Lapatinib Acts against Biofilm Formation and the Hemolytic Activity of Staphylococcus aureus.

Biofilm formation and hemolytic activity are closely related to the pathogenesis of Staphylococcus aureus infections. Herein, we show that lapatinib (12.5 μM) significantly inhibits biofilm formation and hemolytic activity of both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates. Using quantitative reverse transcription PCR, we found that the RNA levels of transcriptional regulatory genes (RNAIII, agrA, agrC, saeR, and saeS), biofilm-formation-related genes (atl, cidA, clfA, clfB, and icaA), and virulence-related genes (cap5A, hla, hld, hlg, lukDE, lukpvl-S, staphopain B, alpha-3 PSM, beta PSM, and delta PSM) of S. aureus decreased after 6 h treatment with lapatinib. Wild-type S. aureus isolates were continuously cultured in vitro in the presence of increasing concentrations of lapatinib for about 140 days. Subsequently, S. aureus isolates with reduced susceptibility to lapatinib (the inhibitory effect of lapatinib on the biofilm formation of these S. aureus isolates was significantly weakened) were selected. Mutations in the genomes of S. aureus isolates with reduced susceptibility to lapatinib were detected by whole-genome sequencing. We identified four genes with mutations: three genes with known functions (membrane protein, pyrrolidone-carboxylate peptidase, and sensor histidine kinase LytS, respectively) and one gene with unknown function (hypothetical protein). In conclusion, this study indicates that lapatinib significantly inhibits biofilm formation and the hemolytic activity of S. aureus.

Introduction

Staphylococcus aureus is one of the main pathogens causing nosocomial and community-acquired infections.[1]S. aureus secretes a variety of virulence factors, including, but not limited to, hemolysin, extracellular protease, leucocidin, and phenol-soluble protein, which aid its ability to invade and damage host cells.[2] Increasing drug resistance in S. aureus, especially methicillin-resistant S. aureus (MRSA), has made clinical treatment of S. aureus infections more challenging.[3] Vancomycin and linezolid are the only antimicrobials that can treat MRSA infections. Disturbingly, global incidences of vancomycin intermediate S. aureus (VISA/hVISA) and linezolid-resistant S. aureus have been identified in recent years.[4,5] Therefore, there is an urgent need to explore and develop new antimicrobials against drug-resistant S. aureus.

In addition to drug resistance and virulence, another important issue is the ability of both methicillin-susceptible and -resistant S. aureus to form biofilms.[6]S. aureus biofilms are usually composed of an exopolymer matrix of exopolysaccharides, proteins, and some micromolecules (e.g., environmental DNA (eDNA)).[7] Thus, the formation of biofilms in S. aureus makes clinical treatment more difficult, resulting in prolonged hospital stays for patients and increased mortality rates.[8] To date, only a few antimicrobials have shown inhibitory effects on S. aureus biofilms.[9]

Lapatinib [4-aniline quinazoline receptor tyrosine kinase inhibitor] is an orally effective quinazoline derivative that inhibits epidermal growth factor receptors 1 (ErbB1) and 2 (ErbB2) in human cells.[10] Lapatinib induces cell cycle arrest and promotes cell apoptosis through inhibition of the MAPK and PI3K/Akt downstream signaling pathways, resulting in an antitumor effect.[11] Lapatinib is mainly used in combination with capecitabine to treat advanced or metastatic breast cancer with ErbB2 overexpression.[12] It was discovered that lapatinib inhibited multiplication of Mycobacterium spp. in macrophages.[13] Dean et al.[14] showed that lapatinib is a strong promoter of Francisella novicida biofilm formation. Inspired by these studies, we explored the effects of lapatinib on the growth and biofilm formation of S. aureus. Interestingly, our preliminary experiments showed that lapatinib inhibits the biofilm formation of S. aureus. Therefore, we sought to explore the characteristics and mechanisms behind this lapatinib-induced inhibition of growth, biofilm formation, and hemolytic activity of S. aureus.

Results

Antimicrobial Activity of Lapatinib Against S. aureus Planktonic Cells

Investigation of the antimicrobial activity of lapatinib against S. aureus planktonic cells revealed that lapatinib at concentrations of 3.125–100 μM had no antimicrobial activity in broth macrodilution (MICs ≥ 100 μM). In contrast, by measuring the OD600 values of S. aureus broth cultures in the presence of different concentrations of lapatinib after 24 h of incubation, a decrease in OD600 values was found in MRSA cultures with high concentrations of lapatinib (Figure ).

Figure 1

Effect of different concentrations of lapatinib on the growth of S. aureus planktonic cells. (a) S. aureus SA113 and CHS101 (MSSA) and (b) YUSA139 and YUSA145 (MRSA) isolates were treated with lapatinib (3.125 μM to 100 μM) for 24 h, and the growth of planktonic cells was determined by optical density at 600 nm (OD600). The data are presented as the average of three independent experiments (mean ± SD). MSSA, methicillin-sensitive S. aureus; MRSA, methicillin-resistant S. aureus; Lap, lapatinib.

Influence of Lapatinib on the Biofilm Formation in S. aureus

This study explored the effect of different concentrations of lapatinib (from 3.125 to 50 μM) on biofilm formation of S. aureus. We found that lapatinib ≥12.5 μM significantly inhibits biofilm formation of four MSSA isolates and four MRSA isolates (Figure ). Subsequently, we observed that lapatinib (at 12.5 μM) significantly inhibits biofilm formation of 10 out of 12 MSSA isolates and all of the 11 tested MRSA isolates (Figure ). Finally, we also verified that lapatinib (at 12.5 μM) significantly inhibits biofilm formation of 46 MSSA isolates and 28 MSSA isolates (Figure S1 and Figure S2). The effect of lapatinib on adherent cells in S. aureus biofilms was also explored. Here, we found that after 24 h of static incubation, the number of adherent cells in S. aureus biofilms was significantly decreased following treatment with lapatinib (at 12.5 or 50 μM; Figure ). We also sought to investigate the ability of lapatinib to eradicate established S. aureus biofilms. The data showed that lapatinib (at 3.125, 12.5, or 50 μM) had no such effect, even combined with vancomycin, linezolid, daptomycin, or rifampin (Table S1, Figure S3, and Figure S4).

Figure 2

Effect of different concentrations of lapatinib on S. aureus biofilm formation. The four MSSA (a) and four MRSA (b) isolates were treated with lapatinib from 3.125 to 50 μM for 24 h. Biofilm biomasses were determined by crystal violet staining. The data are presented as the average of three independent experiments (mean ± SD). Compared to the control, *: P < 0.05; **: P < 0.01; ***: P < 0.001 (Student’s t test). MSSA, methicillin-sensitive S. aureus; MRSA, methicillin-resistant S. aureus; Lap, lapatinib.

Figure 3

Lapatinib significantly inhibits S. aureus biofilm formation at 12.5 μM. The 12 MSSA (a) and 11 MRSA (b) isolates were treated with lapatinib at 12.5 μM for 24 h. Biofilm biomasses were determined by crystal violet staining. The data are presented as the average of three independent experiments (mean ± SD). Compared to the control, *: P < 0.05; **: P < 0.01; ***: P < 0.001 (Student’s t test). MSSA, methicillin-sensitive S. aureus; MRSA, methicillin-resistant S. aureus; Lap, lapatinib.

Figure 4

Effect of lapatinib on adherent cells in S. aureus biofilms. The four MSSA (a) and four MRSA (b) isolates were treated, increasing concentrations of lapatinib (3.125, 12.5, and 50 μM) for 24 h. The adherent cells in S. aureus biofilms were determined by counting the number of CFUs. The data are presented as the average of three independent experiments (mean ± SD). Compared to the control, *: P < 0.05; **: P < 0.01 (Student’s t test). MSSA, methicillin-sensitive S. aureus; MRSA, methicillin-resistant S. aureus; Lap, lapatinib.

Influence of Lapatinib on the Hemolytic Activity of S. aureus

The rabbit erythrocyte lysis assay was used to determine the inhibitory effect of 12.5 μM lapatinib on the hemolytic activity of 10 MSSA and 9 MRSA isolates (Figure ). The data showed that OD550 values of all the experimental isolates decreased after treatment with lapatinib, suggesting that 12.5 μM lapatinib induced a significant inhibitory effect on the hemolytic activity of S. aureus.

Figure 5

Lapatinib (12.5 μM) inhibits the hemolytic activity of S. aureus. The 10 MSSA isolates (a) and 9 MRSA isolates (b) were treated with 12.5 μM lapatinib for 24 h. The 4% rabbit erythrocytes were added to the culture supernatants, and optical density at 550 nm (OD550) was measured following incubation at 37 °C for half an hour. We used 0.1% Triton X-100 as the 100% hemolytic activity control (positive control). The data are presented as the average of three independent experiments (mean ± SD). Compared to the control, *: P < 0.05; ***: P < 0.001 (Student’s t test). MSSA, methicillin-sensitive S. aureus; MRSA, methicillin-resistant S. aureus; Lap, lapatinib.

RNA Expression of S. aureus Genes Downregulated in Lapatinib-Treated Isolates

Inspired by a previous study,[15] we sought to explore the possible mechanism(s) behind lapatinib-induced effects in different S. aureus isolates. We used quantitative reverse transcription PCR (RT-qPCR) to measure RNA expression of S. aureus genes involved in transcriptional regulation, biofilm formation, and virulence following lapatinib treatment. RNA expression of S. aureus transcriptional regulatory genes RNAIII, agrA, agrC, saeR, and saeS in SA113 (MSSA) and YUSA139 (MRSA) lapatinib-treated isolates was significantly downregulated following 6 h of static incubation. We also found that RNA expression of biofilm-formation-related genes (atl, cidA, clfA, clfB, and icaA) and virulence-related genes (cap5A, hla,hld, hlg, lukDE, lukpvl-S, staphopain B, alpha-3 phenol-soluble modulin (PSM), beta PSM, and delta PSM) was significantly downregulated in lapatinib-treated isolate after static incubation for 6 h (Table ).

Table 1

Differential Expression of RNA Genes in Lapatinib-Treated S. aureus SA113 and YUSA139a

	SA113	YUSA139
Transcriptional Regulatory Genes
RNAIII	0.0053	0.0132
agrA	0.2847	0.3089
agrC	0.1419	0.2314
luxS	0.3024	0.6324
sarA	0.3100	0.5324
sigB	0.5339	0.6327
saeR	0.0615	0.1247
saeS	0.2761	0.2687
Biofilm Formation Related Genes
atl	0.0820	0.1038
aur	0.8218	0.6357
ccpA	3.8870	2.6578
cidA	0.2264	0.3574
clfA	0.2153	0.1241
clfB	0.3805	0.2657
fnbA	4.5198	1.6574
fnbB	0.5759	1.2657
icaA	0.0486	0.1085
icaB	0.7244	0.6574
sasG	0.8567	1.1587
Virulence-Related Genes
cap5A	0.0209	0.0568
hla	0.1602	0.1238
hlb	0.8626	0.5347
hld	0.0042	0.0658
hlg	0.0346	0.0863
lukDE	0.0461	0.1268
lukpvl-S	0.1114	0.3574
spa	8.8206	2.3574
sbi	0.1256	2.3654
staphopain B	0.0027	0.0357
alpha-3 PSM	0.0356	0.0785
beta PSM	0.0521	0.0863
delta PSM	0.0058	0.0265
coa	0.5310	0.7852

SA113 and YUSA139 Lap-treated isolate: S. aureus SA113 and YUSA139 isolates were treated with 12.5 μM lapatinib for 6 h under static incubation, and the untreated control isolates were used as the negative control (RNA level = 1.0). The RNA expression of genes in SA113 Lap-treated isolate and YUSA139 Lap-treated isolate were compared to their control isolates. Lap, lapatinib; PSM, phenol-soluble modulins.

Genetic Mutations in S. aureus Isolates with Reduced Susceptibility to Lapatinib

Methicillin-sensitive S. aureus SA113 and CHS101 and methicillin-resistant S. aureus YUSA139 and YUSA145 were subcultured continuously for 140 days on culture media with the addition of different, increasing concentrations of lapatinib. After this treatment, we found that the inhibitory effect of lapatinib on the biofilm formation of S. aureus SA113 (SA113-T1, SA113-T2, and SA113-T3) was significantly weakened (Figure ), indicating reduced susceptibility to lapatinib in treated isolates. However, the inhibitory effect of lapatinib on the biofilm formation of S. aureus CHS101, YUSA139, and YUSA145 isolates was not significantly reduced (Figure S5).

Figure 6

Effect of lapatinib on biofilm formation of lapatinib-induced S. aureus SA113 isolates. (a) S. aureus SA113 isolates were serially subcultured in TSB containing lapatinib. The three isolates (T1–T3) from the generation 55 of S. aureus SA113 isolates were picked, and the inhibitory effect of lapatinib on the biofilm formation of these isolates was determined by crystal violet staining. The data are presented as the average of three independent experiments (mean ± SD). Compared to the control, ***: P < 0.001 (Student’s t test). (b) Inhibition (%) of biofilm formation: (OD570 value of the control – OD570 value of the isolates treated with 12.5 μM lapatinib)/OD570 value of the control. Compared to SA113 (wild-type), ***: P < 0.001 (Student’s t test). Lap, lapatinib; SA113 (wild-type): control isolate, serially subcultured in TSB containing no lapatinib.

The antimicrobial susceptibilities of the S. aureus SA113 control isolate (SA113-wild-type) and the S. aureus isolate with reduced susceptibility to lapatinib (SA113-T1) were measured. We found that S. aureus SA113-wild-type and SA113-T1 isolates were sensitive to all tested antimicrobials (Table S2). The whole genomes of SA113-T1 and SA113-wild-type isolates were sequenced by Illumina HiScanSQ, and genomic analyses of these isolates are summarized in Table S3. We identified four nucleotide mutations in the SA113-T1 isolate leading to nonsynonymous mutations of four amino acids (Table ). These mutations were located in three genes with known functions—a membrane protein, a pyrrolidone–carboxylate peptidase, and a sensor histidine kinase LytS and one gene with unknown function (a hypothetical protein).

Table 2

Mutations in the SA113-T1 Isolate Genome Detected by Whole-Genome Sequencinga

ref_gene_ID	NA mutations	AA mutations	ref_gene_product
CSA113_GM000237	A976G	N326D	WP_047214669.1; membrane protein
CSA113_GM001471	G274A	E92K	WP_000547833.1; pyrrolidone-carboxylate peptidase
CSA113_GM001699	G866A	R289H	K07704; sensor histidine kinase LytS; environmental information processing; signal transduction
CSA113_GM002004	A1216G	S406P	MULTISPECIES: hypothetical protein

S. aureus SA113 isolate was serially subcultured in TSB containing lapatinib. Mutations in the genome of T1 isolates from 55 generations of SA113 isolates were detected by whole-genome sequencing. NA, nucleotide; AA, amino acid.

Discussion

The mechanism of action of lapatinib, a drug traditionally used in the clinic to treat breast cancer, involves the inhibition of ErbB1 and ErbB2.[16] A previous study has found that sorafenib, a tyrosine kinase inhibitor similar to lapatinib, has antibacterial activity against Staphylococcus.[17] SC5005, a derivative of sorafenib, displays a high inhibitory effect on MRSA, but its antibacterial mechanism(s) remain unclear.[18] Interestingly, in this study, we found that while lapatinib has no antibacterial effect on S. aureus it does exhibit a significant dose-dependent manner inhibitory effect on S. aureus biofilm formation. We found that 12.5 μM of lapatinib induced the best inhibition. Moreover, our data also showed that lapatinib shows a strong inhibitory effect on the hemolytic activity of S. aureus.

We also found that lapatinib downregulated the transcription of some genes involved in S. aureus biofilm formation. These included clfA and clfB, which are key players in the attachment and early stage biofilm formation in S. aureus, as well as atl, cidA, and icaA, which are involved in the proliferation and maturation of S. aureus biofilms.[8] The present study shows that lapatinib significantly inhibits biofilm formation and the transcription of some genes involved in biofilm formation in S. aureus. However, lapatinib does not induce the destruction of established S. aureus biofilms, possibly due to the enclosure of established biofilms within an exopolymer matrix of exopolysaccharides, proteins, and some micromolecules (such as eDNA). This restricts the diffusion and penetration of antimicrobials across the biofilm, thus making them very difficult to damage.[19]

Interestingly, we also found that transcription of some virulence-related genes was downregulated following lapatinib treatment. Further, these included genes involved in S. aureus hemolytic activity (i.e., hla, hld, and hlg) as well as other virulence-related genes, such as lukDE, lukpvl-S, sbi, staphopain B, and PSMs. In addition, we found that lapatinib affects the expression of genes involved in transcriptional regulation in S. aureus. These genes are included in the RNAIII/Agr system, LuxS/AI-2 quorum-sensing system, and SaeRS two-component system. These results suggest that the target or target pathway of lapatinib in S. aureus may be the upstream signal regulatory pathway, or it may be an important regulatory factor.

To explore the potential target of lapatinib in S. aureus, S. aureus isolates with reduced susceptibility to lapatinib were selected. The only isolate was S. aureus SA113, and no S. aureus clinical isolates (e.g., CHS101, YUSA139, and YUSA145) showed reduced sensitivity to lapatinib. It may be that the S. aureus SA113 isolate was derived from the defective mutation of the S. aureus 8325 isolate;[20,21] thus, the S. aureus isolates with reduced susceptibility to lapatinib were more easily identified in the S. aureus SA113 isolates than in the clinical isolates. One bioinformatic study suggested that lapatinib has the ability to inhibit ribosomal protein L6 in Staphylococcus.[22] However, we identified four genes with mutations in the S. aureus isolate with reduced lapatinib sensitivity, but ribosomal protein L6 was not implicated in any of these mutations. Among the products of the identified genes was sensor histidine kinase LytS, which is involved in the two-component signaling pathway LytRS. The two-component signal transduction system is one of the primary pathways by which bacteria adapt to the external environment and regulate virulence gene expression, cell wall synthesis, biofilm formation, and bacterial activity.[23] The two-component signaling pathway LytRS is implicated in the vancomycin resistance of S. aureus and may be a potential therapeutic target for S. aureus infections.[24] LytRS plays an important role in the regulation of virulence in S. aureus.[25] Further, LytRS may affect S. aureus biofilm formation by regulating autolysis in S. aureus.[26] Combined with the results of the present study, these data suggest that LytRS may also regulate the adhesion of cells in S. aureus biofilms. However, the role and mechanism(s) of LytRS in S. aureus biofilm formation are not fully understood. Therefore, considering previously published studies as well as our findings, we hypothesize that S. aureus LytS may be a target for lapatinib. However, further research is required to confirm and reveal the detailed mechanisms behind this relationship.

Conclusions

In this study, we found that lapatinib significantly inhibits the biofilm formation and hemolytic activity of S. aureus. Further, we hypothesize that sensor histidine kinase LytS may be a potential target of lapatinib in S. aureus.

Methods

Bacterial Isolates and Culture Conditions

S. aureus ATCC29213, SA113 (ATCC35556), and USA300 FPR3757 (ATCC BAA-1556) isolates were purchased from American Type Culture Collection (ATCC). S. aureus HG003 (NCTC8325 derivative) isolate was obtained from Prof. Di Qu from the Key Laboratory of Medical Molecular Virology of Ministries of Education and Health at Shanghai Medical College of Fudan University. There were 108 S. aureus clinical isolates [isolated from sputum (38), throat swabs (22), blood (18), pus (13), catheters (8), pleural effusion (5), and cerebrospinal fluid (4)] used in this study. All clinical isolates were identified with the Phoenix 100 automated microbiology system (BD, Franklin Lakes, NJ, USA) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IVD MALDI Biotyper, Germany). S. aureus isolates were grown in tryptic soy broth (TSB) and in TSBG [tryptic soy broth (Merck, Germany) with 0.5% glucose] at 37 °C for the biofilm assays.

Antimicrobials and Chemicals

The following antimicrobials and chemicals were obtained from MedChemExpress (MCE, Shanghai, China): oxacillin sodium (catalogue no. HY-B0465, purity: 99.52%), cefazolin sodium (catalogue no. HY-B1078, purity: 98.13%), vancomycin (catalogue no. HY-B0671, purity: 98.25%), linezolid (catalogue no. HY-10394, purity: 99.78%), daptomycin (catalogue no. HY-B0108, purity: 99.90%), rifampin (catalogue no. HY-B0272, purity: 98.07%), minocycline hydrochloride (catalogue no. HY-17412, purity: 99.71%), azithromycin (catalogue no. HY-17506, purity: 98.0%), clindamycin (catalogue no. HY-B1455, purity: 99.15%), and lapatinib (catalogue no. HY-50898, purity: 99.83%).

Antimicrobial Susceptibility Testing with Antibiotics

The susceptibilities of S. aureus isolates to oxacillin, cefazolin, vancomycin, linezolid, daptomycin, rifampin, minocycline, azithromycin, and clindamycin were determined by the minimum inhibitory concentrations (MICs). The MICs were detected by the broth microdilution method, according to the Clinical and Laboratory Standards Institute guidelines (CLSI-M100-S27). The susceptibility of S. aureus isolates to oxacillin was tested on the Phoenix 100 automated microbiology system (BD, Franklin Lakes, NJ, USA) and was reidentified by determining the MICs of oxacillin against these clinical isolates.

Antimicrobial Susceptibility Testing to Lapatinib

Overnight cultures of S. aureus were diluted 1:200 in fresh TSB medium, and different concentrations of lapatinib (3.125, 12.5, 50, and 100 μM) were added to the culture medium (with no lapatinib as a negative control) and then inoculated into a bioscreen honeycomb plate (300 μL/well), cultured at 37 °C with shaking for 24 h. OD600 values were measured using a Bioscreen C system (Lab Systems Helsinki, Finland). MICs of lapatinib (2-fold dilution concentration; 3.125–200 μM) against S. aureus isolates were determined by the broth microdilution method. This experiment was performed at least three independent times.

Investigation of the Effect of Lapatinib on the Formation of S. aureus Biofilms

Biofilm biomasses of S. aureus were determined by the crystal violet staining method based on our previous studies with some modifications.[27,28] After overnight culture, S. aureus was diluted 1:100 with fresh TSBG containing lapatinib (3.125, 6.25, 12.5, 25, and 50 μM) and then inoculated into 96-well polystyrene microtiter plates (1.0–2.0 × 107 CFU/mL, 200 μL/well). TSBG without lapatinib was used as the untreated control. After static incubation for 24 h, the supernatant of 96-well polystyrene microtiter plates was removed, and plates were gently washed with phosphate-buffered saline (PBS) three times, dried at room temperature, and fixed with methanol for 15 min. Methanol was removed, and cells were stained with 0.5% crystal violet for 10 min at room temperature. Crystal violet was dissolved in 95% ethanol, and OD570 was determined. This experiment was performed in triplicate.

Investigation of the Possibility of Disruption of Already Formed S. aureus Biofilms Using Lapatinib

Overnight cultures of S. aureus were diluted 1:100 with fresh TSBG and inoculated into 96-well polystyrene microtiter plates (1.0–2.0 × 107 CFU/mL, 200 μL/well), and after static incubation for 24 h at 37 °C (mature biofilms established), the supernatants were removed and plates washed with 0.9% saline to remove unattached cells. Next, fresh TSBG containing lapatinib (3.125, 12.5, and 50 μM) was added. TSBG without lapatinib was used as the untreated control. After static incubation for 24 h, plates were washed gently three times with PBS, dried at room temperature, and fixed with methanol for 15 min. Methanol was removed, and cells were stained with 0.5% crystal violet for 10 min at room temperature. Crystal violet was dissolved in 95% ethanol, and OD570 was determined. This experiment was performed in triplicate.

Determination of Total CFUs of Adherent S. aureus Cells

Total CFUs of adherent S. aureus cells were determined by the previously applied methodology.[29] Overnight cultures of S. aureus were diluted 1:100 with fresh TSBG containing lapatinib (3.125, 12.5, and 50 μM) and then inoculated into 24-well polystyrene microtiter plates (1.0–2.0 × 107 CFU/mL, 1.5 mL/well). TSBG without lapatinib was used as the untreated control. After static incubation for 24 h, the supernatant was removed, and the adherent cells in biofilms were harvested by scratching the wall of the wells using a flat end toothpick. Finally, the bacteria were centrifuged, and the numbers of CFU were determined. All experiments were performed in triplicate.

Hemolytic Activity Assay

Hemolytic activity of S. aureus was determined according to a previous study with some modifications.[30] After overnight culture, S. aureus was diluted 1:200 in fresh TSB medium containing lapatinib (with no lapatinib as a negative control). After cultured with shaking for 24 h at 37 °C, culture supernatant was collected and filtered with a 0.22 μm filter (Millipore). The filtered culture supernatant was added into rabbit erythrocytes (SBJ-RBC-RAB003, Sbjbio, China) and incubated at 37 °C for half an hour, and finally the optical density at 550 nm (OD550) was determined. The positive control was the 0.1% Triton X-100 (100% hemolytic activity control), and PBS was the negative control. Lapatinib alone without S. aureus was also used as the negative control. This experiment was performed in triplicate.

RNA Expression of S. aureus Genes Detected by RT-qPCR

RNA expression of genes involved in transcriptional regulation, biofilm formation, and virulence of S. aureus were determined by RT-qPCR. Overnight cultures of S. aureus SA113 (MSSA) and YUSA139 (MRSA) isolates were diluted 1:200 with TSBG containing 12.5 μM lapatinib and inoculated into 25 mL polypropylene round culture dishes, and the TSBG without lapatinib was used as the untreated control. After static incubation for 6 h at 37 °C, S. aureus biofilms were harvested from the polypropylene round culture dishes using cell scrapers, and total RNA was collected from the planktonic cells and S. aureus biofilms for RT-qPCR. The total RNA isolation of S. aureus and RT-qPCR was handled as previously described.[27,31] The harvested cells were washed with ice-cold 0.9% saline and homogenized in a Mini-BeadBeater (Biospec, Bartlesville, OK, USA) containing 0.1 mm zirconia-silica beads (grinding centrifuged at 4000 rpm for 1 min and then cooling on ice for 1 min, repeated five times). The total RNA in the homogenized supernatant was purified with an RNeasy Mini Kit (Qiagen) and quantified by an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States).

RT-qPCR was conducted using the SYBR green PCR reagents (SYBR Premix Ex Taq; TaKaRa Biotechnology, Dalian, China) in the Mastercycler RealPlex system (Eppendorf AG, Hamburg, Germany). The housekeeping gene gyrB (DNA gyrase subunit B) was used as an internal reference gene to normalize transcript levels. The reaction conditions of RT-qPCR were as follows: initial incubation at 95 °C for 2 min, followed by 40 cycles of 15 s at 95 °C, and 60 s at 55 °C. The threshold cycle (Ct) numbers were confirmed by the detection system software, and the data were analyzed based on the 2–ΔΔCt method.[32] The untreated control isolates were used as the reference isolates, and RNA expression of genes in S. aureus reference isolates was set as 1.0. RNA expression of genes in lapatinib-treated isolates was compared to their reference isolates. Thus, RNA expression values <1.0 indicate downregulation following lapatinib treatment, whereas values >1.0 are indicative of upregulation. The primers used for RT-qPCR in this study are listed in Table S4. RT-qPCR was performed in triplicate.

In Vitro Reduction of Susceptibility of S. aureus to Lapatinib

According to our previous study where we generated S. aureus isolates with reduced susceptibility to chemicals,[15] wild-type S. aureus SA113, CHS101 (MSSA), YUSA139, and YUSA145 (MRSA) isolates were serially subcultured in TSB containing lapatinib (containing no lapatinib as control isolates). The initial concentration of lapatinib was 12.5 μM, which was then successively increased until the highest concentration of 250 μM. S. aureus isolates in each concentration of lapatinib was cultured for 3–5 passages before inoculated and passaged to the next generation. S. aureus isolates from the last passage of each concentration of lapatinib were collected and subcultured on tryptic soy agar plates without lapatinib for three passages. S. aureus isolates were selected and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IVD MALDI Biotyper, Bruker, Bremen, Germany), and the inhibitory effect of lapatinib on the biofilm formation of these S. aureus isolates was determined by crystal violet staining.

Detection of Mutations in the Genomes of the S. aureus Isolate with Reduced Susceptibility to Lapatinib by Whole-Genome Sequencing

The genomic DNA of S. aureus SA113-T1 isolate (S. aureus isolate with reduced susceptibility to lapatinib) and SA113-wild-type (control isolate, serially subcultured in TSB containing no lapatinib) was extracted by the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The whole-genome sequencing of S. aureus SA113 genomic DNA was performed as previously described.[15] An amount of 1 μg of DNA per isolate was used for the DNA-sequencing sample preparations, and sequencing libraries were produced by a NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s recommendations. The whole genomes were sequenced by Illumina HiScanSQ. The coding gene, repetitive sequences, noncoding RNA, genomics islands, transposon, prophage, and clustered regularly interspaced short palindromic repeat sequences (CRISPR) were predicted. Gene functions were analyzed by GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups), NR (Non-Redundant Protein Database databases), TCDB (Transporter Classification Database), and Swiss-Prot. Genomic alignment between the SA113-T1 isolate and the SA113-wild-type genomes and reference genome (S. aureus NCTC8325, GenBank: NZ_LS483365.1) was finished by the MUMmer and LASTZ tools. SNP (single nucleotide polymorphism) annotations were discovered in the genomic alignment results among samples using the MUMmer and LASTZ.

Statistical Analysis

Data were analyzed by a Student’s t test. P values <0.05 were regarded as statistically significant. All data were analyzed in an SPSS software package (version 19.0, Chicago, IL, USA).