Cinacalcet exhibits rapid bactericidal and efficient anti-biofilm activities against multidrug-resistant Gram-positive pathogens

Summary

Infections caused by Gram-positive bacteria pose a serious threat to global public health. Drug resistance, dormant persister cells, and biofilm formation are the key challenges affecting the efficacy of antibiotics against Gram-positive bacterial infections. In this study, cinacalcet exhibited good inhibitory activity against multidrug-resistant Gram-positive bacteria, with minimum inhibitory concentrations (MICs) ranging from 3.13 μg/mL to 25 μg/mL. Cinacalcet displayed more rapid and stronger bactericidal activity against planktonic and persister cells of Staphylococcus aureus and Enterococcus faecalis compared with the antibiotics vancomycin or ampicillin, as well as potent inhibition and eradication of mature biofilms of methicillin-resistant S. aureus (MRSA) and linezolid-resistant E. faecalis (LRE). In addition, the robust antibacterial activity was demonstrated in vivo by a pneumonia infection model and a biofilm formation and deep-seated infection model. Collectively, these findings indicate that cinacalcet may be a promising new candidate antibiotic to combat infections caused by multidrug-resistant Gram-positive pathogens.

Graphical abstract

Highlights

• •Cinacalcet exhibits rapid bactericidal activity against Gram-positive bacteria
• •Inhibition and eradication of cinacalcet against biofilm of Gram-positive pathogens
• •Cinacalcet inhibits the expression of virulence- and biofilm-related genes
• •A superior anti-infective efficacy of cinacalcet in vivo

Drugs; Microbiology; Bacteriology; Microbiofilms

Introduction

Gram-positive bacteria are one of the most common pathogenic organisms and some of them have gradually become a major cause of hospital-acquired or community-acquired infections.¹ According to the latest report of China Antimicrobial Resistance Surveillance System (CARSS) and China antimicrobial surveillance network (CHINET) in 2022, bacterial resistance to antimicrobial drugs is becoming increasingly serious and prevalent with the widespread use or misuse of antibiotics. Antibiotic resistance is a major reason for poor treatment outcomes or treatment failure of infections; for example, infections attributed to methicillin-resistant Staphylococcus aureus (MRSA) and linezolid-resistant Enterococcus faecalis (LRE) are intensely formidable to cure and recur frequently, especially when invasive prosthetic devices are used.^2,3

In addition to antibiotic resistance, another threat to current antibiotic treatments arises from drug tolerance ascribed to persister cells or biofilm.^4,5 Persisters are a specific subgroup of bacteria that often employ dormant strategies to resist the killing effect of drugs under antibiotic or other stresses, and represent a very low percentage (typically less than 0.1%) that can survive even under antibiotic stress.^6,7 The lack of persister-killing activity is an important reason for the poor efficacy of anti-infective therapy with a various potent antibacterial drug. Bacterial biofilm is formed by bacteria in the process of reproduction and differentiation and adheres to the contact surface and secretes extracellular polymeric substance (EPS) matrix, lipoprotein, and other substances.⁸ Most Gram-positive microorganisms, such as staphylococci and enterococci, tend to form biofilm during the progression of infection.^9,10 Compared with planktonic cells, biofilms can protect biofilm-embedded bacteria from detergent solutions, antimicrobial agents, and environmental stress; hence eradication of biofilm-related infections is overwhelmingly difficult.¹¹ In the current healthcare environment, a multitude of chronic diseases are associated with biofilms, including endocarditis, osteomyelitis, infections of catheters and indwelling devices, gingivitis, and deep-seated infections of soft tissues, and more than 65% of all bacterial infections involve biofilm formation.^12,13,14 Therefore, the development of new antibacterial drugs that can efficiently inhibit drug-resistant bacteria and simultaneously kill persisters and inhibit or even eradicate biofilm formation is a current research hotspot and a serious challenge and urgent task for the clinical treatment of infectious diseases.

Despite the foreseeable crisis of antibiotic use, few new antibiotics are entering the market owing to the long development cycles and rapid acquisition of drug resistance.¹⁵ Drug repurposing is now a widely adopted approach by researchers that can effectively shorten research time and costs by reducing the need for pharmacokinetic and toxicity studies.¹⁶ A successful example of a drug developed using this approach is thioridazine, an antipsychotic compound that demonstrates potent activity against a wide variety of microorganisms including MRSA and methicillin-sensitive S. aureus (MSSA).^17,18 Cinacalcet (CIN), a drug that acts as a calcimimetic agent through the allosteric activation of the extracellular calcium-sensing receptor (CaSR), has been approved by the US Food and Drug Administration (FDA) for the treatment of secondary hyperparathyroidism (SHPT) owing to its safety and efficacy of reducing parathyroid hormone (PTH) levels.^19,20 However, the underlying antibacterial activity of CIN remains unclear. This study aimed to investigate the antibacterial, bactericidal, and anti-biofilm activity of CIN through in vivo and in vitro experiments, and briefly explore its possible mechanism(s) of action.

A series of experiments were designed to investigate the antibacterial and anti-biofilm activity of CIN against Gram-positive bacteria in vitro and in vivo (Figure 1). Superior antibacterial and bactericidal activities of CIN were observed against planktonic bacteria and persister cells, including S. aureus and E. faecalis. Moreover, an inhibitory and eradication effect of CIN on biofilm formation by S. aureus and E. faecalis was detected. Furthermore, in vivo anti-infectious activity of CIN was identified through a pneumonia mouse model and a deep-seated infection mouse model. Findings from the study may provide new insights into the repurposing of CIN for the treatment of Gram-positive bacterial infections.

Figure 1.

Design of experiments to investigate CIN activity against Gram-positive pathogen infections

Schematic demonstration of the antibacterial and anti-biofilm activity of CIN in vitro and in vivo.

Results

Promising antimicrobial activity of cinacalcet against multi-resistant Gram-positive bacteria

The inhibitory activity of CIN against Gram-positive bacteria was measured by agar dilution, and the MIC range of CIN was from 3.13 to 25 μg/mL (Table 1). CIN has favorable activity against MRSA (Table 1) and linezolid-resistant strains (Table 2). However, CIN did not affect the growth of Gram-negative bacteria in this study (Table S1). In addition, the inhibition activity of CIN on the planktonic growth of multi-resistant S. aureus and E. faecalis was further investigated by the bacteria automatic growth curve instrument. The data implied that CIN at concentrations above 12.5 μg/mL could completely inhibit growth of planktonic cells of MSSA CHS101 and MRSA YUSA145 (Figure 2A), and E. faecalis 16C51 and LRE 16C352 (Figure 2B), respectively.

Table 1.

MICs distribution of CIN against Gram-positive bacterial isolates

Species	Strain no.	Cinacalcet MIC distribution (μg/mL)
		1.56	3.13	6.25	12.5	25	MIC50/MIC90
MRSA	44	0	0	0	25 (56.8%)	19 (43.2%)	12.5/25
MSSA	28	0	0	0	22 (78.6%)	6 (21.4%)	12.5/25
E. faecalis	60	0	0	2 (3.33%)	58 (96.67%)	0	12.5/12.5
E. faecium	55	0	0	35 (63.6%)	20 (36.4%)	0	6.25/12.5
S. agalactiae	69	7 (10.1%)	35 (50.7%)	26 (37.7%)	1 (1.4%)	0	3.13/6.25

MIC₅₀/MIC₉₀, the MIC values for 50% or 90% of bacterial growth inhibition.

Table 2.

MICs of CIN against linezolid-resistant isolates

Isolate		Minimal inhibitory concentration (MIC, μg/mL)
		Cinacalcet	Linezolid
E. faecalis	16C83	12.5	8
	16C272	12.5	8
	16C340	12.5	8
	16C352	12.5	8
	16C359	12.5	8
E. faecium	NEFM8	6.25	8
	NEFM39	6.25	8
	EFM43	12.5	16
S. agalactiae	NSGC121	6.25	8
	NSGC122	3.13	8
	NSGC124	3.13	8
	NSGC125	3.13	8

Figure 2.

Efficient inhibition of cinacalcet against planktonic cells

(A) The planktonic cells of MSSA CHS101 and MRSA YUSA145, and (B) E. faecalis 16C51 and LRE 16C352 were treated with 1/8×, 1/4× , 1/2× , 1× MIC of CIN. The CIN MIC for clinical isolates of S. aureus and E. faecalis was 12.5 μg/mL. The OD₆₀₀ of the bacterial cells was then measured by Bioscreen C (Turku, Finland) at 1-h intervals for 16 h. TSB without antimicrobials was used as an untreated control. Data are shown as Mean ± SEM (n = 3). All experiments were performed in triplicate.

Cinacalcet exhibited stronger bactericidal activity against planktonic and persister cells compared with vancomycin, daptomycin, and ampicillin

Time-kill curve assays were used to examine the bactericidal activity of CIN against S. aureus and E. faecalis in the exponential growth phase. After 4× MIC treatment of CIN or daptomycin (DAP) for 3 h, the CFU of S. aureus planktonic cells was reduced by almost 3.5–4.5 log compared with the control, whereas vancomycin (VAN) treatment at 4× MIC slightly reduced the CFU (Figure 3A). When the treatment was extended to 24 h, the CFU of S. aureus significantly reduced to approximately half that of the drug-free control, and the effect was similar to that of DAP. In sharp contrast, VAN, a commonly used antibiotic, had virtually no bactericidal activity against the two strains of S. aureus (Figure 3A). Simultaneously, the bactericidal effect of CIN on E. faecalis 16C51 and 16C352 was tested. CIN exhibited significant bactericidal activity against both strains of E. faecalis after 1 h of treatment and killed approximately 5.5–6.7 log of the planktonic bacteria after 3 h, which is much stronger than the bactericidal activity of ampicillin (AMP) and VAN (Figure 3B). These results fully manifest the rapid and efficient bactericidal activity of CIN against planktonic bacteria, which is essential for the treatment of diseases such as sepsis where there is an urgent need to the kill bacteria.

Figure 3.

Cinacalcet displayed rapid and effective bacterial killing for planktonic and persister cells

(A and B) (A) MSSA CHS101 and MRSA YUSA145, and (B) E. faecalis 16C51 and LRE 16C352 in exponential growth phase were challenged with antimicrobials at 4× MIC. Bacteria were counted on TSB agar plates at 0, 1, 3, and 24 h after challenge.

(C) S. aureus CHS101 and YUSA145 in stationary growth phase were challenged with DAP (50× MIC), VAN (50× MIC), or CIN (10× MIC).

(D) E. faecalis 16C51 and 16C352 in plateau period were treated with AMP (50× MIC), VAN (50× MIC), or CIN (10× MIC). Bacterial counts were determined on TSB agar plates at 0, 24, 48, 72, 96, and 120 h after challenge. The MICs of DAP, VAN, or CIN for S. aureus CHS101 and YUSA145 were 1, 1, or 12.5 μg/mL, respectively. The MICs of AMP, VAN, or CIN for E. faecalis 16C51 and 16C352 were 2, 1, or 12.5 μg/mL, respectively. Bacterial growth in TSB without antimicrobials was used as an untreated control. Data are representative of three independent experiments.

The dormant persister cells of Gram-positive bacteria are predominantly responsible for the stubbornness of chronic diseases.⁴ Therefore, the anti-persister activity of CIN against S. aureus and E. faecalis was further investigated. Bacteria in the stationary phase were used to evaluate the killing efficacy of CIN with 10× MIC. After 24 h of treatment, CIN completely killed S. aureus, whereas the control group of DAP and VAN with a concentration of 50× MIC had almost no bactericidal activity. The DAP control group exhibited relatively robust bactericidal activity after 48 h (Figure 3C). For E. faecalis, CIN treatment also absolutely killed strain 16C352 within 24 h and caused an approximately 6.7 log reduction of strain 16C51, which was in marked contrast to the absence of bactericidal activity with AMP and VAN (Figure 3D). In summary, CIN killed persister cells of S. aureus and E. faecalis faster and more effectively than VAN or AMP, strongly indicating that CIN may have prominent antibacterial activity against the persister cells of Gram-positive bacteria.

Cinacalcet exhibited robust inhibition and eradication activity against bacterial biofilms

Biofilm formation is commonly among Gram-positive bacteria such as staphylococci and enterococci, and biofilms are a difficult challenge affecting the efficiency of antibiotics against antibacterial infection.^10,21 Therefore, biofilm assays were performed to evaluate the inhibition and eradication of CIN against S. aureus and E. faecalis biofilms. CIN with sub-MIC amounts (1/8×–1/2× MIC) could significantly inhibit the biofilm formation of S. aureus in a dose-dependent manner, and especially at 1/2× MIC, where the inhibitory effect on the biofilm of two MSSA strains and two MRSA strains reached approximately 80% (Figure 4A). However, CIN at sub-MIC doses (1/8×–1/2× MIC) did not have an inhibitory effect on biofilm formation of isolates of E. faecalis (Figure 4B). In addition, the eradication of mature biofilms in the S. aureus and E. faecalis isolates was further evaluated by crystal violet staining (Figures 4C and 4D). CIN at 2× and 4× MIC was able to significantly eradicate nearly half or more of S. aureus and E. faecalis biofilms, whereas 8× MIC removed at least 90% of the biofilm. The eradication of mature biofilms of S. aureus and E. faecalis were further confirmed by confocal laser scanning microscopy (CLSM) and cell viability assay. Consistent with the results of crystal violet staining, after 2×, 4× and 8× MIC of CIN treatment, the eradication of the mature biofilms of S. aureus (Figure 4E) and E. faecalis (Figure 4F) was visualized with CLSM. The live bacteria were quantified as a percentage of the total bacteria in each sample, and the survival rate of multidrug-resistant strains YUSA145 or 16C352 after CIN treatment was less than 50%, which was significantly different from control (Figure 4G and Table S2). For cell viability assays, CIN (4× and 8× MIC) treatment of mature biofilms resulted in more than 4 log reduction of bacteria embedded in mature biofilms (Figure 4H). Collectively, these results indicate that CIN has excellent anti-biofilm ability against S. aureus and E. faecalis.

Figure 4.

The inhibition and eradication activity of cinacalcet against biofilms

(A–D) For the inhibition of biofilms of S. aureus (A) and E. faecalis (B), bacteria were treated with CIN at 1/8×, 1/4×, and 1/2× MIC for 24 h. For the eradication of mature biofilms of S. aureus (C) and E. faecalis (D), bacteria were challenged with CIN at 2×, 4×, and 8× MIC.

(E) Mature biofilms of S. aureus isolates CHS101 and YUSA145 were eradicated with CIN.

(F) Mature biofilms of E. faecalis isolates 16C51 and 16C352 were eradicated with CIN. Original magnification ×40; scale bar, 50 μm. Strains were cultured to form mature biofilms, then bacterial cells were challenged by CIN at 2×, 4× and 8× MIC for 48 h. The biofilms were incubated with PI (red-dead) and SYTO 9 (green-live) and photomicrographed by confocal laser microscopy.

(G) The live bacteria were quantified as a percentage of the total bacteria in each sample. Error bars represent SD calculated following error propagation principles.

(H) The number of bacterial in mature biofilms after treatment with CIN. The MICs of CIN for clinical isolates of S. aureus and E. faecalis were 12.5 μg/mL. TSBG without antimicrobial was used as an untreated control (CTRL). The data are representative of three biological replicates. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, compared with the CTRL group.

Cinacalcet possessed the ability to inhibit virulence of S. aureus

RT-qPCR analyses were performed to examine the influence of CIN in regulating biofilm- and virulence-related genes. As illustrated in Figures 5A and 5B, the expression levels of regulatory genes agrA and saeR, biofilm-related genes fnbA, atl, and icaB, and virulence factor genes hla, hlb, hld, hlg, α-psm, sbi, and spa² were reduced in S. aureus after treatment with 1/2× MIC of CIN. Moreover, CIN inhibited the hemolysis and pigmentation of S. aureus. The hemolytic activity of MRSA USA300 was significantly inhibited by CIN at a low concentration of 1.56 μg/mL and reduced by about 70% at 25 μg/mL, with a concentration-dependent manner (Figure 5C). CIN at low concentrations was also highly effective in inhibiting the pigmentation of S. aureus, with 3.13 μg/mL and 6.25 μg/mL reducing pigmentation by approximately 40% and 50%, respectively (Figure 5D). CIN treatment at concentration of 1.56–25 μg/mL for 24 h did not affect the amount of MRSA USA300 (Figure 5E). The decreased virulence might be because of the downregulation of the genes mentioned above.

Figure 5.

Cinacalcet inhibits S. aureus virulence

(A and B) (A) SA113 and (B) CHS101 cultures were incubated with 1/2× MIC of CIN in 6-well microplates for the formation of biofilms. After 24 h, RNA was extracted from the bacteria, and the expression levels of transcriptional regulatory factor, virulence factor, and biofilm-related genes were detected by RT–qPCR. DMSO-treated strains were used as a reference, and house-keeping gene gyrb was used as control (RNA level = 1.0). The MIC values of CIN against SA113 and CHS101 was at 12.5 μg/mL.

(C) CIN strongly inhibits the hemolytic activity of MRSA USA300.

(D) Inhibitory effect of CIN on pigmentation of MRSA USA300.

(E) Planktonic cell numbers of USA300 bacteria treated with CIN (1.56–25 μg/mL) for 24 h. Data are presented as mean ± SEM. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, compared with the untreated group. Experiments were repeated at least three times with three replicates.

Anti-infective efficacy of cinacalcet in vivo

In the above experiments, the antibacterial, anti-biofilm, and bactericidal activities of CIN against bacteria such as S. aureus and E. faecalis were examined in vitro, but the antibacterial effects of CIN in vivo were not known. Hence, a pulmonary infection mouse model using MRSA strain USA300 and an in vivo biofilm formation and deep infection mouse model using E. faecalis ORG1F were designed to assess the antibacterial and anti-biofilm roles of CIN, respectively. The infection models and the strategy of administration are detailed in Figure 6A.

Figure 6.

Cinacalcet displayed high in vivo antimicrobial efficacy in different mouse models

(A) The strategy of administration for mouse model. In the pulmonary infection model, mice were intraperitoneally treated with 10 mg/kg CIN every 24 h for 2 days or 25 mg/kg VAN every 12 h for 2 days after intranasal challenge with lethal or non-lethal doses of MRSA USA300.

(B) Survival was monitored for 3 days after infection with MRSA. Mice were treated with saline, CIN, or VAN after intranasal challenge with a lethal dose (2×10⁸ CFU) of MRSA USA300. N = 10.

(C) CFU of MRSA USA300 in the lungs of mice treated with saline, CIN, or VAN after intranasal challenge with a non-lethal dose (2×10⁷ CFU) of MRSA USA300. N = 5. For the deep-seated infection model, 24 h after inoculation with 2–3×10⁶ CFU of E. faecalis OG1RF, mice were intraperitoneally injected with 40 mg/kg CIN or administered with 100 mg/kg VAN every 12 h for 2 days.

(D) Biofilm formation was investigated by Gram staining of the deeply infected mice after saline, VAN, or CIN treatment. Original magnification ×40; scale bar, 50 μm. Black arrows indicate biofilm bacterial. N = 5.

(E) CFU of E. faecalis OG1RF in the thighs of mice intraperitoneally treated with saline, VAN, or CIN after deep infection. The untreated group was sacrificed 24 h after infection; mice in the other groups were sacrificed 48 h after treatment with saline, VAN, or CIN. Data are presented as mean ± SEM. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, compared with the untreated group.

The pulmonary infection model was performed with lethal and non-lethal doses of S. aureus administered intranasally. The lethal dose mice were used to assess the survival of mice after CIN treatment, and because high doses can cause mouse death, the lungs cannot be taken in time to analyze the number of viable bacteria. In contrast, the low-dose group does not cause mouse mortality and therefore can be used to detect live bacteria counts in the lungs of mice after CIN treatment. The results showed that 60% of mice infected with high-dose S. aureus survived after CIN treatment, which was the same as the survival rate of mice given VAN in the control group and in contrast to the severe mortality rate of 80% in the saline group (Figure 6B). Consistent with the above experiments, CIN treatment reduced the CFU of S. aureus in the lungs by approximately 3.5 log after the low dose of S. aureus, and its effect remained similar to that of VAN (Figure 6C). These results prove that CIN also displays highly effective potential against MRSA in vivo.

To assess whether CIN has an anti-biofilm effect in vivo, E. faecalis OG1RF was injected into the thighs of mice, which were then incubated for 24 h to allow complete biofilm formation, thus establishing a mature model of biofilm formation and deep infection. CIN or VAN was then administered, and Gram staining of the injection site was performed 24 h later. The biofilm was markedly reduced or almost absent in the cross-section of the injected site after CIN or VAN treatment, whereas in the control group without any treatment, a large number of bacteria could be seen growing and aggregating on the biofilm, with attachment to muscle cells (Figure 6D). Meanwhile, muscle tissue from the injection site was homogenized, weighed and spread on agar plates to detect the CFU of viable bacteria. CIN treatment significantly decreased the number of viable bacteria by approximately 3 log, and the antibacterial effect was consistent with that of VAN treatment (Figure 6E). These results suggested the potential of CIN with superior antibacterial ability to be considered for the clinical treatment of antibiotic resistance.

Cinacalcet impaired the permeability of bacterial cell membranes

The destructive effect of CIN on cell membrane permeability and membrane depolarization can be assessed by SYTOX and DiSC3(5) dye assay. The fluorescence intensity of SYTOX was increased 1.2-fold immediately after the addition of 2× MIC of CIN to S. aureus compared with the blank control, and this increase was slightly higher than that of DAP (Figure 7A). There was no obvious change in fluorescence intensity within 60 s, showing that CIN could rapidly disrupt the membrane permeability (Figure 7A). Consistently, DiSC3(5) staining revealed that the fluorescence intensity in S. aureus was enhanced by approximately 6-fold and 10-fold immediately after the addition of 1× MIC or 2× MIC of CIN, respectively, compared with the blank control (Figure 7B). This indicated that CIN exhibited a strong depolarizing effect on the membrane, similar to that of 0.1% Triton X-100. Previous studies have shown that ROS production is accompanied by membrane damage and accelerates bacterial death.^22,23 Therefore, the production of ROS was detected following the treatment of S. aureus with CIN. ROS levels increased after treating bacteria with 4× MIC of CIN for 30 min and were suppressed to normal cellular levels with the addition of the ROS inhibitor acetylcysteine (NAC) at 10 mM (Figure 7C). Once NAC was present, the minimum bactericidal concentrations (MBC) of CIN against S. aureus and E. faecalis were elevated by 2-fold and 4-fold, respectively, reflecting that ROS production may be an important factor in enhancing bactericidal efficacy (Figure 7D). In addition, transmission electron microscopy (TEM) analysis was employed to verify that CIN treatment could kill bacteria by disrupting the cytoplasmic membrane. TEM of CIN-treated (4× MIC) S. aureus and E. faecalis revealed distinct membrane damage and efflux of cytoplasmic components (Figure 7E). The results of this experiment provide clear support for the effective antibacterial activity of CIN.

Figure 7.

The effect of CIN on the disruption of bacterial cytoplasmic membrane

(A and B) Membrane permeability (A) and depolarization (B) by CIN against S. aureus SA113.

(C) Fluorescence intensity of S. aureus SA113 treated with CIN (4× MIC) or the combination of CIN (4× MIC) and NAC (10 mM). Data are presented as mean ± SEM. ∗∗∗p<0.001 (Student’s t test).

(D) An increase in the MBC of CIN for S. aureus SA113 and E. faecalis OG1RF in the presence of NAC (10 mM). N = 3.

(E) TEM characterization of S. aureus SA113 and E. faecalis OG1RF with and without CIN treatment at 4× MIC. Red arrows represent damaged bacterial cell membranes. The MIC of CIN against S. aureus SA113 and E. faecalis OG1RF was 12.5 μg/mL.

Whole-genome sequencing and proteomic analysis of S. aureus treated with cinacalcet

To explore the potential target of CIN in S. aureus, a CIN-induced resistant experiment of S. aureus was conducted (Figure 8A). Biofilm formation was significantly reduced in the CIN-induced strains compared with the wild-type strains of S. aureus SA113 and CHS101 (Figure 8B). The genetic mutations in the CIN-induced tolerant S. aureus SA113 were detected by whole-genome sequencing. A total of 17 genes acquired non-synonymous or stop-gain mutations in the CIN-induced resistant clones of S. aureus SA113, and Table 3 shows the details of these mutations. Among the 17 genes, there were two separate mutations in the gene encoding ClpX protease, which has been reported to play a pivotal role in S. aureus growth and virulence.⁴ Moreover, the genetic mutations located in ClpX of CIN-induced tolerant S. aureus SA113 were further validated through PCR and Sanger sequencing. We reasonably speculate that ClpX may be related to the antibacterial activity of CIN against S. aureus.

Figure 8.

Whole-genome sequencing and proteomic analysis

(A) CIN-induced resistance in S. aureus SA113. The resistant strains were selected under exposure to CIN from 6.25 μg/mL until 400 μg/mL.

(B) Biofilm formation of different clones of resistant strains.

(C) Volcano plots of protein expression levels of S. aureus SA113 treated with 1/2× MIC of CIN. DMSO was used as the control treatment. Blue dots and red dots represent proteins that are downregulated and upregulated by CIN, respectively.

(D) GO pathway terms of the differentially expressed proteins between S. aureus SA113 and S. aureus SA113 treated with CIN.

(E) Protein-protein interaction (PPI) networks were used to analyze the representative proteins and signal transduction pathways affected by 1/2× MIC of CIN treatment in S. aureus SA113. Proteins downregulated or upregulated by CIN in S. aureus are marked in green or red, respectively.

Table 3.

Mutations in clones of S. aureus SA113 detected by whole-genome sequencing

Ref_Gene_ID	Mutation type	NC mutation	AA mutation	Subject description
GM000095	Nonsynonymous	G1188A	V400L	ATP-dependent helicase DinG
GM000237	Nonsynonymous	A976G	N326D	Low specificity L-threonine aldolase
GM000702	Nonsynonymous	C127T	P43S	RNase adapter RapZ
GM000792	Nonsynonymous	C44T	S15L	DUF1129 family protein
GM000960	Nonsynonymous	A175G	N59D	TIGR01741 family protein
GM001072	Nonsynonymous	G1963A	E655K	4ʹ-Phosphopantetheinyl transferase superfamily protein
GM001154	Nonsynonymous	G1963A	E655K	Helix-turn-helix domain-containing protein
GM001197	Nonsynonymous	G1868A	G623D	DNA gyrase subunit B
GM001274	Nonsynonymous	C1069T	R357C	Amidase domain-containing protein
GM001732	Nonsynonymous	G349A	V117I	NADP-dependent oxidoreductase
GM001753	Nonsynonymous	G179A	G60D	Alkaline shock response membrane anchor protein AmaP
GM001782	Nonsynonymous	C41T, T980G	S14F, L327R	ATP-dependent Clp protease ATP-binding subunit ClpX
GM001964	Nonsynonymous	C419T	A140V	Aromatic acid exporter family protein
GM002004	Nonsynonymous	T1216C	S406P	PBSX family phage terminase large subunit
GM002180	Nonsynonymous	G622A	D208N	DNA polymerase III subunit gamma/tau
GM002610	Premature_stop	A319T	K107∗	N-acetyltransferase
GM002670	Premature_stop	C745A	Q249∗	Pur operon repressor

S. aureus SA113 was subcultured in TSB containing 6.25 μg/mL to 400 μg/mL CIN in continuous passages, with concentrations doubled every 10 days. NA, nucleotide; AA, amino acid. An asterisk indicates a stop tanslation

A total of 1,741 proteins were detected by proteomic analysis in S. aureus SA113 (Table S4). CIN treatment resulted in a significant increase in the abundance of 101 proteins, and a significant decrease in the abundance of 111 proteins (Figure 8C), including biofilm-related and virulence factors SaeS, Atl, α-Psam1, α-Psam4, Spa, Sbi, Hld, HlgB, HlgC and Hly. (p≤0.05, 2-fold increase or decrease, Table S5). This was consistent with previous findings of CIN inhibiting virulence factor and biofilm formation in S. aureus. In addition, functional analysis of the differentially expressed proteins was conducted according to the gene ontology (GO) terms of biological process, molecular function, and cellular component (Figure 8D) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (Figure S3), and a protein-protein interaction network (PPI) (Figure 8E) was constructed. The PPI network suggested that a significant enrichment with downregulated S. aureus infection protein, various metabolism processes, and a part of the two-component system regulators were upregulated.

Discussion

The FDA-approved clinical drug CIN is an allosteric activator of CaSR and is commonly used for the treatment of secondary hyperparathyroidism associated with chronic kidney disease or renal failure.¹⁹ The present study showed that CIN not only possess effective antibacterial and anti-biofilm activity against multidrug resistant Gram-positive bacteria in vitro, but also has an efficient and rapid bactericidal capacity superior to antibiotics such as VAN. In addition, CIN treatment achieved the equivalent antibacterial or anti-biofilm effects of VAN in a pneumonia mouse model and a biofilm formation and deep infection mouse model.

Chronic infections caused by Gram-positive bacterial pathogens have become a serious threat to human health owing to recalcitrant persister cells and biofilm formation that exhibit tolerance to conventional antibiotics.⁴ Thus, the dormant persisters and biofilms pose a marked challenge to improving the clinical treatment of chronic infections with Gram-positive bacteria. In this study, CIN not only killed most planktonic cells of S. aureus and E. faecalis more rapidly and effectively at 3 h compared with conventional antibiotics (VAN, DAP, or AMP), but also killed all the persistent cells of S. aureus and E. faecalis isolates within 48 h (Figure 3). This reflects the advantage of CIN over the above antibiotics in terms of rapid bactericidal activity. The remarkable killing ability of CIN may be related to its strong membrane permeability and depolarizing activity (Figure 7). Furthermore, biofilm—a protective extracellular matrix formed by bacteria—has also been linked to the persistence of many infections such as those associated with indwelling devices.¹¹ Indeed, many compounds have exhibited promising anti-biofilm activities, but only a few have been tested or validated by in vivo models, and to date, there are no FDA-approved antibiotics that specifically target bacteria as a complex community.²⁴ However, this study demonstrates that CIN can inhibit and eradicate biofilms in experiments conducted in vivo and in vitro. Thus, it can be concluded that CIN is an effective promising agent for the treatment of refractory chronic infections.

Multidrug resistant Gram-positive pathogenic bacteria have become a major cause of various community- or hospital-acquired infections.²⁵ There are many published studies describing the role of linezolid, a new antibiotic approved in 2000 for the treatment of infections caused by MRSA and VAN-resistant enterococci (VRE).^26,27 To date, resistance to linezolid is prevalent in Gram-positive strains isolated from clinical samples worldwide.²⁸ However, our study discovered that CIN has inhibitory activity against linezolid-resistant isolates, including E. faecalis, E. faecium, and Streptococcus agalactiae (Table 2), demonstrating its broad-spectrum antibacterial activity. The clinical significance of the effect of CIN on the multidrug resistant pathogens requires further investigation.

In this study, whole-genome sequencing and proteomic analysis were applied to gain insights into the molecular mechanism of CIN on S. aureus. The sequencing data revealed that a total of 17 genes were mutated, with two loci mutations in ClpX. ClpX protein belongs to the class Clp/Hsp100 subfamily of AAA+ proteins, which is involved in various cellular processes and plays a pivotal role in the growth of S. aureus.⁴ ClpX is reported to bind to substrate polypeptides through a peptide recognition motif, and then ClpX enters the chamber of the bound ClpP protease through its central pore, harnessing the energy of ATP hydrolysis to force unfolding and translocation of substrate polypeptides for degradation.^29,30 The proteomic analysis also showed that CIN significantly increased the expression of ClpX and putative hemin import ATP-binding proteins (HrtA and HrtB) (Figure 8C and Table S4), and decreased the levels of virulence factor-related proteins in S. aureus, including hemolysin family proteins (Hly, hlb and Hlg), immunoglobulin-binding protein (Sbi) and immunoglobulin G-binding protein A (Spa) (Figure 8C and Table S5). Hemolysin virulence-related proteins are known to be downregulated by mutated ClpX.^31,32 This implies that the antibacterial and anti-biofilm activities of CIN against S. aureus might be associated with the increased expression of ClpX. However, the specific mechanism of CIN against S. aureus needs further research.

In conclusion, this study confirmed that CIN exerts antibacterial and anti-biofilm activity against Gram-positive pathogens by targeting and disrupting bacterial cell membranes. CIN has a good bactericidal effect on the planktonic and persister cells of Gram-positive bacteria. More importantly, in vivo antibacterial activity of CIN was demonstrated in a pneumonia mouse model and a deep-seated infection mouse model. CIN has shown potential to be developed as an antimicrobial agent for the treatment of Gram-positive bacterial infections.

Limitations of the study

This study suggests that CIN displays antimicrobial activity by targeting the cell membrane, and whole-genome sequencing and proteomic analysis indicate that ClpX may be associated with the activity of CIN in S. aureus. However, the exact mechanism by which CIN exerts its antibacterial effects on S. aureus remains largely unknown. Therefore, the detailed molecular mechanism by which CIN exhibits favorable antibacterial activity in Gram-positive bacteria requires further investigation. Furthermore, CIN (60−360 mg/day) reduces serum calcium levels in patients with parathyroid carcinoma,¹⁹ but this effect was not assessed in this study, thus, further research is need to evaluate and investigate the side effects of high-concentration administration.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Experimental models: Organisms/strains
BALB/c	GemPharmatech (Jiangsu, China)	N/A
C57BL/6J	GemPharmatech (Jiangsu, China)	N/A
Bacterial and virus strains
S. aureus	ATCC	USA300
S. aureus	ATCC	SA113
E. faecalis	ATCC	OG1RF
E. faecalis	ATCC	29212
S. aureus	the 6th Affiliated Hospital of Shenzhen University Medical school	Clinical isolates
E. faecalis	the 6th Affiliated Hospital of Shenzhen University Medical school	Clinical isolates
E. faecium	the 6th Affiliated Hospital of Shenzhen University Medical school	Clinical isolates
S. agalactiae	the 6th Affiliated Hospital of Shenzhen University Medical school	Clinical isolates
P. aeruginosa	the 6th Affiliated Hospital of Shenzhen University Medical school	Clinical isolates
K. pneumoniae	the 6th Affiliated Hospital of Shenzhen University Medical school	Clinical isolates
A. baumannii	the 6th Affiliated Hospital of Shenzhen University Medical school	Clinical isolates
Biological samples
S. aureus	the 6th Affiliated Hospital of Shenzhen University Medical school	CHS101
S. aureus	the 6th Affiliated Hospital of Shenzhen University Medical school	CHS128
S. aureus	the 6th Affiliated Hospital of Shenzhen University Medical school	YUSA145
S. aureus	the 6th Affiliated Hospital of Shenzhen University Medical school	YUSA151
E. faecalis	the 6th Affiliated Hospital of Shenzhen University Medical school	16C28
E. faecalis	the 6th Affiliated Hospital of Shenzhen University Medical school	16C51
E. faecalis	the 6th Affiliated Hospital of Shenzhen University Medical school	16C340
E. faecalis	the 6th Affiliated Hospital of Shenzhen University Medical school	16C352
Chemicals, peptides, and recombinant proteins
Cinacalcet	MedChemExpress	HY-70037A
Vancomycin	MedChemExpress	HY-B0671
Daptomycin	MedChemExpress	HY-B0108
Linezolid	MedChemExpress	HY-10394
Ampicillin	MedChemExpress	HY-B0522
Propidium Iodide	Invitrogen	P1304MP
SYTO9	Invitrogen	S34854
SYTOX	Invitrogen	S7020
DiSC3(5)	Invitrogen	D306
DCFH-DA	MedChemExpress	HY-D0940
N-acetyl-L-cysteine	MedChemExpress	HY-B0215
Critical commercial assays
4% rabbit red blood cells	Solarbio	S9452
RNeasy Mini Kit	Qiagen	74104
SYBR Premix Ex Taq II Kit	TaKaRa Biotechnology	RR390W
Gram Stain Kit	Solarbio	G1060
Deposited data
Raw whole-genome sequencing data	This paper	Sequence Read Archive (SRA) database: PRJNA755754 (NCBI: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA755754)
Oligonucleotides
Biofilm-related genes (Table S2)	This paper	N/A
Virulence-related genes (Table S2)	This paper	N/A
House-keeping gene gyrb (Table S2)	This paper	N/A
Software and algorithms
ImageJ	National Institutes of Health, USA	https://imagej.nih.gov/ij/
GraphPad Prism 8.0	GraphPad Software, La Jolla, CA	N/A
Illustrator CC/2021	Adobe Systems	https://www.adobe.com/
OmicsBean	OmicsBean platform	http://www.omicsbean.cn/

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Peiyu Li (lipeiyu199@gmail.com).

Materials availability

The study did not generate any unique reagents.

Experimental model and subject details

Strains

S. aureus strains SA113 (ATCC35556), USA300 (ATCC BAA-1556), and E. faecalis ATCC 29212 and OG1RF (ATCC 47077) were purchased from American Type Culture Collection. The clinical isolates collected from the Shenzhen Nanshan People’s Hospital, the 6th Affiliated Hospital of Shenzhen University Medical School. The microbiology laboratory usage license number is 0305030121, certified by Science and Technology Commission of Guangdong Municipality.

Animals

Mice aged 6–8 weeks (18–22 g) were purchased from GemPharmatech (Jiangsu, China) and housed in a temperature-controlled room. The animal protocols were carried out in accordance with the guidelines for Care and Use of Laboratory Animals of the experiments were approved by the Committee of Animal Ethics of the Ethics Committee of Shenzhen University Medical School. The program (No. A202200872) was performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the ethical standards of Shenzhen Nanshan People’s Hospital and were approved by the Ethics Committee of Shenzhen University.

Method details

Bacterial isolates and growth conditions

Strains of S. aureus (MRSA, n = 44 and MSSA, n = 28), E. faecalis (n = 60), E.faecium (n = 55), and S. agalactiae (n = 69) were isolated from different clinical specimens of individual patients at Shenzhen Nanshan People’s Hospital from 2011 to 2015. The isolates were sourced from urine, secretions, blood, bile, sputum, pus, etc. (Figure S1) and were identified with standard methods using a VITEK 2 system (BioMérieux, Marcy l’ Etoile, France). E. faecalis ATCC 29212 and OG1RF (ATCC 47077), and S. aureus USA300 were tested as quality control strains. Strains of S. aureus and E. faecalis were cultured in tryptic soy broth (TSB), with or without added 0.5% glucose (TSBG). Bacteria were routinely grown at 37°C and 220 rpm. The identified strains were stored at −80°C in 15% glycerol containing with TSB for further investigation.

MIC and MBC assays

The MICs of antimicrobial agents were detected by the broth microdilution method in cation-adjusted Mueller–Hinton broth (CAMHB) according to the Clinical and Laboratory Standards Institute guidelines (CLSI M100, 29th ed.). For the MBC assay, the well of the above MIC test was spread on a TSB agar plate and incubated overnight at 37°C. The MBC was considered as the lowest concentration of an antimicrobial agent needed to kill 99.9% of the bacteria or leave <5 colonies on the plate. Cinacalcet-HCl (CIN), ampicillin (AMP), vancomycin (VAN), and daptomycin (DAP) were purchased from MCE (Princeton, United States).

Bacterial growth curve assay

Strains of S. aureus and E. faecalis were grown to stationary phase (∼16 h) and then diluted (1:200) in TSB. Subsequently, bacteria were cultured at 37°C in the indicated concentration of antibiotics with shaking at 220 rpm and growth was monitored by measuring the OD₆₀₀ at 1 h intervals for 16 h by Bioscreen C (Turku, Finland). All experiments were performed in biological triplicates.

Time-kill dynamic curve assay

For the bactericidal experiment of planktonic cells, overnight-cultured S. aureus and E. faecalis were diluted (∼10⁷ CFU) into TSB and then the antibiotics were added to a final concentration of 4× MIC. A colony count was performed after 0, 1, 3, and 24 h. For the anti-persister assay, strains of S. aureus and E. faecalis were grown to the stationary phase (∼16 h) in TSB. Cells were plated for CFU counts and challenged with the antibiotics AMP, VAN, or CIN (for S. aureus) and DAP, VAN, or CIN (for E. faecalis), respectively. At 0, 24, 48, 72, and 96 h, an aliquot of cells was removed, washed with 0.9% NaCl, and plated to enumerate survivors. Data are representative of three independent experiments.

Biofilm assay

A detailed protocol has been previously reported.³³ For the inhibition of biofilm assay, isolates of S. aureus and E. faecalis were cultured overnight in TSB at 37°C and 220 rpm. The cultures were then diluted 1:200 and inoculated into a polystyrene 96-well plate with 200 μL of TSBG containing a concentration of 1/8, 1/4, or 1/2× CIN. For the eradication of biofilm assay, plates were incubated at 37°C for 24 h to form matured biofilms. The medium was carefully removed, and the wells were gently washed three times with PBS. Subsequently, 200 μL fresh medium containing 2×, 4×, or 8× MIC of CIN was carefully added to each well for 48 h. The biofilms were stained with crystal violet and detected by optical density (OD₅₇₀). The data are representative of three biological replicates.

Biofilm imaging assay

As previously described,⁹ the overnight cultures of isolates of S. aureus or E. faecalis were incubated into a Fluoro Dish and then treated with CIN (2×, 4×, and 8× MIC) for 48 h. The biofilms were washed with PBS and then stained with 5 μM SYTO 9 Green Fluorescent Nucleic Acid Stain (Thermo Fisher Scientific, United States) and 10 μM PI (Thermo Fisher Scientific, United States). The biofilms were visualized and photographed with a confocal fluorescence microscope (Olympus, Japan).

Imaging analysis

Imaging analysis was conducted using ImageJ software and a previously reported method with slight modification.³⁴ The total bacterial was evaluated by calculating the number of pixels corresponding with the viable bacteria in the image (green), then calculating the number of pixels corresponding with the dead (red) bacteria in the image, and finally adding to find the number of pixels corresponding with total bacteria. The live bacteria were quantified as a percentage of the total bacteria in each sample. Data are presented as mean ± SD.

Cell viability assay

The overnight cultures of isolates of S. aureus or E. faecalis were incubated into 6-well plate and then treated with CIN (2×, 4×, and 8× MIC) for 48 h with replaced fresh medium every day. The biofilms were washed with PBS and harvested with cell scraper, and serial dilutions of samples were plated on TSB agr plates for bacterial counting.

Hemolytic activity

MRSA USA300 isolates were inoculated into 6 mL TSB at 1:200 and incubated at 37°C for 24 h with CIN (1.56–25 μg/mL). Bacteria were centrifuged at 4,000 rpm for 10minat 4°C and the supernatant culture medium was collected and passed through a 0.22-μM filter. The filtered supernatant and 1% rabbit red blood cells were mixed in a 1.5 EP tube, and then incubated at 37°C for 15 min. Finally, the mixtures were centrifuged for 15minat 4,000 g, and 100 μL of supernatant was gently transferred into a new 96-well plate and the absorbance was read at 550 nm. All experiments were repeated three times.

Pigment assay

Overnight cultures of S. aureusUSA300 were diluted 1:200 in TSB medium and incubated at 37°C for 24 h with CIN (1.56–25 μg/mL). Subsequently, bacteria were centrifuged at 4,000 rpm for 10 min and washed twice with 1× PBS. The pigment was extracted thrice with methanol. The optical density was detected at 450 nm. The data are representative of three biological replicates.

RNA extraction and RT-qPCR

Overnight cultures of strains of S. aureus were incubated with CIN (1/2× MIC) in 6-wells microplates to form biofilms. After 24 h, the bacteria were harvested by centrifuging and then the RNA was extracted using a RNeasy Minikit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The RNA was reverse-transcribed and used for real-time quantitative PCR. The primers for the biofilm-related genes and virulence factor are listed in Table S3. All experiments were performed in triplicate.

Mouse model of infection

Female mice aged 6–8 weeks were purchased from GemPharmatech and used for this in vivo experiment. In the pulmonary infection model, BALB/c mice were anesthetized and challenged with 2×10⁸ CFU (high-dose group, 10 mice per group) and 2×10⁷ to 4×10⁷ CFU (low-dose group, 5 mice per group) of S. aureus, respectively. The mice were then injected intraperitoneally with saline, VAN, or CIN. VAN and CIN were separately administered at doses of 25 mg/kg every 12 h or 10 mg/kg every 24 h for 2 days. Mice in the high-dose group were used for 72-h survival monitoring. In the low-dose bacterial challenge group, the effect of drug treatment could be perfectly detected as the bacterial load did not cause mouse mortality. Therefore, the lungs of the low-dose group were taken at 48 h post-infection under aseptic anesthesia, homogenized, spread on TSB agar plates according to a serial dilution, and then incubated overnight at 37°C for live bacteria counts.

For the biofilm formation and deep-seated infection model, stationary phase E. faecalis OG1RF was centrifuged and re-suspended in PBS, then 100 μL of 2–3×10⁶ bacteria were injected into the right thigh of each C57BL/6 mouse and the infection was allowed to develop for 24 h before treatment, resulting in the formation of a severe, intractable deep infection (Conlon et al., 2013). VAN was then administered intraperitoneally at 100 mg/kg twice every 12 h for four times, and CIN was administered at 40 mg/kg for twice. Mice in the untreated group were sacrificed 24 h after infection, while mice in the other groups were sacrificed at 48 h. Thigh sites of injected bacteria were aseptically isolated, homogenized, and then diluted and spread on TSB agar for counting to assess their antibacterial effect in vivo. In addition, the isolated thighs of the inoculated bacterial sites were subjected to tissue sectioning followed by Gram staining to observe biofilm formation.

Histologic analyses

The formalin-fixed thighs were embedded with paraffin and sectioned, then Gram staining was performed according to the kit instruction (Solarbio Life Sciences). Briefly, slides were stained with crystal violet for 1 min, washed with distilled water, and then stained with iodine solution for 1 min and washed with distilled water. Next, the slides were decolorized until the crystal violet was washed away, then the sections were washed with water and dehydrated. Slides were counterstained with Gram’s safranin O counterstain, then washed with distilled water and air-dried. Slides were photomicrographed using a Color Camera Nikon DS-Fi3.

Membrane permeability and depolarization assay

S. aureus SA113 and E. faecalis OG1RF were cultured overnight in TSB medium at 37°C. Subsequently, 10⁷ CFU/mL bacteria were added to HEPES medium (5 mM HEPES, 20 mM glucose, pH 7.4) and incubated with 1 μM SYTOX (excitation λ = 488 nm, emission λ = 523 nm) or 1 μM diSC3(5) (excitation λ = 622 nm, emission λ = 673 nm) for 2 h, then KCl was added to a final concentration of 0.1 M to balance cytoplasmic and external K⁺ concentrations. The bacteria were placed in a 96-well plate for a moment until the fluorescence intensity remained stable, then CIN was added to a final concentration of 2× MIC and 4× MIC and the fluorescence intensity was recorded continuously by a high-content instrument. Samples without CIN and with 0.1% Triton X-100 were used as the negative and positive control, respectively.

ROS assay

S. aureus SA113 and E. faecalis OG1RF were cultured overnight in TSB medium at 37°C, then 10⁷ CFU/mL bacteria was diluted into PBS and incubated with 20 μM DCFH-DA. After incubation for 30 min, the bacterial cells were washed twice with PBS to remove the DCFH-DA outside, and then CIN (4× MIC) and/or ROS inhibitor N-acetyl-L-cysteine (10 mM) were added, respectively. The fluorescence intensity was recorded on a high-content instrument (excitation λ = 488 nm, emission λ = 530 nm). The experiment was independently repeated twice.

TEM imaging assay

Overnight cultures of S. aureus SA113 and E. faecalis OG1RF were diluted 1:100 in TSB medium to a cell density of 10⁷ CFU/mL and cultured at 37°C for 4 h. CIN (4× MIC) was added to cells and the mixture was incubated at 37°C for 1 h. DMSO treatment was considered as a negative control. The cells were collected by centrifugation at 4,000 rpm for 5 min, and then washed with PBS and fixed overnight with fixative (4% paraformaldehyde and 0.1 M phosphate buffer) at 4°C. After discarding the supernatant of fixed cells, 0.1 M PB (pH 7.4) was added, and the cells were re-suspended and washed three times in PB for 3 min. The sample was then dehydrated with gradient ethanol (30, 50, 70, 80, 95, 100%) for 20 min. Next, the sample was treated with acetone and EMBed 812 (1:1) for 2hat 37°C, followed by acetone and EMBed 812 (1:2) overnight at 37°C, and then EMBed 812 for 6hat 37°C. The embedding models with resin and samples were polymerized by heating to 65°C for more than 48 h. The resin blocks were cut to 60–80 nm thickness on a LEICA EM UC7 ultrathin slicer, and the sample was fished out onto 50 mesh copper grids with formvar film. After being stainin with 2.6% lead citrate for 8 min, and then rinsed three times with ultra-pure water. The copper grids were dried, placed into the grids board, and then the samples were observed under a transmission electron microscope (TEM, HITACHI HT 7800).

In vitro induction of CIN-tolerant isolates of S. aureus

S. aureus SA113 and CHS101 were serially subcultured in TSB containing CIN at an initial inducing concentration from 6.25 μg/mL to 400 μg/mL. Strains in each concentration were cultured for 10 days before being exposed to the next concentration.^21,35 Three separate isolated clones were picked on day 60 for culture on TSB agar plates and the biofilm formation of the isolated clones was investigated. S. aureus SA113 and CHS101 wild-type (WT) strains were used as controls. Isolated clones were kept frozen at −80°C in glycerol containing 50% TSB.

Whole-genome sequencing detection of mutations in CIN-tolerant clones

Chromosomal DNA extracted from two CIN-induced resistant strains of S. aureus, SA113 and CHS101, was prepared for whole-genome sequencing. Whole genomes of Nexter libraries were constructed and sequenced on the Illumina HiSeq sequencing platform by Novogene Co. Ltd., (Beijing, China). The sequences were compared to the reference genome of S. aureus strain SA113 using the bwa-mem software (version 0.7.5a) with standard parameters. Single nucleotide polymorphisms and indels in resistant strains SA113 and CHS101 were displayed using MUMmer (version 3.23). The data were deposited in the National Center for Biotechnology Information (NCBI) with accession number PRJNA755754 (NCBI: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA755754).

Proteomics analysis

The proteomics analysis was conducted according to a previously reported method with modification.²⁵S. aureus SA113 in exponential phase was treated with 1/2× MIC of CIN (12.5 μg/mL) or DMSO at 37°C for 2 h on a shaker at 200 rpm. Each group contained three biological replicates. The bacteria were harvested by centrifugation at 5,000 g for 10minat 4°C and washed three times with 1× PBS. The cell pellets were then suspended in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime) and protease inhibitor cocktail (P1065, Beyotime), followed by the addition of 0.1 mm diameter Zirconia/Silica beads (catalog number: 11079101z) at 4°C using the Cell Disruption Device (JXFSTPRP-24L, Shanghai Jingxin Industrial Development Co., Ltd.) for bead homogenization (70 HZ lysis for 1 min with 10 s pause, run 4 cycles). Samples were then centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was collected, and the protein concentration was determined by BCA assay and SDS-PAGE. Next, 50 μg total protein was transferred into a 10KD ultrea filter tubes and reduced by 200 μL of 8 M urea solution with 10 mM DTT for 2hat 37°C. The solution was removed by centrifugation (12,000 rpmat 20°C for 10 min), then 200 μL 8M urea solution with 50 mM iodoacetamide (IAA) was added into the pellet and incubated in the dark for 15minat room temperature. The samples were then desalted using Amicon ultracentrifugal filters (10KD, Millipore, Billerica, MA, USA), centrifuged at 12,000 g for 10minat room temperature and washed three times with 200 μL of 8M urea solution containing 25 mM ammonium bicarbonate. Subsequently, 100 μL of 25 mM ammonium bicarbonate containing 1 μg trypsin (protein:trypsin = 50:1) was added to each filter tube and incubated at 37°C for 12 h. The filter tubes were washed twice with 100 μL of 25 mM ammonium bicarbonate by centrifugation at 12,000 rpm for 10 min, and the flow-through peptide fractions were collected and lyophilized. The lyophilized peptide fractions were re-suspended in 40 μL ddH2O containing 0.1% formic acid, 30 μL of which was placed in the inner cannula of the autosampler vial, and a 4 μL aliquot of which was loaded into a C18 (PepMap™Neo Trap Cartridge, 174500, Thermo Scientific) trap column. The online chromatography separation was performed on an Ultimate 3000 RSLCnano (Thermo Scientific). The trapping and desalting procedure was conducted with 20 μL of 100% solvent A (0.1% formic acid). Then, an elution gradient of 5–38% solvent B (80% acetonitrile, 0.1% formic acid) in 60 min was applied on an analytical column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm 100 Å, 164941, Thermo Scientific). DDA (data-dependent acquisition) mass spectrum techniques were used to acquire tandem mass spectrometry (MS) data on a Thermo Fisher Q Exactive plus mass spectrometer fitted with a Nano Flex ion source. Data were acquired using an ion spray voltage of 1.9 kV and an interface heater temperature of 320°C. Thermo Proteome Discoverer was used for protein identification and quantitative analysis of MS/MS data. After searching the target database, the local false discovery rate for peptides was 1.0%, with a maximum of two missed cleavages. In addition to a p-value <0.05, a two-fold cutoff value was used to determine upregulated and downregulated proteins. Differentially expressed proteins were uploaded to the OMICSBEAN database (http://www.omicsbean.com) for volcano plot, heatmap, gene ontology (GO) annotation (including biological process, cellular component, molecular function), KEGG pathway analysis, and PPI networks.

Quantification and statistical analysis

Statistical analysis was conducted using GraphPad Prism software (version 8.0) with analysis of variance (ANOVA) or Tukey’s test for multiple comparisons. The Log-rank (Mantel-Cox) test was employed to analyze the survival rate. Data are shown as mean ± SD. A p-value of <0.05 was considered statistically significant (not significant p>0.05, ∗∗p<0.01, ∗∗∗p<0.001). Most experiments were repeated at least twice with similar results.

Additional resources

Not applicable.

Published: March 11, 2023

Data and code availability

• •This published article includes all datasets generated or analyzed during this study.
• •The raw whole-genome sequencing data was posted in the Sequence Read Archive (SRA) database under accession number PRJNA755754.
• •Any additional information is available from the lead contact upon request.