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

Abstract

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

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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Figures/Tables 12

Table 1

Information of PCR primers for ARGs and integrase genes amplification"

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]

Table 1

Table 2

Fig.1

Table 3

Antibacterial sensitivity testing of 329 E.coli to 24 antibacterial agents"

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

Table 3

Table 4

Summary of antibacterial susceptibility profiles of 329 E.coli"

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)

Table 4

Fig. 2

Table 5

Fig. 3

Table 6

Fig. 4

Table 7

Fig. 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)

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

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毒力基因类别 Types of virluence genes	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
产志贺毒素毒力基因 Shiga toxin-producing virulence gene	stx1	F-ACACTGGATGATCTCAGTGG	614	58	[29]
	stx1	R-CTGAATCCCCCTCCATTATG	614	58	[29]
	stx2	F-CCTGTCAACTGAGCACTTTG	779	58	[29]
	stx2	R-CCATGACAACGGACAGCAGTT	779	58	[29]
非菌毛黏附素 Non-fimbrial adhesin	eaeA	F-ATGCTTAGTGCTGGTTTAGG	248	55	EF079676
非菌毛黏附素 Non-fimbrial adhesin	eaeA	R-GCCTTCATCATTTCGCTTTC	248	55	EF079676
EHEC溶血素 EHEC hemolysin	ehxA	F-CAATAATTGGTGGTGGACCC	583	55	EF088504
EHEC溶血素 EHEC hemolysin	ehxA	R-TTCTTACCTTGGACATCCCG	583	55	EF088504
相变抗原 Phase variation antigen	agn43	F-GACTATGACCGGATTSTGGCAGGCT	499	67	[30]
相变抗原 Phase variation antigen	agn43	R-GTGGCTCCAGCATCARTTGTCAG	499	67	[30]
铁结合蛋白 Iron transport periplasmic-binding protein	sitA	F-AGGGGGCACAACTGATTCTCG	608	57	[31]
铁结合蛋白 Iron transport periplasmic-binding protein	sitA	R-TACCGGGCCGTTTTCTGTGC	608	57	[31]
溶血素 Hemolysin	hlyF	F-GGCGATTTAGGCATTCCGATACTC	599	55	[31]
	hlyF	R-ACGGGGTCGCTAGTTAAGGAG	599	55	[31]
	hlyA	F-AACAAGGATAAGCACTGTTCTGGCT	1177	55	[32]
	hlyA	R-ACCATATAAGCGGTCATTCCCGTCA	1177	55	[32]
耶尔森菌强毒力岛 Yersinia high-pathogenicity island	fyuA	F-TGATTAACCCCGCGACGGGAA	880	55	[32]
耶尔森菌强毒力岛 Yersinia high-pathogenicity island	fyuA	R-CGCAGTAGGCACGATGTTGTA	880	55	[32]
纤维素合成酶基因 Cellulose synthase operon genes	bcsA	F-GTATCGGTAGAAAGCAAACAGG	185	55	[33]
	bcsA	R-GAACGGTACACGAGAAGAGG	185	55	[33]
	bcsB	F-CTCGTGTACCGTTCAGGATTTCT	388	55	[33]
	bcsB	R-CAGCCCAACTTCATTACCCAT	388	55	[33]
Ⅰ型菌毛亚基 Type Ⅰ fimbriae	fimC	F-GGAAATAACATTCTGCTTGC	288	56	[34]
Ⅰ型菌毛亚基 Type Ⅰ fimbriae	fimC	R-TTTGTTGCATCAAGAATACG	288	56	[34]
外膜蛋白基因 Outer membrane protein	ompT	F-ATCTAGCCGAAGAAGGAGGC	559	55	[34]
外膜蛋白基因 Outer membrane protein	ompT	R-CCCGGGTCATAGTGTTCATC	559	55	[34]
铁离子摄取相关基因 Iron uptake-related gene	irp2	F-AAGGATTCGCTGTTACCGGAC	280	55	[34]
铁离子摄取相关基因 Iron uptake-related gene	irp2	R-TCGTCGGGCAGCGTTTCTTCT	280	55	[34]
热敏肠毒素 Heat-stable enterotoxin	LT	F-ATGAGTACTTCGATAGAGG	279	55	[34]
热敏肠毒素 Heat-stable enterotoxin	LT	R-ATGGTATTCCACCTAACGC	279	55	[34]

抗菌药物类别 Classes of antibacterial agents	基因名称 Gene name	动物来源 Animal origin	耐药基因阳性菌株数 Strain number of ARGs positive
抗菌药物类别 Classes of antibacterial agents	基因名称 Gene name	动物来源 Animal origin	整合酶阳性 Integrase genes positive	整合酶阴性 Integrase genes negative
四环素类 Tetracyclines	tetA	牦牛 Yaks	8	7
	tetA	藏猪Tibetan pigs	24	15
	tetB	牦牛 Yaks	3	4
	tetB	藏猪Tibetan pigs	10	13
	tetD	牦牛 Yaks	7	7
	tetD	藏猪Tibetan pigs	12	11
	tetM	牦牛 Yaks	0	3
	tetM	藏猪Tibetan pigs	1	1
磺胺类 Sulfonamides	Sul1	牦牛 Yaks	13	79
	Sul1	藏猪Tibetan pigs	7	15
	Sul2	牦牛 Yaks	20	19
	Sul2	藏猪Tibetan pig	16	25
	Sul3	牦牛 Yak	3	17
	Sul3	藏猪Tibetan pig	16	25
喹诺酮类 Quinolones	qnrA	牦牛 Yak	0	2
	qnrA	藏猪Tibetan pigs	4	2
	qnrB	牦牛 Yaks	1	9
	qnrB	藏猪Tibetan pigs	10	14
	qnrS	牦牛 Yaks	8	18
	qnrS	藏猪Tibetan pigs	40	20
	qepA	牦牛 Yaks	0	2
	qepA	藏猪Tibetan pigs	0	0
β-内酰胺类 β-lactams	bla_TEM	牦牛 Yaks	10	3
	bla_TEM	藏猪Tibetan pigs	22	6
	bla_DHA	牦牛 Yaks	28	16
	bla_DHA	藏猪Tibetan pigs	10	13
酰氨醇类 Amphenicols	floR	牦牛 Yaks	16	34
	floR	藏猪Tibetan pigs	40	20
	cmlA	牦牛 Yaks	1	12
	cmlA	藏猪Tibetan pigs	21	5
	cmlB	牦牛 Yaks	1	3
	cmlB	藏猪Tibetan pigs	5	5
氨基糖苷类 Aminoglycosides	aac(3’)-Ⅳ	牦牛 Yaks	4	18
	aac(3’)-Ⅳ	藏猪Tibetan pigs	9	17
	rmtB	牦牛 Yaks	4	20
	rmtB	藏猪Tibetan pigs	10	16
	aadA2	牦牛 Yaks	2	35
	aadA2	藏猪Tibetan pigs	18	13
	aac(6’)-Ib	牦牛 Yaks	16	64
	aac(6’)-Ib	藏猪Tibetan pigs	18	18
	aph(3’)-Ⅶ	牦牛 Yaks	14	31
	aph(3’)-Ⅶ	藏猪Tibetan pigs	21	18

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