Prevalence and molecular characterization of multi-resistant Escherichia coli isolates from clinical bovine mastitis in China

Figures & data

Table 1. Primers sequences and PCR product sizes of genes for antimicrobial resistance.

Primers	Primer sequence (5′–3′)	Size of amplification (bp) Ref.
Intl1	F: CCTCCCGCACGATGATC	280 [12]
	R: TCCACGCATCGTAAGGC
Intl2	F: CGTGCTGGAGGGAAAGAC	207 [12]
	R: CATGACGGTAAGGGTGGG
Intl3	F: AGTGGGTGGCGAATGAGTG	600 [12]
	R: TGTTCTTGTATCGGCAGGTG
5′-CS	GGCATCCAAGCAGCAAG	Uncertain [1]
3′-CS	AAGCAGACTTGACCTGA
blaSHV	F: TCTCCCTGTTAGCCACCCTG	593 [1]
	R: CCACTGCAGCAGCTGCCGTT
blaTEM	F: GTATCCGCTCATGAGACAATA	717 [1]
	R: AGAAGTGGTCCTGCAACTTT
blaCTX	F: ACAGCGATAACGTGGCGATG	443 [1]
	R: TCGCCCAATGCTTTACCCAG
gyrA	F: GAGGGATAGCGGTTAGATGAGC R: CCGTTCACCAGCAGGTTAGG	525 [1]
parC	F: AATGAGCGATAGGCAGAGCG R: TTGGCAGACGGGCAGGTAG	487 [1]
gyrB	F: GCCTTTCTTCACTTTGTACAGCG R: GTGACGGCGGTACTCACCTG	794[1]
parE	F: CTGACCGAAAGCTACGTCAACC	892 [1]
	R: CGTTCGGCTTGCCTTTCTTG
qnrA	F: CAACTTGAGTGGCCAATGCC	211 [27]
	R: GACTCCTTCGAGGTTGACCC
qnrS	F: ACGACATTCGTCAACTGCAA	417[27]
	R: TAAATTGGCACCCTGTAGGC
qnrB	F: GGMATHGAAATTCGCCACTG	264 [27]
	R: TTTGCYGYYCGCCAGTCGAA
qnrC	F: GGGTTGTACATTTATTGAATC	447 [27]
	R: TCCACTTTACGAGGTTCT
qnrD	F: CGSGATCAATTTACGGGGAATA	582 [27]
	R: AACAAGCTGAAGCGCCTG
qepA	F: GCAGGTCCAGCAGCGGGTAG	199 [28]
	R: CTTCCTGCCCGAGTATCGTG
aac(6′)-Ib-cr	F: TTGCGATGCTCTATGAGTGGCTA	482 [28]
	R: CTCGAATGCCTGGCGTGTTT

Table 2. In vitro susceptibilities of 105 E. coli strains isolated from CBM to 22 antimicrobial agents.

Antibacterial agents	No. (%) of resistant strains^a
	R	I	S
Streptomycin	38 (36.2)	—	67(63.8)
Kanamycin	34 (32.4)	2 (1.9)	69 (65.7)
Gentamicin	31 (29.5)	6 (5.7)	67 (63.8)
Amikacin	26 (24.8)	6 (5.7)	73 (69.5)
Amoxicillin	63 (60.0)	6 (5.7)	36 (34.3)
Ampicillin	73 (69.5)	3 (2.9)	29 (27.6)
Cefazolin	66 (62.9)	10 (9.5)	29 (27.6)
Ceftiofur	10 (9.5)	1 (1)	94 (89.5)
Cefalotin	34 (32.4)	30(28.6)	41 (39.0)
Tetracycline	80 (76.2)	7(6.7)	18 (17.1)
Doxycycline	50 (47.6)	13 (12.4)	42 (40.0)
Oxytetracycline	75 (71.4)	0 (0)	30 (28.6)
Colistin	27 (25.7)	—	78 (74.3)
Chloramphenicol	43 (41.0)	4 (3.8)	58 (55.2)
Florfenicol	48 (45.7)	—	57 (54.3)
Enrofloxacin	29 (27.6)	3 (2.9)	73 (69.5)
Norfloxacin	29 (27.6)	7 (6.7)	69 (65.7)
Lomefloxacin	29 (27.6)	11 (10.5)	65 (61.9)
Ciprofloxacin	30 (28.6)	3 (2.8)	72 (68.6)
Sulphadiazine	104 (99.0)	—	1 (1.0)
Sulphamethoxydiazine	105 (100)	0 (0)	0 (0)
Trimethoprim	105 (100)	0 (0)	0 (0)

^aR: resistant; I: intermediate; S: susceptible

Figure 1. Multidrug resistant levels of E. coli isolates from CBM.

Figure 2. Antimicrobial resistant profile of E. coli isolates from CBM.

Table 3. Antibiotic resistance phenotypes and resistance-determining regions in E. coli isolates from dairy cattle with CBM.

Strain number	Resistance phenotypes^a	Gene cassettes of class 1 Integron^b		β-Lactamase gene^c
10	STR, AMC, TET, DOX, OXY, AMP, SMD, ENR, NOR, LOM, CIP, CET, SAD, TMP	Integrase1	dfr2d-catB3-aadA1	blaTEM
22	STR, CEZ, CHL, AMC, AMP, FOR, TET, DOX, OXY, SAD, SMD, TMP	Integrase1	dfrA1-aadA1	blaTEM
23	STR, KAN, GEN, AMC, AMP, CEZ, CET, TET, DOX, OXY, CST, CHL, ENR, NOR, LOM, CIP, SAD, SMD, TMP	Integrase1	dfrA1-catB2-aadA1	blaTEM,,blaCTX
25	STR, KAN, AMK, CEZ, CST, CHL, AMP, TET, OXY, CET, SAD, SMD, TMP, AMC	Integrase1	aadA1	blaTEM
26	CST, CHL, AMP, TET, OXY, AMC, FOR, SAD, SMD, TMP	Integrase1	aadA1, dfrA1-aadA1, dfrA17-aadA5	blaTEM
27	STR, KAN, CEZ, CST, FOR, AMC, AMP, TET, DOX, OXY, SAD, SMD, TMP, ENR, NOR, LOM, CIP	Integrase1	dfrA17-aadA5	blaTEM
28	KAN, GEN, AMK, AMC, AMP, CEZ, TET, DOX, OXY, CST, CHL, ENR, NOR, LOM, CIP, SAD, SMD, TMP	Integrase1	aadA1, dfr2d-catB3-aadA1, dfrA17-aadA5	blaTEM
30	STR, CHL, DOX, OXY, FOR, SAD, SMD, TMP	Integrase1	aadA1	blaTEM
34	CEZ, CST, CET, FOR, CHL, TET, OXY, SAD, SMD, TMP	Integrase1	dfrA17-aadA5	blaTEM
35	CHL, FOR, CET, AMP, TET, DOX, OXY, SAD, SMD, TMP	Integrase1	aadA1	blaTEM
41	STR, KAN, GEN, AMK, AMC, AMP, CEZ, CET, TET, DOX, OXY, CST, CHL, FOR, ENR, NOR, LOM, CIP, SAD, SMD, TMP	Integrase1	aadA1, dfr2d-catB3-aadA1	blaTEM,blaCTX
44	STR, KAN, GEN, AMK, AMC, AMP, CEZ, CET, TET, DOX, OXY, SAD, SMD, TMP	Integrase1	dfrA17-aadA5	blaTEM
45	STR, AMC, AMP, TET, DOX, OXY, SAD, SMD, TMP	Integrase1	dfrA17-aadA5	blaTEM
50	STR, KAN, GEN, AMK, AMC, AMP, CEZ, CET, TET, DOX, OXY, CST, CHL, FOR, SAD, SMD, TMP	Integrase1	dfr2d-catB3-aadA1, dfrA17-aadA5	blaTEM,blaCTX
54	STR, KAN, GEN, AMC, AMP, CEZ, CEF, CET, TET, DOX, OXY, CST CHL, FOR, NOR, LOM, SAD, SMD, TMP	Integrase1	aadA1	blaTEM,blaCTX
55	STR, AMC, AMP, TET, DOX, OXY, SAD, SMD, TMP	Integrase1	aadA1, dfrA17-aadA5	blaTEM
56	STR, KAN, GEN, AMK, CEZ, CET, AMP, TET, DOX, CST, OXY, SAD, SMD, TMP	Integrase1	aadA1	blaTEM
59	STR, AMK, CEZ, CST, CHL AMP, TET, OXY, SAD, SMD, TMP	Integrase1	dfrA1- catB2-aadA1	blaTEM
67	AMC, CHL, TET, OXY, AMP, CET, ENR, NOR, LOM, CIP, SAD, SMD, TMP	Integrase1	aadA1, dfrA17-aadA5	blaTEM
73	STR, KAN, GEN, CEZ, CET, AMC, AMP, TET, DOX, OXY, CHL, FOR, SAD, SMD, TMP	Integrase1	dfrA1-aadA1	blaTEM
81	STR, KAN, GEN, AMC, AMP, TET, OXY, CST, CHL, CIP, SAD, SMD, TMP	Integrase1	aadA1, dfrA1- catB2-aadA1	blaTEM,blaCTX
82	STR, AMK, CEZ, CST, CHL, SAD, SMD, TMP	Integrase1	dfrA1- catB2-aadA1	blaTEM
84	STR, KAN, AMK	Integrase1	aadA1	blaTEM
85	STR, KAN, AMP, CEZ, OXY, CST, CHL, ENR, NOR, LOM, CIP, SAD, SMD, TMP	Integrase1	dfr2d-catB3-aadA1	blaTEM
86	CEZ, OXY, FOR, SAD, SMD, TMP	Integrase1	dfrA17-aadA5	blaTEM
87	CEZ, TET, AMP, OXY, ENR, SAD, SMD, TMP	Integrase1	aadA1, dfrA1-aadA1, dfrA17-aadA5	blaTEM
89	CEZ, TET, SAD, SMD, TMP	Integrase1	aadA1	blaTEM
93	CEZ, CEF, CST, TET, OXY, CHL, FOR, SAD, SMD, TMP	Integrase1	dfrA17-aadA5	blaTEM
94	STR, KAN, GEN, AMC, AMP, CEF, TET, DOX, OXY, FOR, NOR, SAD, SMD, TMP, NOR	Integrase1	dfrA1-aadA1	blaTEM
100	CEZ, CST, CHL, SAD, SMD, TMP	Integrase1	dfr2d-catB3-aadA1	blaTEM
103	KAN, CEZ, TET, DOX, OXY, CST, FOR, SAD, SMD, TMP	Integrase1	dfrA17-aadA5	blaTEM
104	STR, AMC, AMP, CEZ, TET, DOX, OXY, CHL, FOR, SAD, SMD, TMP	Integrase1	dfrA17-aadA5, dfr2d-catB3-aadA1	blaTEM

^aSTR: streptomycin; KAN: kanamycin; GEN: gentamicin; AMK: Amikacin; AMC: Amoxicillin; AMP: ampicillin; CEZ: cefazolin; CEF: ceftiofur; CET: cefalotin; DOX: doxycycline; TET: tetracycline; OXY: oxytetracycline; CST: colistin; SAD: sulphadiazine; SMD: sulphamethoxydiazine; TMP: Trimethoprim; CHL: chloramphenicol; FOR: florfenicol; ENR: enrofloxacin; NOR: norfloxacin; LOM: lomefloxacin; CIP: ciprofloxacin

^bIntegraseI: integrase gene; aadA1: aminoglycoside-3′-adenylytransferase gene; dfrA17-aadA5: dihydrofolate reductase gene and aminoglycoside-3′-adenylytransferase gene; dfrA1-aadA1: dihydrofolate reductase gene and aminoglycoside-3′-adenylytransferase gene; dfrA1-catB2-aadA1: dihydrofolate reductase gene and chloramphenicol acetyltransferase gene and aminoglycoside-3′-adenylytransferase gene

^cbla_TEM: TEM-type ESBL gene; bla_CTX: CTX-type ESBL gene

Table 4. Fluoroquinolone susceptibility and substitutions in GyrA, GyrB, ParC and ParE QRDRs in E. coli isolates from dairy cattle with CBM.

Isolate	ENR^a	NOR^b	CIP^c	LOM^d	GyrA^e	ParC^f	GyrB^g	ParE^h	Gene cassettes of class 1 Integron^i		β-Lactamase gene^j
1	8	16	16	8	S83L	S58I	P385A
9	64	32	64	32	S83L, D87N	S58I, R138V,	P385A	S458A
14	64	32	64	32	S83L, D87N	S58I, R138D,	P385A
27	64	32	32	16	D87N	S58I, P140T,	P385A	S458A	Integrase1	dfrA17-aadA5	blaTEM
40	16	32	16	16		S58I,	P385A
61	16	32	64	16	S83L, D87N	S58I, N141K	P385A	S458A
62	16	16	32	16		R138V, N141K	P385A	S458A
69	8	32	32	16	S83L, D87N	S58I	P385A
73	16	64	32	16	S83L, D87N	S58I, R138D	P385A	S458A	Integrase1	dfrA1-aadA1	blaTEM
80	8	16	4	16		R138V	P385A
86	16	64	32	16	S83L, D87N	S58I, R138V	P385A		Integrase1	dfrA17-aadA5	blaTEM
96	16	32	8	16		S58I	P385A
98	16	32	8	16		R138V, N141K	P385A	S458A	Integrase1	dfr2d-catB3-aadA1	blaTEM
100	32	8	64	16	S83L, D87N	S58I, P140T, N141K	P385A
105	8	16	4	16		S58I

^aENR: enrofloxacin minimal inhibition concentration ≥8 resistance

^bNOR: norfloxacin minimal inhibition concentration ≥16 resistance

^cCIP: ciprofloxacin minimal inhibition concentration ≥4 resistance

^dLOM: lomefloxacin minimal inhibition concentration ≥8 resistance

^eNumber of amino acid substitution sites: S83I: substitution of serine to leucine at amino acid 83; D87N: aspartic acid to asparagine

^fNumber of amino acid substitution sites: S58I: serine to isoleucine; R138V: arginine to valine; R138D: arginine to aspartic acid; P140T: proline to threonine; N141K: aspartic acid to lysine

^gNumber of amino acid substitution sites: P385A: proline to alanine

^hNumber of amino acid substitution sites: S458A: serine to alanine

ⁱIntegraseI, integrase gene; aadA1: aminoglycoside-3′-adenylytransferase gene; dfrA17-aadA5, dihydrofolate reductase gene and aminoglycoside-3′-adenylytransferase gene; dfrA1-aadA1: dihydrofolate reductase gene and aminoglycoside-3′-adenylytransferase gene; dfrA1-catB2-aadA1: dihydrofolate reductase gene and chloramphenicol acetyltransferase gene and aminoglycoside-3′-adenylytransferase gene

^jbla_TEM: TEM-type ESBL gene; bla_CTX: CTX-type ESBL gene

Partridge SR, Tsafnat G, Coiera E, Iredell JR. Gene cassettes and cassette arrays in mobile resistance integrons. FEMS Microbiol Rev. 2009;33(4):757–784.

 PubMed Web of Science ®Google Scholar

Zhao H-X, Zhao J-L, Shen J-Z, et al. Prevalence and molecular characterization of fluoroquinolone resistance in Escherichia coli isolates from dairy cattle with endometritis in China. Microb Drug Resist. 2014;20(2):1–13.

 PubMed Web of Science ®Google Scholar

Park CH, Robicsek A, Jacoby GA, Sahm D, Hooper DC. Prevalence in the United States of AAC (6C aIb-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother. 2006;50(11):3953–3955.

 PubMed Web of Science ®Google Scholar

Richter SN, Frasson I, Bergo C, Manganelli R, Cavallaro A, Palù G. Characterisation of qnr plasmid-mediated quinolone resistance in Enterobacteriaceae from Italy: association of the qnrB19 allele with the integron element ISCR1 in Escherichia coli. Int J Antimicrob Agents. 2010;35(6):578–583.

 PubMed Web of Science ®Google Scholar

Data availability statement

Upon reasonable request, the corresponding author will provide all documents, data, and protocols that support the study’s findings. The links and accession numbers of the target genes are as follows: gyrA (https://www.ncbi.nlm.nih.gov/nuccore/X06373.1/;X06373), parC (https://www.ncbi.nlm.nih.gov/nuccore/M58408.1/;M58408), GyrB (https://www.ncbi.nlm.nih.gov/nuccore/AE000447.1/; AE000447), ParE (https://www.ncbi.nlm.nih.gov/nuccore/M58409.1/; M58409).