Bacterial strains and culture conditions
Bacterial samples were collected from a throat swab. D19T was obtained from the oropharyngeal mucosa of a healthy 6-year-old child. Staphylococcus aureus CGMCC10201, P. aeruginosa CGMCC10104, E. coli CGMCC10003, K. pneumoniae CGMCC31001, P. vulgaris CGMCC1.1651, E. coli cGMCC1.8726, A. baumannii CGMCC1.10395, and suppurative Streptococcus cGMCC9801 were purchased from the Shanghai Industrial Strain Preservation Center, China.
Streptococcus D19T (BHI; HyClone Laboratories, Logan, UT, USA) was incubated in a culture medium at 37°C for 24 h, and then stored at -40°C for later use. The pathogens used in the antagonism and adhesion inhibition experiments were cultured in BHI medium for 6–8 h.
Isolation and identification of the strain
A throat swab sample was collected from the oropharynx of a healthy 6-year-old child from Shenyang, China, to isolate strain D19T. The sample was stored at -80°C until the isolation and culture of D19T. The culture was placed in 1 mL normal saline, mixed, and diluted to 10− 3 in a vortex mixer. Thereafter, 0.1 mL of the culture was evenly coated on BHI agar containing 5% defibrillated sheep blood. After incubation at 37°C for 24 h, white α haemolytic colonies were extracted from the medium and purified using the continuous streaking technique. The phenotypic, phylogenetic, and genomic characteristics were analysed. The strain was identified using polymerase chain reaction (PCR). The total DNA was extracted and the primers used for 16S rRNA gene amplification were 27F/1492R (5'-agagtttgatcmtggctcag-3' and 5'-ggytaccttgttacgactt-3'). PCR was performed using a reaction mixture of volume 30 µL. The specific operation was as follows: DNA degeneration (94°C, 5 min), modification (94°C, 30 s), annealing (56°C, 30 s), extension (72°C, 1 min 40 s), and final stretch (72°C, 8–10 min). The PCR products were analysed using 1.5% agarose gel electrophoresis, and the amplicons were outsourced (Shanghai, China) for sequencing. The identification results showed that the isolate was a gram-positive, catalase-negative strain of a new Streptococcus species, closely associated with oral S. pneumoniae subspecies dentisaniDSM27088, and named it Streptococcus shenyangsis sp. nov.[13].
Resistance to acid and bile salts
The pH tolerance assay reported by Jia et al. (2019) was used with slight modifications. Overnight D19T culture of density 108 CFU/mL was inoculated in either BHI broth at 3% (v/v) and the pH was adjusted to 2.0, 3.0, 4.0, and 5.0 with 1 M HCl or BHI broth containing 0.5% (w/v) and 1% (w/v) bile. Uninoculated BHI broth (pH 7.0) served as the control. The inoculated broths were incubated at 37°C for 24 h. Absorbance of the samples was spectrophotometrically (UV-6800, 5%; Lichen Technology Co., Ltd., China) measured at 600 nm every 2 h.
Antimicrobial activity
Strain D19T was cultured on fibrillated sheep blood agar plate for 24 h, and monoclonal colonies were inoculated in BHI liquid medium and cultured on a 37°C shaking table at 180 rpm for 24 h. The final concentration of the culture was adjusted to 1 × 109 CFU/mL. Simultaneously, the indicator strains (S. aureus, P. aeruginosa, E. coli, K. pneumoniae, K. vulgaris, E. coli, A. baumannii, and S. pyogenes) were cultured to the logarithmic phase, and their concentrations were adjusted to 1 × 106 CFU/mL. Finally, the inhibitory effect of strain D19T on the indicator strains was observed using the Oxford cup method. Specifically, 100 µL of indicator bacterial solution was coated on a nutritional agar plate with sterile cotton swabs. Four Oxford cups were placed on the agar plate with a pair of sterile tweezers at equal distances. Thereafter, 200 µL of culture suspension of each antagonistic strain was added into two cups and BHI medium of equal amount was added into the other two cups as controls. Finally, the culture plate was placed horizontally in a 37°C incubator for 18 h. Three plates were used for each indicator strain.
Antibiotic susceptibility
The antibiotic susceptibility of D19T was determined using the disc diffusion assay according to the 2018 recommendations of the Clinical Laboratory Standards Institute[14]. Colonies were selected and cultured for 24 h on agar plates containing 5% (w/v) defibrinated sheep blood and suspended in 5 mL of sterile normal saline solution with a turbidity of 0.5 McFarland. Bacterial suspension was applied to MH medium with sterile cotton swabs. The antibiotics tested were penicillin (30 µg), ampicillin (10 µg), cefepime (30 µg), cefotaxime (30 µg), ceftriaxone (10 µg), linezolid (30 µg), clindamycin (2 µg), chloramphenicol (30 µg), and vancomycin (30 µg). All assays were performed in triplicates.
Cytotoxicity assay
The cytotoxicity of D19T in 16-HBE cells was determined using the colourimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)[15]. The cells were seeded in a 96-well tissue culture plate at a density of 8 × 104 /well until confluence. Thereafter, D19T was added at MOIs (bacteria:16-HBE cells) of 0.2:1, 2:1, and 20:1 before coincubation at 37°C under 5% CO2 for 24 h. Subsequently, 10 µL of MTT (5 mg/mL) was added to each well, and the suspensions were incubated at 37°C under 5% CO2 for 4 h. After incubation, 150 µL of dimethyl sulfoxide was added to each well and the formazan crystal product was completely dissolved by shaking for 10 min. The absorbance (A) of sample in each well was measured at 570 nm using a microplate reader (TECAN Infinite 200 PRO; Beijing Long yue Biological Technology Development Co., Ltd., CA, USA) at 570 nm. Cell viability was calculated as follows:
Cell viability (%) = (Asample/Acontrol) × 100 (1);
where, Asample is the absorbance of 16-HBE cells co-incubated with D19T and Acontrol is the absorbance of D19T alone.
Bacterial fluorescent labelling
The bacteria were labelled according to a previously reported method[16]. The bacterial strains were cultured to the middle and late periods of exponential growth and centrifuged at 4°C for 10 min at 5000 × g. Thereafter, the supernatant was discarded; the pellet was washed twice with phosphate-buffered saline (PBS; pH 7.4), centrifuged at 4°C for 10 min at 5000 × g, and mixed with carbonate buffer (0.5 mol/L, pH 9.5) to prepare 0.5 × 109 CFU/mL bacterial suspensions. FITC solution was added to a final concentration of 50–100 µg/mL in the bacterial suspension; the solution was stirred at room temperature for 1–2 h and centrifuged at 5000 × g for 10 min. Subsequently, the supernatant was discarded, and the pellet was washed twice with PBS and centrifuged at 5000 × g for 10 min. The precipitated bacteria were suspended in PBS to obtain a bacterial suspension of concentration 1 × 109 CFU/mL. The labelling was confirmed using fluorescence microscopy, and the bacterial suspension was stored at 4°C, protected from light, until further use.
To observe bacterial fluorescence signal, 5 µL of the labelled bacterial suspension was applied on to a slide, covered with a cover glass, and bacterial fluorescence image was observed under a fluorescence microscope; a clear bacterial fluorescence field indicated successful labelling.
Cell culture assays
16-HBE cell line was purchased from Shenyang Medical College (Liaoning, China). The cells were cultured in endothelial cell medium (ECM; HyClone) containing 5% (v/v) foetal bovine serum, 1% (w/v) penicillin/streptomycin solution, and 1% (w/v) endothelial cell growth supplements at 37°C under 5% CO2. The cells were then cultured in a 25-cm2 flask containing 0.25% (v/v) trypsin-EDTA solution (Sigma-Aldrich Corp., St. Louis, MO, USA) at 37°C for 13 min and centrifuged (4000 × g, 4°C, 1 min). The cells were inoculated in a six-well tissue culture plate at a density of 8 × 104/well and sub-cultured at 37°C under 5% CO2 for 3 days until fusion.
Adhesion experiment
The method described by Jia et al.[15] was used. Cell cultures without bacteria were used as the blank controls. The experimental group contained 100 µL of culture medium and 100 µL fluorescent-labelled D19T and S. aureus (1 × 109 CFU/mL) in 96-well plates. The plates were placed in an incubator at 37°C under 5% CO2 for 2 h, and then, washed with sterile PBS for three times. The unattached bacterial cells were eluted, and 0.1 mL of trypsin was added to each well and the plates were incubated for 13 min. After the complete exfoliation of cells, 0.4 mL of the complete culture medium was added to terminate the reaction. The liquid was collected, and its fluorescence intensity was measured using a microplate reader. The excitation wavelength was set at 495 nm and emission wavelength was set at 530 nm. Relative fluorescence intensity was determined using 10 replicates for each group.
Calculation formula:
Adhesion rate of bacteria (%) = A/A0 × 100;
where, A is the relative fluorescence intensity of the cell suspension after the adherence of D19T and S. aureus cells to 16-HBE cells and elutes, and A0 is the relative fluorescence intensity of the cell suspension before the adherence of D19T and S. aureus cells to 16-HBE cells.
Competitive test
D19T cells (unlabelled), 16-HBE cells, and pathogenic bacteria (FITC-labelled) were incubated at 37°C under 5% CO2 for 2 h. Thereafter, the samples were washed with sterile PBS twice to remove non-adherent cells and allowed to stand in dark.
Exclusion test
D19T cells (unlabelled) and 16-HBE cells were incubated at 37°C under 5% CO2 for 1 h. Thereafter, the cells were washed with sterile PBS twice to remove non-adherent cells. Subsequently, pathogenic bacteria (FITC-labelled) was added, and the samples were incubated at 5% CO2 under 37°C for 1 h.
Displacement assay
Pathogenic bacteria (FITC labelled) and 16-HBE cells were co-incubated at 37°C under 5% CO2 for 1 h and washed with sterile PBS twice to remove non-adherent bacterial cells; thereafter, D19T cells (unlabelled) were added, and the samples were incubated at 37°C under 5% CO2 for 1 h.
After the above treatment, 0.1 mL of pancreatic enzyme was added to each culture well and the samples were incubated for 13 min. After complete shedding of cells, 0.4 mL of complete culture medium was added to terminate the reaction. The suspension was collected, and its fluorescence intensity was measured using an enzyme-linked immunosorbent assay. Each group had six replicates and each culture well had one replicate.
Calculation formula: Adhesion rate (%) = A2 / A1 × 100;
where, A1 represents the relative fluorescence intensity of S. aureus adhering to 16-HBE cells in the presence of strain D19T and A2 represents the relative fluorescence intensity of S. aureus adhering to 16-HBE cells in the presence of strain D19T.
Statistical Analysis
All experiments were conducted independently, at least in triplicates. Data are presented as mean ± standard deviation. Data were analysed using a one-way ANOVA, and Duncan’s test was used to compare overall differences (P < 0.05).