Bacterial strains and identification
A total of 200 non-repetitive E. coli strains from urinary tract infection (UTI) patient urine were obtained from Affiliated Hospital of Wenzhou Medical University in Wenzhou, China in 2018. All bacteria were identified by the Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS; bioMérieux, Lyons, France).
Minimum Inhibitory Concentrations Of Triclosan
We measured the MICs of triclosan according to previously study, isolates with MICs ≥ MIC90 (the concentration required to inhibit growth by 90% isolates; MIC90 = 0.5 µg/ml) were classified as resistance [20]. E. coli ATCC 25922 served as the quality control strain.
Antimicrobial Susceptibility Test
Antimicrobial susceptibility test was also performed for 10 clinical conventional antibiotics by agar dilution method, and the results were interpreted by the latest guidelines from the Clinical and Laboratory Standards Institute (CLSI).
Detection of fabI gene mutation by PCR
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 [17]. 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 Table S1 (see Additional file 1).
Efflux Pump Inhibition Test
To test efflux pump activity of triclosan resistant E. coli strains, efflux pump inhibitor CCCP was trialed. The resistant strains were tested on agar plates with the presence or absence of 10 µg/ml CCCP by the agar dilution method. Compared with triclosan alone, MICs value of triclosan decreased ≥ 4 was confirmed having an inhibitory effect when triclosan was used in combination with 10 µg/ml CCCP [21]. In addition, the 10 µg/ml concentration was determined as the ideal concentration using the agar dilution method.
Expression Levels Of Efflux Pumps By Rt- Qpcr
Except detecting fabI expression, 14 efflux pump encoding genes were also checked using RT-qPCR, including the ABC transporters system encoding genes ydcT, ydcU, ydcV, ydcS, cysP and cysU, the Arac-regulator genes marA and soxS, the RND efflux pump encoding genes yhiv and acrBDF; the MdfA efflux Tolc encoding genes mdfA; and the NorE efflux pump encoding gene norE.
Briefly, triclosan resistant strains with active efflux pump 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, the quantification of efflux pump genes was performed by the 2˗ΔΔCt method. Compared to the control strain ATCC 25922, an expression ≥ 2 indicated up-regulation according to previous study [22]. Specific RT-qPCR primers were listed in Supplementary Table S1 (see Additional file 1). All experiments were performed in triplicates and the data was displayed as the mean ± SD values in Supplementary Table S2 (see Additional file 1).
Genotyping By Mlst
All the triclosan non-susceptible isolates were typed using MLST method. The sequences of 8 housekeeping genes (trpB, uidA, dinB, icdA, pabB, polB, put and trpA) 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 Pfge
To confirm and analyze the clonal relatedness of 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 [23]. 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 ≥ 85% was considered to analyze genetic relatedness [24]. Salmonella standard strain H9812 was taken as the positive control.
Serial Passage Experiment
In order to determine whether triclosan exposure in vitro increased bacterial resistance, as previously described, serial passage experiment was conducted for triclosan susceptible isolates DC8361, DC8363, DC8400, DC8413 and DC8510[25]. 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 3 ml 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 ≥ 0.5 µg/ml were obtained.
Next, the stability of triclosan resistance was confirmed by continuous passage in vitro. 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.