成:数据处理、实验操作;万鹏:方案设计、数据分析、文稿审查;胡健欣:方案设计、数据分析、文稿审查;黄鸿昊:方案设计、文稿审查;李杰:方案设计、文稿审查;熊文广:方案设计、项目管理、监督指导;曾振灵:方案设计、项目管理、监督指导、文稿审查及编辑。
利益冲突
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
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