Open Access

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

Ke Li
Ke Li
, 
Mingyuan Hou
Mingyuan Hou
, 
Lin Zhang
Lin Zhang
, 
Mengyue Tian
Mengyue Tian
, 
Ming Yang
Ming Yang
, 
Li Jia
Li Jia
, 
Yanyan Liang
Yanyan Liang
, 
Dongmin Zou
Dongmin Zou
, 
Ruonan Liu
Ruonan Liu
 and 
Yuzhong Ma
Yuzhong Ma
   | Nov 04, 2022
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Published Online: Nov 04, 2022
Page range: 571 - 579
Received: Mar 23, 2022
Accepted: Sep 28, 2022
DOI: https://doi.org/10.2478/jvetres-2022-0055
Keywords
© 2022 K. Li et al. published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

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
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

Fig. 2

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)
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
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
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