The S. aureus clinical isolates used in this study were collected from the different inpatients of Shenzhen Nanshan People’s Hospital (a tertiary hospital with 1200 beds) between 2010 and 2016. The in vitro induction S. aureus clinical isolates, including CHS221, CHS165, 149, CHS759, CHS810, CHS820, have been used in our previous study . N315 (GenBank accession number: BA000018) and MS4 (GenBank accession number: CP009828) are two reference S. aureus isolates reserved in our lab, which are MRSA and sensitive to Tige, Omada. The parental MSSA isolates CHS237, CHS632, CHS62, CHS239 and their heteroresistant derived clones CHS237-E1, CHS6 32-E1, CHS62-E1, CHS239-E1, included in this study, have been described in our previous research for the investigation of Erava heteroresistance . The S. aureus isolates, including SE4, SE7, SE13, CHS545 and CHS569, were used as reference isolates for the overexpression experiments as described previously . The minimum inhibitory concentration (MIC) of antimicrobials and multilocus sequence typing of the isolates used in this study were determined in our previous research , and were summarized in Table S2.
All procedures involving human participants were performed in accordance with the ethical standards of Shenzhen University and the 1964 Helsinki declaration and its later amendments, or comparable ethical standards. For this type of study, formal consent is not required.
Antimicrobial susceptibility and population analysis profiling (PAP) development
Omada, Tige and Erava were purchased from the Medicines Company (Med Chem Express, Monmouth Junction, NJ) and their MICs were determined by the agar dilution method according to CLSI guidelines . Because the CLSI guidelines provide no recommendation for the Erava MIC susceptibility breakpoints against S. aureus, we adopted an MIC susceptibility breakpoint of 0.5 mg/L, the value recommended for tigecycline nonsusceptibility in Gram-positive bacteria and defined heteroresistance as growth in 0.5mg/L Erava [7, 20]. PAP experiments were conducted as previously [21, 22]. Briefly, 50μL aliquots (~ 108 colony forming units/ml) were spread onto Müller-Hinton agar plates containing serially diluted Erava (0.5, 1.0, 2.0, 3.0 mg/L). Colonies were counted after 24h of incubation at 37°C. Erava heteroresistance was defined as the observation of subpopulations isolated from the Erava-containing plates able to grow in the presence of 0.5mg/L Erava (detection limit, ≥ 5 colony forming units/plate).
Polymerase chain reaction (PCR) and sequence alignment
Genomic DNA of all clinical isolates was extracted and used as templates for PCR amplification in lysis buffer for microorganisms to direct PCR (Takara Bio Inc., Japan). The presence of 30S ribosomal subunit mutations, including five separate copies (RR1, RR2, RR3, RR4, RR5) of the 16S rRNA gene, the genes encoding the 30S ribosomal proteins S3 and S10, were analyzed by PCR and sequence alignment as described previously , and the PCR primers were listed in Table S3.
In vitro induction of Erava resistance
Eight parental S. aureus isolates, including six clinical isolates (MSSA: CHS221, CHS165, 149; MRSA: CHS759, CHS810, CHS820) and two reference strains (MRSA: N315 and MS4), were used to select Erava-resistant isolates in vitro as described previously . Briefly, these isolates were subcultured serially in Mueller-Hinton broth containing gradual increasing Erava concentrations with the initial concentration being MIC followed by successive increases to 2×, 4×, 8×, 16×, 32×MICs , with four passages at each concentration. Isolates from the passages of each concentration were stored at -80°C in Mueller-Hinton broth containing 40% glycerol for further analysis, including Tet-target site genetic mutation detection and MICs determination.
Quantitative real time PCR (RT-qPCR) analysis
Total RNA of S. aureus isolates was extracted with a RNeasy mini kit (QIAGEN GmbH, Hilden, Germany) and RT-qPCR was performed with an SYBR Premix Ex Taq II kit in a Mastercycler EP Realplex system (Eppendorf, Hamburg, Germany) according to the manufacturer’s instructions (Takara Bio Inc., Shiga, Japan). The internal control gene gyrB was used to normalize gene expression. Threshold cycle (Ct) numbers were determined by detection system software and analyzed with the 2−△△Ct method. The RT-qPCR primers used were described previously and as shown in Table S4 . RT-qPCR was performed in triplicate at least three times.
The overexpression vectors pRS00550, pRS01625, pRS03535, ptetK were constructed by integrating the candidate gene fragments, including USA300HOU_RS00550 (encodes a Na/Pi cotransporter family protein), USA300HOU_RS01625 (encodes a branched-chain amino acid transport system II carrier protein), USA300HOU_RS03535 and USA300HOU_Tet(K), each into separate pIB166 vectors as described previously . Then, the vectors were transformed separately each into three to five Erava-sensitive isolates and confirmed by PCR and Sanger sequencing. The overexpression was induced by 2 μM CdCl2 and verified by RT-qPCR. All strains, plasmids, and primers used for overexpression analysis are listed in Tables S5 and S6.
Continuous data were analyzed by Student’s t-tests with SPSS software package (version 17.0, Chicago, IL). P values < 0.05 were regarded as statistically significant.