川西北高原牦牛和藏猪源大肠杆菌生物被膜表型、耐药基因、整合酶基因和毒力基因检测

doi:10.3864/j.issn.0578-1752.2021.23.018

摘要/Abstract

摘要：

【目的】对329株采自川西北高原牦牛和藏猪源大肠杆菌进行生物被膜形成能力、抗生素耐药基因、整合酶基因、毒力基因和遗传谱系分型分析,以期了解其耐药现状、毒力特性和优势遗传谱系分布等生物学特征。【方法】利用麦康凯培养基和15e肠杆菌科细菌生化编码鉴定管对牦牛和藏猪粪便和胃肠道内容物样本进行大肠杆菌分离和鉴定;采用改良结晶紫半定量染色法和微量肉汤稀释法分别对分离菌株进行生物被膜形成能力鉴定及其对24种抗菌药物的敏感性试验;采用普通PCR或多重PCR方法对28个耐药基因、2个整合酶基因、15个毒力基因进行检测和遗传谱系分型分析。【结果】（1）从471份牦牛、藏猪粪便和胃肠道内容物样本分离鉴定329株大肠杆菌,分离率为78.9%。（2）329株大肠杆菌大多表现出弱或无生物被膜形成能力,仅2株为强成膜能力表型（其中牦牛和藏猪源各1株）。（3）329株大肠杆菌对24种抗菌药物多表现出一定的耐药性并呈现多重耐药现象,其中对磺胺甲口恶唑、磺胺二甲嘧啶、链霉素、氯霉素、氨苄西林、利福平和土霉素耐药率较高,对氨基糖苷类（卡那霉素、阿米卡星和壮观霉素）、β-内酰胺类（头孢噻呋、头孢曲松、头孢唑啉）、喹诺酮类（萘啶酸、沙拉沙星、恩诺沙星、环丙沙星、达氟沙星、左氧氟沙星）和多黏菌素B 等敏感。（4）除cat1、cat2、bla_CMY-2、bla_SHV、tetC、tetG、tetX等7个耐药基因外,其他21个耐药基因检测结果均为阳性,其中以aac(6')-Ib最为流行,其次是sul1和floR,检出率均在30%以上。藏猪源大肠杆菌对喹诺酮类抗生素耐药与qnrA相关,牦牛源大肠杆菌对β-内酰胺类耐药性与bla_TEM和bla_DHA相关。（5）整合酶基因intⅠ1和intⅠ2的检出率分别为30.09%（99/329)和4.56%（15/329),其中10株大肠杆菌（牦牛源2株,藏猪源8株)同时检测到intⅠ1和intⅠ2。（6）毒力基因agn43、sitA、ompT、eaeA、bcsA、fimC、LT、fyuA和irp2均有阳性检出,但stx1、stx2、ehxA、bcsB、hlyA和hlyE未检测到; 329株大肠杆菌共存在38种不同的毒力谱型,其中285株至少携带除agn43和bcsA外7个毒力基因中的1个,最多携带6个毒力基因。（7）21个耐药基因中,A型和B1型分布的耐药基因种类较B2型和D型丰富;A型中sul3、qnrS、tetM耐药基因分布最广,B1型中sul1和aac(6')-Ib分布最广,不存在tetM和qnrA;7个毒力基因主要分布于A型和B1型,fimC、sitA和ompT主要分布于A型和B1型,eaeA、fyuA和irp2的主要分布于B1型,LT主要分布于A型(仅1株分布于D型)。【结论】329株大肠杆菌耐药较为严重,且耐药基因谱型和毒力谱型呈现多样化,本研究能够为川西北高原牦牛和藏猪源大肠杆菌病治疗、发病机制探讨及抗菌药物合理使用提供数据支持和理论依据。

关键词: 大肠杆菌, 生物被膜, 抗生素耐药基因(ARGs), 整合酶基因, 毒力基因, 遗传背景

Abstract:

【Objective】To improve the understanding of biological properties of drug resistance status, virulence characteristics and the predominant phylogroups of Escherichia coli (E.coli) isolated from yaks and Tibetan pigs in northwest Sichuan Plateau, biofilm-forming ability, antibacterial resistance genes, virulence genes, integrase genes and phylogenetic analyses were carried out in current study.【Method】Fecal samples and gastrointestinal contents from yaks and Tibetan pigs were collected for E.coli isolation and identification using MacConkey agar and 15e enterobacteriaceae bacterial biochemical coding identification tube. Modified semi-quantitative crystal violet staining method and microdilution broth method were used for biofilm-forming ability and antibacterial sensitivity testing to 24 antibacterial agents, respectively. Meanwhile, the detection of 28 antibacterial resistance genes, 2 integrase genes and 15 virulence genes and phylogenetic analyses were performed by conventional PCR or multiple PCR method. 【Result】The results showed that: (1) 329 strains of E.coli were isolated and identified from 471 feces and gastrointestinal samples collected from yaks and Tibetan pigs, and the isolation rate was 78.9%. (2) Most of the 329 strains of E.coli performed weak or absent biofilm-forming ability, and only 2 strains showed strong biofilm formation phenotype (one isolated from yak and the other isolated from Tibetan pig). (3) Most of the 329 strains of E.coli revealed drug resistance to 24 antibacterial agents and were multi-drug resistant, among which, the drug resistance rates to Sulfamethoxazole, Sulfadimidine, Streptomycin, Chloramphenicol, Ampicillin, Rifampicin, and Oxytetracycline were relatively high, and were sensitive to Aminoglycosides (Kanamycin, Amikacin, Spectinomycin), β-lactams(Ceftiofur, Ceftriaxone, Cefazolin), Quinolones (Nalidixic acid, Sarafloxacin, Enrofloxacin, Ciprofloxacin, Danofloxacin, Levofloxacin) and Colistin B. (4) Twenty-one antibacterial resistance genes (ARGs) were detected positive and the other seven ARGs (cat1, cat2, bla_CMY-2, bla_SHV, tetC, tetG, and tetX) were detected negative, among which aac(6')-Ib was the most prevalent gene, followed by sul1 and floR, with the detection rates over 30%. There existed correlation between drug resistance to quinolones antibiotics and qnrA in Tibetan pig-derived E.coli, and for yak-derived E.coli, the strains resistant to β-lactam antibiotics existed correlation between bla_TEM and bla_DHA. (5) The detection rates of integrase genes intⅠ1 and intⅠ2 were 30.09% (99/329) and 4.56% (15/329), respectively. And integrase genes intⅠ1 and intⅠ2 were simultaneously detected in 10 isolates (2 yak-derived and 8 Tibetan pig-derived, respectively). (6) Virulence genes of agn43, sitA, ompT, eaeA, bcsA, fimC, LT, fyuA and irp2 were all positively detected, but stx1, stx2, ehxA, bcsB, hlyA, and hlyE were not detected. There existed 38 different virulence genotypes in 329 strains of E.coli and 285 of which carried at least one of the seven virulence genes except for agn43 and bcsA, some strains carried six virulence genes at most. (7) Among the 21 ARGs, types of ARGs in phylogroup A and B1 were more abundant than those of phylogroup B2 and D; In phylogroup A, sul3, qnrS, tetM were the most widely distributed ARGs, and for phylogroup B1 most widely distributed ARGs were sul1 and aac(6')-Ib, without tetM and qnrA; Seven virulence genes were mainly distributed in phylogroup A and B1, fimC, sitA and ompT genes were mainly distributed in phylogroup A and B1, and eaeA, fyuA and irp2 were the mainly distributed genes in phylogroup B1; LT was mainly distributed in phylogroup A (only one distributed in phylogroup D).【Conclusion】 In summary, the resistance status of 329 strains of E.coli was serious, revealing various drug resistance profiles and virulence genotypes. The current study could provide data support and theoretical basis for yak and Tibetan pig colibacillosis treatment, mechanism of pathogenesis and rational use of antibacterial agents in northwest Sichuan Plateau.

Key words: E. coli, biofilm, antibacterial resistance genes (ARGs), integrase genes, virulence genes, phylogenetic background

陈朝喜,李宇涵,谭敏,汪露,黄志宏. 川西北高原牦牛和藏猪源大肠杆菌生物被膜表型、耐药基因、整合酶基因和毒力基因检测[J]. 中国农业科学, 2021, 54(23): 5144-5162.

CHEN ChaoXi,LI YuHan,TAN Min,WANG Lu,HUANG ZhiHong. Biofilm-Forming Phenotype, Antibacterial Resistance Genes, Integrase Genes and Virulence Genes Detection of Escherichia coli Isolated from Yaks and Tibetan Pigs in Northwest Sichuan Plateau[J]. Scientia Agricultura Sinica, 2021, 54(23): 5144-5162.

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图/表 12

表1

耐药基因和整合酶基因扩增引物信息"

抗菌药物类别 Classes of antibacterial agents	基因名称 Gene name	引物序列（5′→3′） Primer sequence (5′→3′)	扩增片段大小 Amplification size (bp)	退火温度 Annealing temperature (℃)	参考文献或序列号 Reference or sequence number
四环素类 Tetracyclines	tetA	F-CACTATGGCATTCTGCTGGC	948	60	[18]
	tetA	R-CATAGATCGCCGTGAAGAGG	948	60	[18]
	tetB	F-GCCCAGTGCTGTTGTTGTC	553	60	[18]
	tetB	R-AAGACCAAGACCCGCTAATG	553	60	[18]
	tetC	F-TCCTGCTCGCTTCGCTACT	730	58	[18]
	tetC	R-TGGTCGTCATCTACCTGC	730	58	[18]
	tetD	F-AAACCATTACGGCATTCTGC	787	56	[19]
	tetD	R-GACCGGATACACCATCCATC	787	56	[19]
	tetG	F-CGGTCTTATGGGTGCTCTA	721	58	[18]
	tetG	R-CCTTGCTTGTTACTGAC	721	58	[18]
	tetM	F-TTATCAACGGTTTATCAGG	397	55	[20]
	tetM	R-CGTATATATGCAAGACG	397	55	[20]
	tetX	F-CAATAATTGGTGGTGGACCC	468	56	[21]
	tetX	R-TTCTTACCTTGGACATCCCG	468	56	[21]
磺胺类 Sulfonamides	sul1	F-TCAGACGTCGTGGATGTCG	393	57	[22]
	sul1	R-CGAAGAACCGCACAATCTCG	393	57	[22]
	sul2	F-CCTGTTTCGTCCGACACAGA	435	59	[23]
	sul2	R-GAAGCGCAGCCGCAATTCAT	435	59	[23]
	sul3	F-AGATGTGATTGATTTGGGAGC	443	53	[24]
	sul3	R-TAGTTGTTTCTGGATTAGAGCCT	443	53	[24]
喹诺酮类 Quinolones	qnrA	F-TCAGCAAGAGGATTTCTCA	627	55	[25]
	qnrA	R-GGCAGCACTATTACTCCCA	627	55	[25]
	qnrB	F-ACGATGCCTGGTAGTTGTCC	469	55	[25]
	qnrB	R-ACGACATTCGTCAACTGCAA	469	55	[25]
抗菌药物类别 Classes of antibacterial agents	基因名称 Gene name	引物序列（5′→3′） Primer sequence (5′→3′)	扩增片段大小 Amplification size (bp)	退火温度 Annealing temperature (℃)	参考文献或序列号 Reference or sequence number
	qnrS	F-ACGACATTCGTCAACTGCAA	417	55	[25]
	qnrS	R-TAAATTGGCACCCTGTAGGC	417	55	[25]
	qepA	F-GCAGGTCCAGCAGCGGGTAG	299	56	[25]
	qepA	R-CTTCCTGCC CGAGTATCGTG	299	56	[25]
β-内酰胺类 β-lactams	bla_DHA	F-AACTTTCACAGGTGTGCTGGGT	387	59	[26]
	bla_DHA	R-CCGTACGCATACTGGCTTTGC	387	59	[26]
	bla_TEM	F-ATAAAATTCTTGAAGACGAAA	1080	52	[26]
	bla_TEM	R-GACAGTTACCAATGCTTAATC	1080	52	[26]
	bla_CMY-2	F-ATGATGAAAAAATCGTTATGC	1143	57	[26]
	bla_CMY-2	R-TTGCAGCTTTTCAAGAATGCG	1143	57	[26]
	bla_SHV	F-CACTCAAGGATGTATTGTG	885	53	[26]
	bla_SHV	R-TTAGCGTTGCCAGTGCTCG	885	53	[26]
酰氨醇类 Amphenicols	floR	F-CTGAGGGTGTCGTCATCTAC	673	54	[23]
	floR	R-GCTCCGACAATGCTGACTAT	673	54	[23]
	cmlA	F-CGCCACGGTGTTGTTGTTAT	394	59	[23]
	cmlA	R-GCGACCTGCGTAAATGTCAC	394	59	[23]
	cmlB	F-ACTCGGCATGGACATGTACT	284	57	[23]
	cmlB	R-ACGGACTGCGGAATCCATAG	284	57	[23]
氨基糖苷类 Aminoglycosides	cat1	F-CTTGTCGCCTTGCGTATAAT	508	53	[23]
	cat1	R-ATCCCAATGGCATCGTAAAG	508	53	[23]
	cat2	F-AACGGCATGATGAACCTGAA	547	53	[23]
	cat2	R-ATCCCAATGGCATCGTAAAG	547	53	[23]
	aac(3’)-Ⅳ	F-GGCCACTTGGACTGATCGAG	409	58	X01385
	aac(3’)-Ⅳ	R-GCGGATGCAGGAAGATCAAC	409	58	X01385
	aadA2	F-GGTGCTAAGCGTCATTGAGC	470	57	AB154408
	aadA2	R-GCTTCAAGGTTTCCCTCAGC	470	57	AB154408
	rmtB	F-ACATCAACGATGCCCTCAC	472	53	AB103506
	rmtB	R-AAGTTCTGTTCCGATGGTC	472	53	AB103506
	aac(6’)-Ib	F-TTGCGATGCTCTATGAGTGGCTA	482	56	[27]
	aac(6’)-Ib	R-CTCGAATGCCTGGCGTGTTT	482	56	[27]
	aph (3’)-Ⅶ	F-TCCACAGGATGGCAAGATCC	690	55	AY260546
	aph (3’)-Ⅶ	R-TTCAACGGGAAACGTCTTGC	690	55	AY260546
整合酶基因 Integrase genes	intⅠ1	F-CCTCCCGCACGATGATC	280	57	[28]
	intⅠ1	R-TCCACGCATCGTCAGGC	280	57	[28]
	intⅠ2	F-TTATTGCTGGGATTAGGC	233	52	[28]
	intⅠ2	R-ACGGCTACCCTCTGTTATC	233	52	[28]

表1

表2

图1

表3

329株大肠杆菌对24种抗菌药物的药物敏感性试验"

抗菌药物 Antibacterial agents	耐药率 Antibiotic resistance rate (%)			中介率 Antibiotic mediate rate (%)			敏感率 Antibiotic sensitivity rate (%)
抗菌药物 Antibacterial agents	牦牛 Yaks	藏猪 Tibetan pigs	小计 Sub-total	牦牛 Yaks	藏猪 Tibetan pigs	小计 Sub-total	牦牛 Yaks	藏猪 Tibetan pigs	小计 Sub-total
链霉素Streptomycin	100.0	100.0	100.0	0.0	0.0	0.0	0.0	0.0	0.0
庆大霉素Gentamicin	27.7	46.3	34.7	5.3	4.9	5.2	67.0	48.8	60.2
妥布霉素Tobramycin	1.5	1.6	1.5	4.4	4.9	4.6	94.2	93.5	93.9
卡那霉素Kanamycin	0.0	0.0	0.0	4.9	17.1	9.4	95.1	82.9	90.6
大观霉素Spectinomycin	0.0	0.0	0.0	57.3	73.2	63.2	42.7	26.8	36.8
阿米卡星Amikacin	0.0	0.0	0.0	0.0	0.0	0.0	100.0	100.0	100.0
氯霉素Chloramphenicol	96.6	97.6	97.0	1.5	1.6	1.5	1.9	0.8	1.5
氟苯尼考Florphenicol	9.7	30.9	17.6	38.8	36.6	38.0	51.5	32.5	44.4
磺胺二甲嘧啶Sulfadimidine	100.0	100.0	100.0	0.0	0.0	0.0	0.0	0.0	0.0
磺胺甲噁唑 Sulfamethoxazole	100.0	100.0	100.0	0.0	0.0	0.0	0.0	0.0	0.0
利福平Rifampicin	52.9	62.6	56.5	24.3	31.7	27.1	22.8	5.7	16.4
多黏菌素B Colistin B	3.4	1.6	2.7	5.8	8.9	7.0	90.8	89.4	90.3
土霉素Oxytetracycline	41.3	83.7	57.1	6.8	4.1	5.8	51.9	12.2	37.1
多西环素Doxycycline	25.2	48.8	34.0	11.2	25.2	16.4	63.6	26.0	49.5
萘啶酸Nalidixic acid	11.2	17.9	13.7	0.0	0.0	0.0	88.8	82.1	86.3
沙拉沙星Sarafloxacin	7.8	3.2	6.1	9.2	9.8	9.4	83.0	87.0	84.5
恩诺沙星Enrofloxacin	6.8	4.9	6.1	4.4	8.9	6.1	88.8	86.2	87.8
环丙沙星Ciprofloxacin	5.8	4.9	5.5	0.0	0.8	0.3	94.2	94.3	94.2
达氟沙星Danofloxacin	5.8	3.3	4.9	8.3	13.0	10.0	85.9	83.7	85.1
左氧氟沙星 Levofloxacin	4.4	2.4	3.6	1.0	0.0	0.6	94.7	97.6	95.7
氨苄西林Ampicillin	27.7	49.6	35.9	1.5	1.6	1.5	70.9	48.8	62.6
头孢噻呋Ceftiofur	1.9	8.9	4.6	0.5	1.6	0.9	97.6	89.4	94.5
头孢曲松Ceftriaxone	1.9	12.2	5.8	1.0	0.8	0.9	97.1	87.0	93.3
头孢唑啉Cefazolin	0.5	0.0	0.3	0.0	0.0	0.0	99.5	100.0	99.7

表3

表4

329株大肠杆菌耐药谱型汇总"

耐药表型 Antibacterial susceptibility profiles	菌株数 Strain numbers (%)
耐药表型 Antibacterial susceptibility profiles	牦牛 Yaks	藏猪 Tibetan pigs	小计 Sub-total
AMP/CHL/CIP/CRO/CTF/DAN/DOX/ENR/GEN/LVX/NAL/OXY/RIF/SAR/SMX/SM2/STR/TOB	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/CRO/CTF/DAN/ENR/FLR/GEN/LVX/NAL/OXY/RIF/SAR/SMX/SM2/STR/TOB	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/CZO/DAN/DOX/ENR/FLR/GEN/LVX/NAL/OXY/RIF/SAR/SMX/SM2/STR	2 (0.97)	1 (0.80)	3 (0.91)
AMP/CHL/CIP/CZO/DAN/DOX/ENR/FLR/GEN/LVX/NAL/RIF/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/CZO/DAN/DOX/ENR/GEN/LVX/NAL/OXY/RIF/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/CZO/DAN/ENR/FLR/GEN/LVX/NAL/OXY/RIF/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/CZO/DAN/DOX/ENR/GEN/LVX/NAL/OXY/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/CZO/DAN/ENR/GEN/LVX/NAL/OXY/RIF/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/CZO/DAN/ENR/GEN/LVX/NAL/OXY/SAR/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CIP/DAN/DOX/ENR/GEN/LVX/NAL/OXY/SAR/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CIP/CZO/DAN/ENR/GEN/NAL/OXY/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/DAN/DOX/ENR/GEN/NAL/OXY/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CRO/CTF/CZO/DOX/FLR/GEN/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/DOX/ENR/FLR/GEN/OXY/RIF/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CIP/CZO/DOX/FLR/GEN/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CIP/DAN/ENR/GEN/NAL/OXY/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CRO/CTF/CZO/DOX/NAL/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CRO/CTF/DOX/GEN/FLR/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/DOX/FLR/GEN/NAL/OXY/RIF/SMX/SM2/STR	2 (0.97)	0 (0.00)	2 (0.61)
AMP/CHL/CRO/CTF/DOX/FLR/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CRO/CZO/DOX/FLR/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CRO/DOX/FLR/GEN/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CRO/CTF/GEN/NAL/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CTF/DOX/GEN/OXY/POL/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CZO/DOX/FLR/GEN/OXY/POL/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/DOX/FLR/GEN/OXY/RIF/SMX/SM2/STR	0 (0.00)	2 (1.63)	2 (0.61)
AMP/CHL/CZO/DOX/GEN/OXY/POL/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/CRO/CZO/DOX/GEN/NAL/OXY/POL/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/CZO/DOX/FLR/GEN/NAL/OXY/SMX/SM2/STR/TOB	0 (0.00)	1 (0.80)	1 (0.30)
CHL/DOX/ENR/FLR/GEN/NAL/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CRO/CTF/DOX/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/DOX/FLR/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/DOX/FLR/OXY/POL/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/DOX/FLR/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/DOX/GEN/OXY/RIF/SMX/SM2/STR	2 (0.97)	1 (0.80)	3 (0.91)
AMP/CHL/CZO/FLR/GEN/OXY/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/DOX/GEN/FLR/OXY/RIF/SMX/SM2/STR	3 (1.46)	2 (1.63)	5 (1.52)
AMP/CHL/DOX/GEN/OXY/RIF/STR/SMX/SM2/TOB	1 (0.49)	0 (0.00)	1 (0.30)
耐药表型 Antibacterial susceptibility profiles	菌株数 Strain numbers (%)
耐药表型 Antibacterial susceptibility profiles	牦牛 Yaks	藏猪 Tibetan pigs	小计 Sub-total
CHL/CZO/DOX/GEN/NAL/OXY/RIF/SMX/SM2/STR	1 (0.49)	1 (0.80)	2 (0.61)
AMP/CHL/CRO/CTF/DOX/GEN/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CRO/CTF/DOX/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CTF/DOX/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/DOX/FLR/OXY/SMX/SM2/STR	1 (0.49)	1 (0.80)	2 (0.61)
AMP/CHL/CZO/DOX/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/FLR/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CZO/GEN/RIF/OXY/SMX/SM2/STR	2 (0.97)	1 (0.80)	3 (0.91)
AMP/CHL/DOX/FLR/GEN/OXY/SMX/SM2/STR	0 (0.00)	2 (1.63)	2 (0.61)
AMP/CHL/DOX/FLR/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/DOX/GEN/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/FLR/GEN/OXY/RIF/SMX/SM2/STR	2 (0.97)	1 (0.80)	3 (0.91)
AMP/CHL/GEN/OXY/RIF/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/CZO/DOX/FLR/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/CZO/DOX/GEN/NAL/OXY/SMX/SM2/STR	1 (0.49)	1 (0.80)	2 (0.61)
CHL/CZO/DOX/GEN/OXY/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/DOX/GEN/FLR/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CRO/CTF/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/CRO/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/DOX/ FLR/OXY/SMX/SM2/STR	3 (1.46)	0 (0.00)	3 (0.91)
AMP/CHL/DOX/GEN/OXY/SMX/SM2/STR	3 (1.46)	3 (2.44)	6 (1.82)
AMP/CHL/DOX/OXY/RIF/SMX/SM2/STR	1 (0.49)	1 (0.80)	2 (0.61)
AMP/CHL/GEN/FLR/OXY/SMX/SM2/STR	0 (0.00)	3 (2.44)	3 (0.91)
AMP/CHL/GEN/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
AMP/CHL/GEN/OXY/RIF/SMX/SM2/STR	6 (2.91)	1 (0.80)	7 (2.13)
AMP/CHL/NAL/OXY/RIF/SMX/SM2/STR	0 (0.00)	2 (1.63)	2 (0.61)
CHL/CRO/FLR/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/CTF/DOX/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/CZO/DOX/OXY/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/CZO/GEN/OXY/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/DOX/FLR/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/DOX/GEN/NAL/OXY/SMX/SM2/STR	2 (0.97)	1 (0.80)	3 (0.91)
CHL/DOX/GEN/OXY/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/GEN/FLR/OXY/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CTF/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/CZO/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/DOX/OXY/SMX/SM2/STR	0 (0.00)	2 (1.63)	2 (0.61)
AMP/CHL/OXY/RIF/SMX/SM2/STR	4 (1.94)	0 (0.00)	4 (1.22)
AMP/CHL/GEN/OXY/SMX/SM2/STR	0 (0.00)	8 (6.50)	8 (2.43)
AMP/GEN/OXY/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/CIP/FLR/RIF/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/CZO/OXY/TOB/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
耐药表型 Antibacterial susceptibility profiles	菌株数 Strain numbers (%)
耐药表型 Antibacterial susceptibility profiles	牦牛 Yaks	藏猪 Tibetan pigs	小计 Sub-total
CHL/DOX/FLR/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/DOX/GEN/OXY/SMX/SM2/STR	2 (0.97)	2 (1.63)	4 (1.22)
CHL/DOX/NAL/OXY/SMX/SM2/STR	2 (0.97)	0 (0.00)	2 (0.61)
CHL/DOX/OXY/RIF/SMX/SM2/STR	10 (4.85)	1 (0.80)	11 (3.34)
CHL/GEN/OXY/POL/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/FLR/GEN/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/GEN/NAL/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/GEN/OXY/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
GEN/OXY/RIF/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/GEN/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/OXY/SMX/SM2/STR	0 (0.00)	2 (1.63)	2 (0.61)
AMP/CHL/RIF/SMX/SM2/STR	3 (1.46)	0 (0.00)	3 (0.91)
AMP/DOX/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/CZO/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/DOX/OXY/SMX/SM2/STR	4 (1.94)	10 (8.13)	14 (4.26)
CHL/ENR/RIF/SMX/SM2/STR	0 (0.00))	1 (0.80)	1 (0.30)
CHL/FLR/OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
CHL/FLR/RIF/SMX/SM2/STR	2 (0.97)	0 (0.00)	2 (0.61)
CHL/GEN/OXY/SMX/SM2/STR	1 (0.49)	2 (1.63)	3 (0.91)
CHL/NAL/OXY/SMX/SM2/STR	0 (0.00)	3 (2.44)	3 (0.91)
CHL/POL/RIF/SMX/SM2/STR	3 (1.46)	0 (0.00)	3 (0.91)
CHL/OXY/RIF/SMX/SM2/STR	5 (2.43)	1 (0.80)	6 (1.82)
DOX/GEN/OXY/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
DOX/OXY/RIF/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
AMP/CHL/SMX/SM2/STR	4 (1.94)	2 (1.63)	6 (1.82)
CHL/CRO/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/DOX/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/ENR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/FLR/SMX/SM2/STR	2 (0.97)	0 (0.00)	2 (0.61)
CHL/GEN/SMX/SM2/STR	1 (0.49)	1 (0.80)	2 (0.61)
CHL/OXY/SMX/SM2/STR	1 (0.49)	9 (7.32)	10 (3.04)
CHL/SAR/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
CHL/RIF/SMX/SM2/STR	36 (17.48)	4 (3.25)	40 (12.16)
CHL/SMX/SM2/STR	57 (27.67)	9 (7.32)	66 (20.06)
GEN/SMX/SM2/STR	1 (0.49)	0 (0.00)	1 (0.30)
OXY/SMX/SM2/STR	0 (0.00)	1 (0.80)	1 (0.30)
SMX/SM2/STR	2 (0.97)	1 (0.80)	3 (0.91)

表4

图2

表5

图3

表6

图4

表7

图5

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毒力基因谱型 Virulence genotypes	菌株数 Strain number (%)
毒力基因谱型 Virulence genotypes	牦牛 Yaks	藏猪 Tibetan pigs	小计 Sub-total
eaeA/fimC/fyuA/irp2/ompT/sitA	2(0.97)	7(5.69)	9(2.74)
eaeA/fimC/fyuA/irp2/LT/sitA	0(0.00)	1(0.80)	1(0.30)
eaeA/fimC/fyuA/irp2/ompT	0(0.00)	1(0.80)	1(0.30)
eaeA/fyuA/irp2/ompT/sitA	1(0.49)	0(0.00)	1(0.30)
fimC/fyuA/irp2/ompT/sitA	1(0.49)	0(0.00)	1(0.30)
eaeA/fimC/fyuA/irp2	0(0.00)	1(0.80)	1(0.30)
eaeA/fimC/irp2/ompT	1(0.49)	1(0.80)	2(0.61)
eaeA/fimC/LT/ompT	0(0.00)	1(0.80)	1(0.30)
eaeA/fimC/LT/sitA	1(0.49)	1(0.80)	2(0.61)
eaeA/fimC/ompT/sitA	5(2.43)	11(8.94)	16(4.86)
fimC/fyuA/irp2/ompT	0(0.00)	1(0.80)	1(0.30)
eaeA/fyuA/irp2/sitA	1(0.49)	0(0.00)	1(0.30)
fimC/irp2/LT/sitA	1(0.49)	0(0.00)	1(0.30)
fimC/irp2/ompT/sitA	0(0.00)	1(0.80)	1(0.30)
eaeA/fimC/fyuA	1(0.49)	0(0.00)	1(0.30)
eaeA/fimC/ompT	5(2.43)	6(4.88)	11(3.34)
eaeA/fimC/sitA	6(2.91)	9(7.32)	15(4.56)
eaeA/ompT/sitA	0(0.00)	2(1.63)	2(0.61)
fimC/fyuA/irp2	1(0.49)	1(0.80)	2(0.61)
fimC/fyuA/ompT	1(0.49)	0(0.00)	1(0.30)
fimC/irp2/LT	3(1.46)	3(2.44)	6(1.82)
fimC/irp2/ompT	0(0.00)	1(0.80)	1(0.30)
fimC/irp2/sitA	0(0.00)	1(0.80)	1(0.30)
fimC/LT/ompT	1(0.49)	2(1.63)	3(0.91)
fimC/LT/sitA	14(6.79)	3(2.44)	17(5.17)
fimC/ompT/sitA	17(8.25)	7(5.69)	24(7.29)
LT/ompT/sitA	0(0.00)	1(0.80)	1(0.30)
eaeA/fimC	14(6.79)	13(10.57)	27(8.21)
eaeA/sitA	0(0.00)	5(4.07)	5(1.52)
fimC/LT	4(1.94)	2(1.63)	6(1.82)
fimC/ompT	25(12.14)	10(8.13)	35(10.64)
fimC/sitA	28(13.59)	5(4.07)	33(10.03)
ompT/sitA	2(0.97)	0(0.00)	2(0.61)
eaeA	0(0.00)	5(4.07)	5(1.52)
fimC	5(2.43)	3(2.44)	58(17.63)
LT	2(0.97)	0(0.00)	2(0.61)
ompT	2(0.97)	0(0.00)	2(0.61)
sitA	10(4.85)	7(5.69)	17(5.17)
ND	31(15.05)	13(10.57)	44(13.37)

菌株来源 Strain origin	菌株数 Strain number	遗传谱系百分比 Percentage of phylogroup (%)
菌株来源 Strain origin	菌株数 Strain number	A	B1	B2	D
牦牛源Yak origin	206	74(35.92)	103(50.0)	17(8.25)	12(5.82)
藏猪源Tibetan pig origin	123	89(72.36)	26(21.14)	3(2.44)	5(4.07)
总计Total	329	163(49.54)	129(39.21)	20(6.08)	17(5.18)