Sixty SIHP strains were isolated from patients admitted in the second affiliated hospital of Soochow University between 2013 and 2015. Duplicate samples from each patient were taken for analysis. These isolates were then cultured on Columbia sheep blood agar plates (CHROMagar Company, Shanghai, China) at 35°C in an atmosphere containing 5% CO₂ (v/v). Strains identity as S. aureus were confirmed using the Phoenix-100 automated microbiology system (Becton, Dickinson and Company, USA). The control S. aureus strains, with complete hemolytic phenotype, were isolated from patients in our hospital during the same time period. Here, S. aureus with the complete hemolytic phenotype was called SCHP strains. The S. aureus ATCC25923 reference strain (Shanghai Center for Clinical Laboratory, China) has the complete hemolytic phenotype and acted as the control strain as well. The Medical Ethics Committee of Second Affiliated Hospital of Soochow University approved this study and all isolates were collected with patient consent in this study.

The S. aureus strains were cultured on commercial blood agar plates from different companies (CHROMagar, Autobio Diagnostics Co., Ltd., China and BioMérieux, China) as well as self-prepared sheep blood agar plates using Columbia blood agar powder (OXIDE, UK). The bacteria were cultured at 35°C in an atmosphere containing 5% CO₂ (v/v) for 24 h and then underwent serial passage. The hemolytic phenomenon was then observed. Clinically-isolated strains with complete hemolytic phenotype and the ATCC25923 reference strain were also observed as a control for comparative analyses.

Total RNA extraction from the SIHP and the ATCC25923 reference strain was undertaken as previously described (Qin et al., 2014). Initially the cells were lysed using lysostaphin, lysozyme, and proteinase K. RNA was then extracted and purified using RNeasy Mini Kit (Qiagen) according to the manufacturer's recommended protocol. RNA quality and concentration was evaluated using the NanoDrop1000. RNA was then reverse-transcribed to cDNA using a reverse transcription kit (Thermo Fisher Scientific Inc., USA) according to the manufacturer's instructions. Briefly qPCR was conducted as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, elongation at 72°C for 40 s, a total of 45 cycles. Each sample had three technical and three biological repeats. The transcriptional profile of each of the four hemolysin genes and the 16s rRNA gene was determined using the 2^−ΔΔCt method. The level of transcription was determined relative to the expression of the 16s rRNA gene. The sequences of four hemolysin genes (hla, hlb, hlgC, and hld) were retrieved from the GenBank database. Primer 5.0 software was used to design the primers (Table 1) and these primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

SPSS 17.0 was used for data analysis. Measurement data is presented as x̄ ±s. T-test for two independent samples was used to compare the relative expression of each of the four hemolysin genes. Statistical significance was defined as p < 0.05.

The concentration of the SIHP and the reference strain S. aureus ATCC25923 were adjusted to 5.0 McFarland using a turbidity meter. Total protein was then extracted from each sample, adjusted to the same concentration and underwent electrophoresis on an SDS-PAGE gel. The proteins were then transferred to a nitrocellulose membrane and the membrane was blocked with 5% skim milk for 1 h. Goat anti-staphylococcal α-hemolysin polyclonal antibodies (Abcam) were added at a final concentration of 2 μg/mL. The reaction mixture was then incubated at 4°C overnight. After the membrane was washed, 1:1000 diluted HRP-labeled rabbit anti-goat IgG antibodies were added and incubated at 37°C for 2 h. Finally, chemiluminescent substrates were added for color development. The expression of α-hemolysin was finally observed under the imager.

The microtiter broth dilution method was used to perform antimicrobial susceptibility screening. The procedure was done according the operational manual of the Phoenix-100 automated microbiology system. The results from this screening were interpreted using the M100-S24 criteria introduced by the Clinical and Laboratory Standards Institute (CLSI; Clinical and Laboratory Standards Institute, 2014). Methicillin-resistant S. aureus (MRSA) status was determined using the MIC (minimum inhibitory concentration) of two antibiotics. For the SIHP strains ≥4 μg/mL was the MIC for oxallicin, and ≥8 μg/mL was the MIC for cefoxitin.

All 60 SIHP strains were tested for the drug-resistance gene mecA and virulence gene tst. Boiling extraction of genomic DNA was conducted following cellular lysis using lysostaphin. The mecA and tst genes were then amplified from the genomic DNA using the DreamGreen Taq kit. PCR conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 1 min. PCR products were sequenced and analyzed by BLAST to validate to be the expected products. The sequences of drug-resistance gene mecA and virulence gene tst of S. aureus were retrieved from the GenBank database. Primer 5.0 software was used to design the primers (Table 1) and these primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

To compare the survival of S. aureus SIHP strains in macrophages with that of tst negative S. aureus SCHP strains, the human monocyte cell line THP-1 was maintained in RPMI-1640 containing 10% (v/v) FBS at 37°C in an atmosphere containing 5% (v/v) CO₂. For macrophage infection, THP-1 cells were seeded at 5×10⁵ cells per well in 24-well tissue-culture dishes and induced to differentiate by 10⁻⁷ M phorbol 12-myristate 13-acetate (PMA) for 48 h. Approximately 3×10⁸ colony-forming units (CFU) of logarithmic phase (OD₆₀₀ 0.5–0.6) bacteria were pelleted by centrifugation, washed twice with PBS, and resuspended in 1 ml of RPMI-1640. Then bacteria were added to the cell monolayer at a MOI of 20:1, and centrifuged for 5 min at 1000 rpm. Infected cells were incubated for 20 min at 37°C, then were washed three times with pre-warmed PBS (pH 7.4) and incubated for an additional 1 h in medium containing 100μg/ml gentamicin to kill extracellular bacteria. Cells were washed and lysed to quantify the intracellular bacterial as the time zero sample (T₀). Additional cells were collected after 12 or 24 h of incubation in the presence of fresh supplemented tissue-culture medium containing 12 μg/ml gentamicin. The growth of bacteria in THP-1 cells was determined by dividing the number of intracellular bacteria at 12 h or 24 h by the number at time 0 (T₁₂/T₀ or T₂₄/T₀). The experiment was repeated independently three times.

The culture supernatants of THP-1 macrophages infected for 12 h by bacteria were collected. The cytokine levels were measured by FACSCalibur flow cytometry (BD) using a CBA Human Thl/Th2 Cytokine Kit II (BD) according to the manufacture's instruction.

MLST was performed as previously described, including internal fragments of the following seven housekeeping genes: arcC (carbamate kinase), aroE (shikimate dehydrogenase), glpF (glycerol kinase), gmk(guanylate kinase), pta (phosphate acetyltransferase), tpi (triosephosphate isomerase), and yqiL (acetyl coenzyme A acetyltransferase) (Heym et al., 2002). PCR amplification of these seven housekeeping genes was done with the primers which sequences were given at the MLST website (http://pubmlst.org/saureus/info/primers.shtml). Amplicons were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). MLST alleles and sequence types (ST) were determined for each strain with the S. aureus MLST website scheme (Profile/ST query) (http://pubmlst.org/perl/bigsdb/bigsdb.pl?db=pubmlst_saureus_seqdef).

As shown in Figures 1A,B, the complete hemolytic ring (β-hemolytic phenotype) was observed in control S. aureus strains after culturing strains on blood agar plates for 24 h. However, different hemolytic phenotypes (called incomplete hemolytic phenotype here) were displayed in SIHP strains. Moreover, the incomplete hemolytic phenotype could be observed in the SIHP strains which grown in different environmental conditions including microaerophilic, aerobic and anaerobic conditions respectively (Figures 1C–E). In addition, after 10 serial passages, this incomplete hemolytic phenotype was still maintained (Figure 1F).

As shown in Figure 2, the expression levels of the four hemolysin genes hla, hlb, hlgC, and hld in SCHP strains exhibited no statistically significant differences (p > 0.05) compared to that of control strains ATCC25923. However, the expression levels of hla, hlgC, and hld in SIHP strains were significantly suppressed by 50-, 16.7-, and 8.3-folds respectively (p < 0.05) compared with that of control strains ATCC25923 and SCHP strains, while the expression of hlb in SIHP strains was significantly increased 7.7-folds compared to the controls (p < 0.05).

The α-hemolysin encoded by gene hla is a pore-forming toxin secreted by S. aureus and its molecular weight is 33 kDa (Andrey et al., 2015; Al Laham et al., 2015). Western blot analysis showed that the expression of α-hemolysin in SIHP strains was 40-folds lower than that of the SCHP and control strains, which is consistent with the qRT-PCR data (Figure 3).

Drug susceptibility tested using microtiter broth dilutions demonstrated that the MIC values for oxacillin and cefoxitin were all >2 and >8 μg/mL respectively in 60 SIHP stains. According to the CLSI M100-S24 guidelines, all 60 SIHP strains are classified as MRSA strains. PCR also detected the mecA gene in all 60 strains.

The tst gene is an important virulence factor in S. aureus. PCR showed that all 50 of the SIHP strains carried tst genes.

We compared the intracellular survival abilities of SIHP strains and tst negative SCHP strain in macrophages. By counting the number of bacteria recovered from the plates, we found that intracellular survival ability of SIHP strain was much higher than that of tst negative S. aureus strain after infected THP-1 derived macrophages 12 or 24 h (Figure 4). Preliminarily, we speculated that SIHP strains possess a potential high virulence, and it was suggested that SIHP should be paid more attention in hospital.

To compare the secretion differences of cytokines and chemokines in THP-1 derived macrophages infected by SIHP strain and tst negative S. aureus strain, we detected pro-inflammatory cytokines by flow cytometry in the culture supernatant of macrophages infected by SIHP strains and tst negative S. aureus strain. The results showed significantly higher induction of IL-2, IL-6, and IL-17A in SIHP strains, but not by tst negative S. aureus, suggesting that SIHP strains induce cytokine/chemokine responses in the macrophages (Figure 5). The other cytokines IL-4, IL-10, INF-γ, and TNF secreted by macrophages were no obvious differences between SIHP strains and tst negative S. aureus affected groups.

We determined the multilocus genotype of 60 SIHP isolates collected from patients admitted in our hospital between 2013 and 2014. The seven-locus scheme recommended in the S. aureus MLST database was applied. MLST analysis showed that all of SIHP strains were classified into ST5 clone.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.