Bacterial strains and identification
A total of 200 uropathogenic non-repetitive E. coli strains were obtained from the patients with urinary tract infections from Affiliated Hospital of Wenzhou Medical University in 2018. All bacteria were identified by the Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS; bioMérieux, Lyons, France).
Antimicrobial susceptibility testing
Agar dilution method was used to detect the minimum inhibitory concentrations (MICs) of clinical conventional antibiotics, including ampicillin, ciprofloxacin, levofloxacin, cefepime, ceftazidime, ertapenem, imipenem, gentamycin, nitrofurantoin and tobramycin, and the results were interpreted in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) 2019 [25]. Furthermore, antimicrobial susceptibility testing of triclosan was performed by agar dilution method and according to previously study [19], isolates with an MICs value of > MIC90 (Minimum drug concentrations at which 90% of strains were inhibited; MIC90 = 0.5 µg/ml) were classified as triclosan-resistant. E. coli ATCC 25922 served as the quality control strain for susceptibility testing.
Serial passage experiments
In order to determine whether there was cross-resistance between triclosan and other antibiotics in E. coli, serial passage experiments were conducted in vitro for triclosan-susceptible isolates DC8361, DC8363, DC8400, DC8413 and DC8510 as previously described [26]. Specifically, the isolates were cultivated on Maconkey ager plate and cultured overnight at 37 ºC to obtain a single isogenic strain, which was then inoculated into 3ml fresh LB broth with different concentrations of triclosan at 37 ºC overnight, the ticlosan gradient concentrations were 0.0625, 0.125, 0.25, 1, 2, 4, 8, 16 and 32 µg/ml. Culture supernatants with bacteria growing in the highest triclosan concentrations were aspirated and continuously passaged in new triclosan gradients, after only four days of triclosan exposure, triclosan-mutant strains with MICs of not less than 0.5 µg/ml were obtained.
Mutant Stability testing
The stability of triclosan resistance was confirmed by continuous passage in vitrofor triclosan-mutant strains. Briefly, the triclosan-mutant strains were cultured in 3 ml fresh LB broth without triclosan at 37 ºC for 24 hours. Every 24 hours, 30 µl of overnight culture supernatants were transferred to another 5 ml tube containing 2.97 ml fresh LB broth without triclosan. After six cycles, the MICs of triclosan as well as ten antibiotics were tested in triplicate respectively using the same method described previously.
Efflux pump inhibition testing
To test efflux pump activity of triclosan-resistant E. coli, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was trialed. The resistant strains were tested on agar plates with the presence or absence of efflux pump inhibitor CCCP by the agar dilution method as described [25]. Test concentrations of CCCP were ensured with consideration to determine the optimal subminimal inhibitory concentrations (sub-MICs) that could inhibit the overexpression of efflux pump without affecting the growth of bacteria. Compared with triclosan alone, MICs value of triclosan decreased by four times or more was confirmed having an inhibitory effect when triclosan was used in combination with the efflux pump inhibitors (CCCP, 10 μg/ml) [27].
Polymerase chain reaction (PCR) detection of mutations in fabI gene
Genome DNA of triclosan-resistant E. coli strains,as well as randomly selected equal numbers of triclosan-susceptible strains, were extracted using the Biospin Bacterial Genomic DNA Extraction kit (Bioflux, Tokyo, Japan) according to the manufacturer's instructions. Then, fabI gene and 14 known drug efflux pump encoding genes (ydcT, ydcU, ydcV, ydcS, cysP, cysU, marA, soxS, yhiv, acrB, acrD, acrF,mdfA and norE) were amplified by PCR with the specific oligonucleotide primers, positive PCR products were directly sequenced by Shanghai Genomics Institute Technology Co. Ltd [15]. Genetic mutations were further analyzed by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi; GenBank accession number: NC000913.3). Primer of fabI gene was listed in supplementary materials, Tables 3.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Analysis of the transcriptional levels was undertook using qRT-PCR on six different ABC transporters system encoding genes ydcT, ydcU, ydcV, ydcS, cysP and cysU; two Arac-regulator genes marA and soxS; four RND efflux pump encoding genes of the yhiv and acrBDF; the fabI gene which encoding for the enacyl carrier protein reductase during the bacterial fatty acid; the MdfA efflux Tolc, mdfA; and the NorE efflux pump encoding gene of the norE. Briefly, triclosan-resistant strains with overexpressing of the efflux pump activity were included, ATCC 25922 served as the control strain. The strains above were inoculated in fresh Luria broth (LB) and allowed to grow to logarithmic phase (OD600 = 0.6). The total cellular RNA of these cultures was extracted using the Bacterial RNA Miniprep Kit (Biomiga, Shanghai, China) according to the manufacturer's recommendation. Subsequently, the purified RNA was subjected to reversely transcribed into cDNA by means of the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA), amplification was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×) (Takara, Japan). In the PCR reaction, a global gene gapA and housekeeping gene 16S rRNA were used as corresponding internal control to calculate for quantification of transcriptional expression level of the efflux pump genes by the 2˗ΔΔCt method. According to Fatma Ibrahim Sonbol et al., the increase of 2-fold or greater indicated overexpression compared to the control strain ATCC 25922 [21]. Specific qRT-PCR primers were listed in supplementary materials, Tables 3. All experiments were performed in triplicates and the datas were displayed as the mean ± SD values (listed in supplementary materials, Tables 2).
Genotyping by Multilocus sequence typing (MLST)
All the triclosan non-susceptible isolates were typed by using MLST method. In short, the sequences of eight housekeeping gene (trpB, uidA, dinB, icdA, pabB, polB, put and trpA) of E. coli were amplified with specific primers available at the MLST database (https://bigsdb.pasteur.fr/index.html), and sequence types (STs) were evaluated by comparing the allelic profiles to the MLST database.
Strain typing by pulse field gel electrophoresis (PFGE)
To confirm and analyze the clonal relatedness of the triclosan-resistant isolates, PFGE was also used for analysis the clonal relatedness of the triclosan-resistant isolates according to the PulseNet protocols published by the US Centers for Disease Control and Prevention (CDC) with minor modifications. The cell suspensions treated with protease K were incubated with XbaI restriction enzyme at least for 2 hours at 37 ºC to digest the DNA fragments. Then PFGE was performed using a CHEF-MAPPER XA PFG system (Bio-Rad, USA) for 18 hours. The detailed running condition were as follows: initial switch time value of 2.16 sec, final switch time of 54.17 sec at a gradient of 6 V/cm at a 120° included angle [28]. Next, the electrophoretic banding patterns were visualized by GelDoc XR gel imaging system (Bio-Rad, USA) and further analyzed by Quantity One (Bio-Rad Laboratories, USA). The Unweighted Pair Group Method with Arithmatic Mean (UPGMA) with optimization set at 1.5% to create the dendrogram, cut off line at 85% was considered to analyze genetic relatedness [29]. Salmonella standard strain H9812 was taken as the positive control.