Analysis of antimicrobial resistance and genetic correlations of Escherichia coli in dairy cow mastitis

Li, Ke; Hou, Mingyuan; Zhang, Lin; Tian, Mengyue; Yang, Ming; Jia, Li; Liang, Yanyan; Zou, Dongmin; Liu, Ruonan; Ma, Yuzhong

doi:10.2478/jvetres-2022-0055

Figures & Tables

Full minimum spanning tree using the goeBURST algorithm (n = 40). Each square represents a single sequence type (ST), and the circumference is proportional to the number of isolates within each ST. Grey regions represent a clonal complex. The numbers above the lines (1–5) represent the number of different alleles between the two ST types. The major nodes are indicated by in olive green

Molecular phylogenetic and antimicrobial resistance analysis of 40 E. coli isolates. The evolutionary tree was inferred using the maximum likelihood method based on the Tamura–Nei model. The bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analysed. The branches of the evolutionary tree were named with ID, location, and ST type of E. coli isolates. The same clonal complexes were highlighted in the same colour area. The height of the blue bar graph on the periphery of the evolutionary tree represents the number of drug-resistant genes (2, 3, 4, 5, 6) carried by E. coli isolates. The diameter of different antibiotic inhibition zones (6–34 mm) was displayed as a heat map where red represents high-resistance diameters (trending to susceptible) and blue represents low-resistance diameters (trending to resistant)

Pearson’s correlation coefficients (r) of resistance genes and corresponding antibiotics

Resistance gene	Antimicrobials
Resistance gene	AMP	AMX	CRO	CFZ	GEN	STR	NER	AMI	EM	DOX	SXT	CIP	ENR
blaSHV	0.231	0.317	0.333	0.053	-	-	-	-	-	-	-	-	-
blaOXA	−0.114	0.248	0.124	−0.059	-	-	-	-	-	-	-	-	-
aac(2ʹ)	-	-	-	-	−0.27 3	−0.12 5	−0.083	0.035	-	-	-	-	-
aacA4	-	-	-	-	0.331	0.035	0.281	−0.053	-	-	-	-	-
aadA	-	-	-	-	0.427	0.353	0.105	−0.059	-	-	-	-	-
erm(B)	-	-	-	-	-	-	-	-	−0.04 7	-	-	-	-
tet(A)	-	-	-	-	-	-	-	-	-	−0.156	-	-	-
tet(B)	-	-	-	-	-	-	-	-	-	−0.347	-	-	-
sul1	-	-	-	-	-	-	-	-	-	-	- 0.174	-	-
sul2	-	-	-	-	-	-	-	-	-	-	- 0.343	-	-
qnrB	-	-	-	-	-	-	-	-	-	-	-	−0.106	0.035

Susceptibility of 40 E_ coli strains to 13 antibiotics commonly used in China

Antibiotic	Distribution of E. coli strains (number of strains/%)			Decision criteria/Diameter of inhibitory zone (mm)
	R¹	I	S	R	I	S
Ampicillin	21/52.5%	6/15.0%	13/32.5%	≤13	14–16	≥17
Amoxicillin	18/45.0%	3/7.5%	19/47.5%	≤13	14–17	≥18
Ceftriaxone	19/47.5%	1/2.5%	20/50.0%	≤19	20–23	≥24
Cefazolin	31/77.5%	8/20.0%	1/2.5%	≤19	20–22	≥23
Gentamicin	13/32.5%	0	27/67.5%	≤12	13–14	≥15
Streptomycin	12/30.0%	5/12.5%	23/57.5%	≤11	12–14	≥15
Neomycin	1/2.5%	15/37.5%	24/60.0%	≤11	12–16	≥17
Amikacin	1/2.5%	1/2.5%	38/95.0%	≤14	15–16	≥17
Erythromycin	17/42.5%	21/52.5%	2/5.0%	≤13	14–22	≥23
Doxycycline	14/35.0%	5/12.5%	21/52.5%	≤10	11–13	≥14
Trimethoprim- sulfamethoxazole	22/55.0%	0	18/45.0%	≤12	13–16	≥17
Ciprofloxacin	7/17.5%	0	33/82.5%	≤15	16–20	≥21
Enrofloxacin	6/5.0%	11/27.5%	23/57.5%	≤15	16–23	≥24

Primer sequences, product sizes, annealing temperature and references used for the PCR in the study

Gene	Primer sequence (5′–3′)	Product size (bp)	Annealing temperature (℃)	Reference or GenBank accession no.
blaTEM	ATAAAATTCTTGAAGACGAAA GACAGTTACCAATGCTTAATC	643	53	(25)
blaSHV	TTTGTCGCTTCTTTACTCGCCTTTA GCCAGATCCATTTCTATCATGCCTA	198	56	DQ247972
blaOXA	TCAACTTTCAAGATCGCA GTGTGTTTAGAATGGTGA	591	53	(25)
aac(2')	ACTGTGATGGGATACGCGTC CTCCGTCAGCGTTTCAGCTA	482	54	(26)
aacA4	CTTCAGGATGGCAAGTTGGT TCATCTCGTTCTCCGCTCAT	286	55	(26)
aadA	CTGGAGGTCACTGTCGTGC CCGTGGATTGCCAAAGGTC	274	55	X68089
erm(B)	AAAACTTACCCGCCATACCA TTTGGCGTGTTTCATTGCTT	126	53	MN461246
erm(C)	GCTCGTGTCATTTCTGGGAGT AGCCTAGCAGCCATTTCTATC	375	53	GQ483470
tet(A)	CGGAGCAGAAACAAGAAAGCG GGATCAGGACCGGATACACCAT	345	57	(26)
tet(B)	CATTAATAGGCGCATCGCTG TGAAGGTCATCGATAGCAGG	391	53	(26)
sul1	GCCTGGAACTGCTGCTGATGC TCGCCTGCCAAACCGAACTCT	314	59	(27)
sul2	GCGCTCAAGGCAGATGGCATT GCGTTTGATACCGGCACCCGT	793	57	(27)
qnrB	GATCGTGAAAGCCAGAAAGG ACGATGCCTGGTAGTTGTCC	513	55	(25)
adk	ATTCTGCTTGGCGCTCCGGG CCGTCAACTTTCGCGTATTT	583	54	(20)
fumC	TCACAGGTCGCCAGCGCTTC GTACGCAGCGAAAAAGATTC	806	54	(20)
gyrB	TCGGCGACACGGATGACGGC ATCAGGCCTTCACGCGCATC	911	60	(20)
icd	ATGGAAAGTAAAGTAGTTGTTCCGGCACA GGACGCAGCAGGATCTGTT	878	54	(20)
mdh	ATGAAAGTCGCAGTCCTCGGCGCTGCTGGCGG TTAACGAACTCCTGCCCCAGAGCGATATCTTTCTT	932	60	(20)
purA	CGCGCTGATGAAAGAGATGA CATACGGTAAGCCACGCAGA	816	54	(20)
recA	CGCATTCGCTTTACCCTGACC TCGTCGAAATCTACGGACCGGA	780	58	(20)

Sequence types, resistance phenotypes and resistance genes in 40 E_ coli strains

ID	Location	MLST	Resistance phenotypes	Resistance genes
1	Xushui	ST10	AMP-AMX-CRO-CFZ-GEN-STR-EM-DOX-SXT-ENR	aadA, qnrB
2	Xushui	ST10	AMP-AMX-CRO-CFZ-GEN-STR-NER-EM-DOX-SXT-CIP-ENR	aadA, tet(B)
3	Xushui	ST359	AMP-AMX-CRO-CFZ-GEN-STR-NER-EM-DOX-SXT-CIP-ENR	blaSHV, aadA
4	Xushui	ST10	CFZ-EM	aadA, tet(B), sul2
5	Xushui	ST10	AMP-AMX-CRO-CFZ-STR-NER-EM-DOX-SXT-CIP-ENR	aadA, sul2
6	Xushui	ST1585	AMP-AMX-CRO-CFZ-GEN-STR-NER-EM-DOX-SXT-CIP-ENR	aacA4, aadA
7	Xushui	ST359	AMP-AMX-CRO-CFZ-GEN-STR-NER-AMI-EM-DOX-SXT-CIP-ENR	blaSHV, blaOXA, aadA
8	Xushui	ST359	AMP-AMX-CRO-CFZ-GEN-NER-EM-DOX-SXT-ENR	blaOXA, aacA4, aadA
9	Xushui	ST359	AMP-AMX-CRO-CFZ-GEN-STR-EM-DOX-SXT-CIP-ENR	blaSHV, blaOXA, aadA, tet(B)
10	Xushui	ST10	CFZ-EM	aadA, sul2
11	Xushui	ST10	CFZ-EM	aadA, tet(B), sul2
12	Xushui	ST359	AMP-AMX-CRO-CFZ-GEN-NER-EM-SXT-ENR	blaOXA, aadA, sul2
13	Xushui	ST1125	AMP-AMX-CRO-CFZ-STR-SXT	aadA, tet(B)
14	Xushui	ST1585	AMP-AMX-CRO-CFZ-EM-DOX-SXT-ENR	blaSHV, aadA, tet(B)
15	Xushui	ST327	AMP-AMX-CRO-CFZ-EM-DOX-ENR	aadA, tet(B)
16	Xushui	ST937	AMP-AMX-CRO-CFZ-STR-EM-SXT-ENR	aadA, tet(B)
17	Qingyuan	ST10717	AMP-CFZ-STR-NER-EM-DOX-SXT	aac(2ʹ), aadA, erm(B), tet(A), tet(B)
18	Qingyuan	ST942	CFZ-NER-EM-DOX	erm(B), tet(A), sul1, sul2
19	Qingyuan	ST446	AMP-CFZ-GEN-STR-NER-EM-DOX-SXT	aac(2ʹ), aadA, erm(B), tet(A), tet(B)
20	Qingyuan	ST1310	AMP-AMX-CRO-CFZ-GEN-STR-NER-AMI-EM	aac(2ʹ), erm(B), tet(A), tet(B), sul2
21	Qingyuan	ST515	AMP-AMX-CRO-CFZ-STR-EM-DOX-SXT-ENR	aac(2ʹ), erm(B), tet(A), sul1
22	Qingyuan	ST48	AMP-CFZ-NER-EM	aac(2ʹ), erm(B), tet(A), tet(B), sul1
23	Qingyuan	ST10	CFZ-NER-EM	aac(2ʹ), tet(A), sul2
24	Quyang	ST1252	AMP-AMX-CRO-CFZ-STR-EM-SXT-ENR	aac(2ʹ), erm(B), tet(A), tet(B)
25	Quyang	ST1079	AMP-CFZ-EM	tet(A), tet(B)
26	Quyang	ST154	CFZ	aac(2ʹ), erm(B), tet(A), tet(B), sul2
27	Quyang	ST1585	AMP-AMX-CRO-CFZ-GEN-STR-NER-EM-DOX-SXT-CIP-ENR	aadA, erm(B)
28	Quyang	ST1167	AMP-AMX-CRO-CFZ-STR-NER-EM-DOX-SXT	aadA, erm(B), tet(A)
29	Mancheng	ST1610	AMP-CFZ-EM	aac(2ʹ), tet(A)
30	Mancheng	ST10	NER-EM	aac(2ʹ), tet(A), sul1, sul2
31	Mancheng	ST2741	CFZ-EM	aac(2ʹ), aadA, tet(A), tet(B)
32	Mancheng	ST2741	CFZ-EM	aac(2ʹ), tet(A), tet(B)
33	Mancheng	ST48	AMP-CFZ-GEN-STR-EM-DOX-SXT-ENR	aac(2ʹ), aadA, erm(B), tet(A), tet(B), sul1
34	Mancheng	ST10	AMP-AMX-CRO-CFZ-EM-DOX-SXT-ENR	aac(2ʹ), erm(B), tet(A), tet(B), sul1, sul2
35	Mancheng	ST906	AMP-AMX-CRO-CFZ-EM-SXT	aac(2ʹ), tet(A), tet(B)
36	Mancheng	ST48	CFZ-EM	aac(2ʹ), aadA, erm(B), tet(B), sul1, qnrB
37	Mancheng	ST48	CFZ-EM	erm(B), tet(B), sul1
38	Mancheng	ST48	CFZ-EM	aadA, erm(B), tet(B), sul1
39	Mancheng	ST906	CFZ-EM	aac(2ʹ), tet(B)
40	Mancheng	ST48	AMP-AMX-CFZ-GEN-EM-DOX-SXT	aadA, erm(B), sul1

Analysis of antimicrobial resistance and genetic correlations of Escherichia coli in dairy cow mastitis

Figures & Tables

Fig. 1

Fig. 2

Pearson’s correlation coefficients (r) of resistance genes and corresponding antibiotics

Susceptibility of 40 E_ coli strains to 13 antibiotics commonly used in China

Primer sequences, product sizes, annealing temperature and references used for the PCR in the study

Sequence types, resistance phenotypes and resistance genes in 40 E_ coli strains

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