Isolation and identification of sul3 positive Escherichia coli
142 strains of Escherichia coli were identified from 150 samples of bacteria collected from farms and pet hospital in Nanning from 2015 to 2017. All the strains were identified by MaConkey’s agar and Eosin methylene blue agar, and sequence detection with primers of 16 s rRNA and sul3 gene.
Each sample was cultured at 37℃ for 16–18 h in McConkey medium and 18–24 h in Eosin methylene blue agar. DNA was extracted by boiling method. The primer of 16 s rRNA and sul3 is reference the previous description (Table 1). The PCR product were sent to the company for sequencing, and uploaded to NCBI for BLST confirmation of suspected isolates and sul3 carrier. The 30% glycerol sample (V/V) and DNA sample of sul3 positive E. coli were stored at -20 ℃.
Mlst Typing Detection
A total of 46 strains of sul3 positive E. coli were detected, PCR amplification was conducted using 7 pairs of primers (adk, fumC, gyrB, icd, mdh, purA and recA) (Table 6). The positive products were sent to the company for sequence determination, and the results were uploaded to the MLST website (https://pubmlst.org) to obtain the ST type. (NOTE: Table 6 is longer than a page of A4, so we put it at the end of this document. You can put Table 6 at the end of this paragraph)
Table 6
Primers used in this study
| Gene | Primer sequence (5’→3’) | Product size (bp) | Annealing temp (℃) | References |
| 16Sr RNA | F: AGAGTTTGATCCTGGCTCAG | 1466 | 55 | [38] |
| R: ACGGCTACCTTGTTACGACTT |
| TEM | F: AGGAAGAGTATGATTCAACA | 511 | 52.5 | [38] |
| R: CTCGTCGTTTGGTATGGC |
| SHV | F: GGTTATGCGTTATATTCGCCTGTG | 56.5 | 1031 | [38] |
| R: TTAGCGTTGCCAGTGCTCGATCA |
| CTX-M1 | F: GGTTAAAAAATCACTGCGTC | 864 | 56 | [39] |
| R: TTGGTGACGATTTTAGCCGC |
| CTX-M9 | F: ATGGTGACAAAGAGAGTGCA | 870 | 50 | [40] |
| R: CCCTTCGGCGATGATTCTC |
| CTX-MU | F: ATGTGCAGTACCAGTAAAGT | 593 | 56 | [41] |
| R: TGGGTRAAGTARGTCACCAGAA |
| OXA-1 | F: TTGAAGGAACTGAAGGTTGT | 651 | 54 | [35] |
| R: CCAAGTTTCCTGTAAGTGCG |
| armA | F: AGGTTGTTTCCATTTCTGAG | 591 | 55 | [42] |
| R: TCTCTTCCATTCCCTTCTCC |
| rmtA | F: CTAGCGTCCATCCTTTCCTC | 635 | 60 | [43] |
| R: TTTGCTTCCATGCCCTTGCC |
| rmtB | F: ATCAACGATGCCCTCACCTCC | 631 | 61 | [42] |
| R: TTCCACGCCCGCCTAAACT |
| aac(6’)-Ib | F: CAAGAGTCCGTCACTCCATACA | 396 | 61 | [44] |
| R: ATGGAAGGGTTAGGCATCACT |
| aac(3’)-II | F: ACTGTGATGGGATACGCGTC | 237 | 60 | [45] |
| R: CTCCGTCAGCGTTTCAGCTA |
| tetA | F: GCTACATCCTGCTTGCCTTC | 210 | 60 | [46] |
| R: CATAGATCGCCGTGAAGAGG |
| tetB | F: TTGGTTAGGGGCAAGTTTTG | 659 | 65 | [46] |
| R: GTAATGGGCCAATAACACCG |
| tetM | F: GTGGACAAAGGTACAACGAG | 406 | 55 | [46] |
| R: CGGTAAAGTTCGTCACACAC |
| qnrA | F: CAAGAGGATTTCTCACGCCAG | 628 | 67 | [35] |
| R: AATCCGGCAGCACTATTACTCC |
| qnrB | F: ATGACGCCATTACTGTATAA | 562 | 57 | [35] |
| R: GATCGCAATGTGTGAAGTTT |
| flor | F: GTCATTCCTCACCTTCATCCTAC | 243 | 60 | [47] |
| R: GACACCAGCACTGCCATTG |
| mcr-1 | F: ATGATGCAGCATACTTCTGTG | 1626 | 65 | [48] |
| R: TCAGCGGATGAATGCGGTG |
| oqxA | F: GATCAGTCAGTGGGATAGTTT | 670 | 56 | [49] |
| R: TACTCGGCGTTAACTGATTA |
| oqxB | F: TTCTCCCCCGGCGGGAAGTAC | 512 | 68 | [49] |
| R: CTCGGCCATTTTGGCGCGTA |
| sul1 | F: GGCTGGTGGTTATGCACTCA | 263 | 64 | [50] |
| R: CGAGACCAATAGCGGAAGC |
| sul2 | F: ACGCAAGCCTATGCCTTGTCG | 234 | 62 | [50] |
| R: TTGCGTTTGATACCGGCACCC |
| sul3 | F: CGTAAATATAACCACCGAT | 326 | 55 | [50] |
| R: CCAAGCCTGAATAAATCTCA |
| fosA3 | F: GCGTCAAGCCTGGCATTTT | 258 | 55 | [38] |
| R: GCCGTCAGGGTCGAGAAA |
| ERIC-2 | AAGTAAGTGACTGGGGTGACGC | Variable | 50 | [51] |
| Adk | F: CTCGCCATTAACCGTTTCAG | 739 | 55 | [49] |
| R: CCAGATCAGCGCGAACTTCA |
| FumC | F: TCACAGGTCGCCAGCGCTTC | 769 | 64 | [49] |
| R: TCCCGGCAGATAAGCTGTGG |
| gyrB | F: ATCGGCGACACGGATGAC | 816 | 66 | [49] |
| R: GTCCATGTAGGCGTTCAGG |
| lcd | F: CCGGCACAAGGCAAGAAGATC | 857 | 59.5 | [49] |
| R: GGACGCAGCAGGATCTGTT |
| mdh | F: GCCTTCAGGTTCAGAACTCTCTCT | 798 | 55 | [49] |
| R: TTCTGTTCAAATGCGCTCAGG |
| PurA | F: CGCGCTGATGAAAGAGATGA | 817 | 66 | [49] |
| R: CATACGGTAAGCCACGCAGA |
| recA | F: CGCATTCGCTTTACCCTGACC | 731 | 55 | [49] |
| R: GTCGAAATCTACGGACCGGAAT |
Antibiotic Sensitivity Experimentt
The minimum inhibitory concentration (MIC) of antimicrobial agents against sul3 positive E. coli was used by the broth dilution method recommended which was recommended by Clinical and Laboratory Standards Institut (CLSI). The tested antimicrobial agents included penicillin, ceftazidime, ceftriaxone, meropenem, amikacin, streptomycin, tetracycline, ciprofloxacin, gatifloxacin, chloramphenicol, fosfomycin and colistin. The results of antibiotic sensitivity were also judged according to the break-point standard established by CLSI. The E. coli of ATCC 25922 was used for the quality control of antibiotic sensitivity test.
Antimicrobial Gene Detection
There were 24 antimicrobial genes, including the β-lactam (TEM, CTX-M9, CTX-MU and OXA-1), aminoglycosides (armA, rmtA, rmtB, aac(6')–Ib and aac(3')-II), tetracyclines ( tetA, tetB and tetM), quinolones (qnrA and qnrB ), sulfonamides (Sul1 and sul2), and other classes ( flor, mcr-1, oqxA, oqxB, and fosA3 ) (Table 6).
Conjugative Experiment
The conjugative experiment was conducted by filter membrane method. the Escherichia coli C600, which did not produce acid and has rifampicin resistance, was used as the recipient bacteria. And sul3-positive isolates were used as the donor bacteria. The transconjugants were screened from McConkey medium with a concentration of 6000 µg/mL sulfamethazine and 3500 µg/mL rifampicin.
The suspected transconjugants were subjected to PCR and antibiotics sensitivity tests to confirm whether the plasmid transfer was successful, and then ERIC-PCR was used to determine the correlation between the transconjugants and C600, with the ERIC-primers as described previously[35] (Table 1). It also detects whether other resistant genes are co-transmitted.
Growth Curve
We used absorbance method to observe the change of the growth status of transconjugants and C600, specific as follows. After shaking culture at 37℃ overnight, the bacterial solution was added to fresh LB broth according to the ratio of 1:1000. A total of 16 time points, 3 ml was taken from each time point for OD600 absorbance detection. The observation lasted for 24 hours and need repeated 3 times in parallel.
In Vitro Competitive Test
In vitro competition experiments refer to previous descriptions[36]. First, two kinds of bacteria were cultured to 0.5 McFarland, then took 100 µL for each mixed in a 1:1 ratio and added to 10 mL LB broth, and incubated for 16 h at 37℃, 220 r/min. After diluted 106 times, 100 µL bacterial solution was respectively coated with streptomycin 60 µg/mL LB agar and streptomycin free LB agar, and cultured overnight at 37℃. The total CFU and streptomycin resistant CFU were counted, and the competition index of without resistant CFU and streptomycin resistant CFU was calculated, and the parallel repetition was 3 times.
Plasmid Stability
According to the previous description of plasmid stability[37], the transconjugants was shaken in LB medium at 37℃ for 12 h, and then inoculated in new LB medium and shaken at 37℃ for 12 h again, repeat every 12 h. Each time was counted as one generation, and the procedure was repeated for 60 generations. Every 10 generations, part of the bacterial solution was diluted and coated with agar medium, 24 colonies of bacteria were randomly selected. DNA was extracted by boiling method. Then the PCR of sul3 was performed to determine the positive rate of sul3.