Bacterial strains and identification
A total of 200 non-repetitive E. coli strains isolated from the urine samples of urinary tract infection (UTI) patients admitted to the Affiliated Hospital of Wenzhou Medical University in Wenzhou, China in 2018 were collected. The isolates were identified by using 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 in accordance with a previous study; isolates with MICs ≥MIC90 (the concentration required to inhibit growth by 90% isolates; MIC90 = 0.5 µg/mL) were considered to be resistant [20]. E. coli ATCC 25922 was used as the quality control strain.
Antimicrobial susceptibility test
A total of 200 E. coli isolates were subjected to antimicrobial susceptibility testing for 10 clinical conventional antibiotics by the agar dilution method, such as ampicillin (AMP), ciprofloxacin (CIP), levofloxacin (LVX), cefepime (FEP), ceftazidime (CAZ), ertapenem (ETP), imipenem (IPM), gentamicin (GEN), nitrofurantoin (NIT), and tobramycin (TOB). Our results were interpreted by the latest guidelines from the Clinical and Laboratory Standards Institute (CLSI).
Detection of fabI mutation by PCR
Genome DNA of triclosan-resistant E. coli strains as well as randomly selected equal numbers of triclosan-susceptible strains were extracted by using the Biospin Bacterial Genomic DNA Extraction Kit (Bioflux, Tokyo, Japan) in accordance with the manufacturer’s instructions. Then, fabI was amplified by PCR with specific oligonucleotide primers, and the positive PCR products were directly sequenced by the Shanghai Genomics Institute Technology Co. Ltd [17]. Next, gene mutations were further analyzed according to the GenBank accession number NC000913.3 of the E. coli genome assembly used in the BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) comparisons [21]. Moreover, 14 known drug efflux pump encoding genes (ydcT, ydcU, ydcV, ydcS, cysP, cysU, marA, soxS, yhiv, acrB, acrD, acrF, mdfA, and norE) were also amplified with the RT-qPCR primers in order to ensure that the strain carried the gene for subsequent RT-qPCR experiments. The PCR and RT-qPCR primers used are listed in the Supplementary Table S1 (see Additional File 1).
Efflux pump inhibition test
To test the efflux pump activity of triclosan-resistant E. coli strains, efflux pump inhibitor CCCP was tested. The resistant strains were tested on agar plates without or with 10 μg/mL CCCP by the agar dilution method. Compared with for triclosan alone, the MICs value of triclosan combination with 10 μg/mL CCCP decreased to ≥4, which confirmed a positive inhibitory effect [22]. In addition, the concentration of 10 μg/mL was determined as the optimal sub-minimum inhibitory concentrations (sub-MICs) that could inhibit the overexpression of efflux pump without affecting the growth of bacteria using the agar dilution method.
Expression levels of efflux pumps by RT- qPCR
In addition to detecting the fabI expression, 14 efflux pump encoding genes were also examined by RT-qPCR, which included the ABC transporters system encoding the 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 an active efflux pump were also tested, while ATCC 25922 served as the control strain. The abovementioned strains were also inoculated in fresh Luria broth (LB) and allowed to grow to the logarithmic phase (OD600 = 0.6). The total cellular RNA of these cultures was extracted by using the Bacterial RNA Miniprep Kit (Biomiga, Shanghai, China) according to the manufacturer’s recommendation. Subsequently, the purified RNA was reverse transcribed into cDNA via the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA) and amplified by using the TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×) (Takara, Japan). In the PCR reaction, a global gene gapA and the housekeeping gene 16S rRNA were used as the corresponding internal controls, and the quantification of efflux pump genes was performed by the 2˗ΔΔCt method. An expression of ≥2 in comparison with that of the control strain ATCC 25922 indicated an upregulation, which is in accordance with a previous report [21]. Specific RT-qPCR primers are listed in the Supplementary Table S1 (see Additional file 1). All experiments were performed in at least 3 biological replicates, and the data were expressed as the mean ± SD (Supplementary Table S2; see Additional file 1).
Genotyping by MLST
All triclosan non-susceptible isolates were typed using the 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 the sequence types (STs) were evaluated in comparison with the allelic profiles to the MLST database [23].
Strain-typing PFGE
To confirm and analyze the clonal relatedness among the triclosan-resistant isolates, PFGE was performed in accordance with the PulseNet protocols published by the US Centers for Disease Control and Prevention (CDC) with some minor modifications. Briefly, the cell suspensions were treated with protease K and incubated with the XbaI restriction enzyme for at least 2 h at 37ºC to digest the DNA fragments. Then, PFGE was performed using the CHEF-MAPPER XA PFG system (Bio-Rad, USA) for 18 h. The detailed running condition were as follows: initial switch time value of 2.16 s and a final switch time of 54.17 s at a gradient of 6 V/cm at a 120° included angle [24]. Next, the electrophoretic banding patterns were visualized by the 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 at the cut-off line ≥85% was considered to analyze the genetic relatedness [25]. The standard Salmonella strain H9812 was considered as the positive control.
Serial passage experiment
In order to determine whether triclosan exposure in vitro increased the bacterial resistance, as previously described, serial passage experiment was conducted for triclosan-susceptible isolates DC8361, DC8363, DC8400, DC8413, and DC8510 [26]. Specifically, the isolates were cultivated on Macconkey agar plate and cultured overnight at 37ºC to obtain a single isogenic strain, which was then inoculated into 3-mL fresh LB broth with different concentrations of triclosan at 37ºC for overnight, and the ticlosan gradient concentrations were 0.0625, 0.125, 0.25, 1, 2, 4, 8, 16, 32, 64, and 128 µg/mL. Culture supernatants with bacterial growth in the highest triclosan concentrations were aspirated and continuously passaged in fresh triclosan gradients, and after only 12 days of triclosan exposure, triclosan-mutant strains with MICs ≥32 µg/mL were obtained.
Next, the stability of triclosan resistance was confirmed via continuous passage in vitro. Briefly, the triclosan-mutant strains were cultured in 3-mL fresh LB broth without triclosan at 37ºC for 24 h. Every 24 h, 30 µL of overnight culture supernatants were transferred to another 5-mL tube containing 2.97-mL of fresh LB broth without triclosan. After 12 days, the MICs of triclosan and antibiotics and the expression levels of efflux pump genes were tested in triplicate, respectively, using the same method described previously.