Source of strains
S. aureus reference strain USA300 was a gift from Prof. Lan of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. All 26 strains of MRSA were isolated from inpatient specimens of Shanghai Eighth People’s hospital from October 2016 to March 2017. All strains were identified using the VITEK 2 compact microbiology analysis system (BioMérieux Industry, France). In addition, we confirmed the strains were MRSA by PCR for detection of the mecA gene. (mecA primer: mecA-R:GTAGAAATGACTGAACGTCCGATAA, mecA-F:TTACAGAGTTAACTGTTACC, with a size of 310 bp) The 26 isolated strains of MRSA were numbered as MRSA01~MRSA26.
BBR and antibiotics
BBR was purchased from Tianzheng Pharmaceutical Co., LTD (Northeast pharmaceutical group, batch number: 0361611030) and was prepared with dimethyl sulfoxide (DMSO) to 32 mg/mL solution. After filtration and sterilization with 0.22 um filter membrane, BBR solution was separated into sterilized EP tubes and stored at 4℃ for reserve. GEN (Xinchen Pharmaceutical Co., LTD, batch number: 1607252211), AMI (Shanghai Xinyijinzhu Pharmaceutical Co., LTD, batch number: 1611107), and LEV (Yangtze River Pharmaceutical Co., LTD, batch number: 16080231) were all purchased from the pharmacy of Shanghai Eighth People’s hospital. These antibiotics were freshly prepared with sterile water to a concentration of 1024 µg/mL.
Multilocus sequence typing (MLST)
Isolates were screened using a previously described method [16] to detect the following seven housekeeping genes: carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glp), guanylate kinase (gmk),phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), and acetyl coenzyme A acetyltransferase(yqiL). The sequences of the PCR products were compared with the existing sequences available from the MLST website (http://www.mlst.net) for S. aureus [17],and the allelic number was determined for each sequence.
Susceptibility testing of the BBR and antimicrobial agents
According to the 2018 standard of the Clinical and Laboratory Standards Institute (CLSI)[18], the MIC of GEN, LEV, AMI and BBR on MRSA was determined by broth microdilution. GEN, LEV, AMI, and BBR solutions were serially diluted to a final concentration of 8, 16, 32, 64, 128, 256, 512 and 1024 μg/mL with M-H broth. Different concentration of antibiotics or BBR solution was added to a 96-well plate. The bacterial suspension was prepared with 0.5 McFarland standard, then diluted to 1:1000 with M-H broth. A 50 μL bacterial suspension was added to each well of the 96-well plate and incubated in a Heal Force CO2 incubator (Likang, Shanghai, China) at 37oC for 24 h. The minimum drug concentration without bacterial growth was MIC.
Synergy Testing
5 strains (MRSA01~MRSA05) of MRSA resistant to GEN, 17 strains (MRSA01~MRSA04, MRSA06~MRSA18) resistant to LEV and 6 strains (MRSA02, MRSA04, MRSA07~MRSA10) resistant to AMI were selected for the combined bacteriostatic test. BBR solution was diluted to 4 dilutions (32, 64, 128, and 256 μg/ml) with M-H broth. GEN, LEV and AMI were diluted with M-H broth to 8 increasing concentrations (8, 16, 32, 64, 128, 256, 512, and 1024 μg/ml). Bacterial suspensions were prepared with 0.5 McFarland standard, then diluted to 1:1000 with M-H broth. 50 μl of BBR solution (32, 64, 128, and 256 μg/ml) and GEN, LEV, AMI solution (8, 16, 32, 64, 128, 256, 512, and 1024 μg/ml) were arranged in a 96-well plate, and 100 μl bacterial suspension was added to the sterile microporous plate.The final concentrations of BBR were 8, 16, 32, and 64 μg/mL, respectively. The final concentrations of GEN, LEV and AMI were 2, 4, 8, 16, 32, 64, 128, and 256 μg/mL, respectively. All plates were incubated at 37oC for 24 h. After 24 h incubation, the minimum drug concentration without bacterial growth was considered to be the MIC. The interaction was judged by calculating the fractional inhibitory concentration (FICIs). These were calculated as follows:
See Formula 1 in the Supplemental Files.
FICI ≤ 0. 5, 0.5 < FICI ≤1, 1 < FICI ≤2, 2 ≤ FICI represents synergy, additivity, indifference, and antagonism, respectively[19].
Concentration-killing curve
MRSA02 strain, which was resistant to all 3 antibiotics, was selected for the concentration-killing curve test. BBR and antibiotic solutions were diluted to final concentrations (16, 32, 64, 128, 256, 512, and 1024 μg/mL) with M-H broth. 50 μL BBR or antibiotic solutions were added to each well of a 96-well plate. Bacterial suspensions were prepared with 0.5 McFarland standard, then diluted to 1:1000 with M-H broth. 50 μL bacterial suspensions were added to the 96-well plate. The initial OD value of each well was measured by an enzyme-linked immunometric meter (Biotek) for 3 times and the average value was set as the base value. After incubating at 37oC for 24 h, the OD value was measured again for 3 times and the average value was calculated. The growth of bacteria was determined by the difference of OD values measured before and after 24 h of incubation.
Time-killing curves analysis
MRSA02 strain, which was resistant to all 3 antibiotics, was selected for the time-killing curve test. BBR solution was diluted to 4 concentrations (32, 64, 128, and 256 μg/mL) with M-H broth. GEN, LEV, and AMI were diluted with M-H broth to 8 concentrations (8, 16, 32, 64, 128, 256, 512, and 1024 μg/mL). Bacterial suspensions were prepared as before. 50 μl of BBR solution (32, 64, 128 and 256 μg/mL) and GEN, LEV, AMI solution (8, 16, 32, 64, 128, 256, 512 and 1024 μg/mL) were arranged in a 96-well plate. 100 μL bacterial suspensions were added to the sterile microporous plate. The final concentrations of BBR were 8, 16, 32, and 64 μg/mL, respectively, while the final concentrations of GEN, LEV, and AMI were 2, 4, 8, 16, 32, 64, 128, and 256 μg/mL, respectively. The initial OD value of each well was measured by an enzyme-linked immunometric meter (Biotek) at 0 h for 3 times, and the average value was set as the base value. The plate was then incubated at 37oC, and the OD values were measured (repeated 3 times) at 4, 8, 12, 16, 20 and 24 h time points, respectively. The average OD value of each time point was calculated. The growth of bacteria was determined by the difference of OD values.
Conductivity test
MRSA02 strain was selected for the conductivity test. Measuring the conductivity of culture medium with a conductivity meter is to measure the ionic concentration of culture medium. The higher the ion concentration is, the greater the conductivity. BBR solution at 16, 128, and 1024 µg/mL were added to the bacterial suspension culture at logarithmic phase. The final concentrations of BBR solution were 8, 64, and 512 µg/mL, respectively. 5 mL of suspension was taken and centrifuged at 2800×g for 10 min at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h, respectively. Conductivity of the supernatant was measured by DDS-11A conductivity meter (Leici, Shanghai) after a 20-time dilution of the supernatant. Absolute ethanol was taken as the control group. The test was repeated 3 times, and the average value was obtained.
TEM examination
MRSA02 strain was selected for the TEM examination. Bacterial suspensions were prepared with 0.5 McFarland standard. After culture with 8, 64, and 512 µg/mL BBR solution at 37oC for 24 h, 10 mL solution was taken and centrifuged at 12000×g for 10 min, and the supernatant was removed to collect 0.5 mL sediment. After fixation with 2% glutaraldehyde PBS fixing solution at 4oC for 2 h, the sediment was washed twice with PBS. After fixed with 1% osmium acid-PBS fixing solution at 4oC for 2 h, cells were washed twice with PBS. Bacteria were dehydrated step by step with ethanol, replaced with propylene oxide and immersed in epoxy resin. They were then sliced with a LKB V ultrathin section machine and stained with lead citrate. The changes in MRSA cell wall were observed by an H-7650 transmission electron microscopy (HITACH, Japan).
RNA isolation, mRNA enrichment and sequencing
After culturing S. aureus USA300 strain to logarithmic phase, USA300 was cultured with BBR of different concentration for 3 h, and total RNA was extracted. The samples were divided into 3 groups: normal control group (group A), high concentration group (group B, 1/2 MIC, 64 ug/mL), and low concentration group (group C, 1/8 MIC, 16 ug/mL). Each group had 3 repetitive samples. Total RNA was extracted from bacterial cells using RNeasy Mini kit (Qiagen)[20]. Qubit 2.0 RNA detection kit was used to quantify total RNA accurately to determine the amount of total RNA added to the library. rRNA was removed by kit and fragmentation buffer was added to the obtained mRNA to make the fragments short. The fragmented RNA was used as template to synthesize the first strand of the DNA with random hexamers, and the second strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. The product was purified by QiaQuick PCR kit and eluted by EB buffer. After terminal repair, base A and sequencing connector were added, the target fragments were collected by agarose gel electrophoresis, and amplified by PCR. The whole library was prepared and the library was sequenced by Illumina HiSeq2500[21].