Table 1
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) |
Table 2
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 |
Table 3
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 |
Table 4
Pearson’s correlation coefficients (r) of resistance genes and corresponding antibiotics
| 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 |
[i] AMP – ampicillin; AMX – amoxicillin; CRO – ceftriaxone; CFZ– cefazolin; GEN– gentamicin; STR – streptomycin; NER – neomycin;
AMI – amikacin; EM – erythromycin; DOX – doxycycline; SXT – trimethoprim-sulfamethoxazole; CIP – ciprofloxacin; ENR – enrofloxacin Pearson’s correlation coefficients (r) shown in bold are significant at P < 0.05
A positive r indicates a positive association between the two variables, whereas a negative r indicates a negative association
- represents antimicrobials without corresponding antibiotic resistance genes
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
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)