To investigate the inhibition mechanism of copper ions on Streptococcus mutans–Veillonella parvula dual biofilm.
Methods
S. mutans–V. parvula dual biofilm was constructed and copper ions were added at different concentrations. After the biofilm was collected, RNA-seq and qRT-PCR were then performed to get gene information.
Results
The coculture of S. mutans and V. parvula formed a significantly better dual biofilm of larger biomass than S. mutans mono biofilm. And copper ions showed a more significant inhibitory effect on S. mutans–V. parvula dual biofilm than on S. mutans mono biofilm when copper ions concentration reached 100 µM, and copper ions showed a decreased inhibitory effect on S. gordonii–V. parvula dual biofilm and S. sanguis–V.parvula dual biofilm than on the two mono biofilms as the concentration of copper ions increased. And common trace elements such as iron, magnesium, and zinc showed no inhibitory effect difference on S. mutans–V. parvula dual biofilm. The RNA-seq results showed a significant difference in the expression of a new ABC transporter SMU_651c, SMU_652c, SMU_653c, and S. mutans copper chaperone copYAZ. SMU_651c, SMU_652c, and SMU_653c were predicted to function as nitrite/nitrate transporter-related proteins, which suggested the specific inhibition of copper ions on S. mutans–V. parvula dual biofilm may be caused by the activation of S. mutans reactive nitrogen species.
Conclusions
Streptococcus mutans and Veillonella parvula are symbiotic, forming a dual biofilm of larger biomass to better resist the external antibacterial substances, which may increase the virulence of S. mutans. While common trace elements such as iron, magnesium, and zinc showed no specific inhibitory effect on S. mutans–V. parvula dual biofilm, copper ion had a unique inhibitory effect on S. mutans–V. parvula dual biofilm which may be caused by activating S. mutans RNS when copper ions concentration reached 250 µM.
Zhang Yun and Liu Xianghong contributed equally to this work and should be considered co-first authors
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Abkürzungen
S. mutans
Streptococcus mutans
V. parvula
Veillonela parvula
RNS
Reactive nitrogen species
qRT-PCR
Quantitative real-time PCR
Background
Dental caries is one of the most common oral diseases endangering human health. Its occurrence and development are closely related to dental biofilm [1]. Streptococcus mutans (S. mutans) is a major causative bacterium of dental caries. Crucial to S. mutans’ cariogenicity is the ability to attach to the tooth surface and interact with other bacteria to form a tenacious, well-structured biofilm [2, 3]. Veillonela parvula (V. parvula) is a gram-negative oral commensal [4, 5], which has been reported to help S. mutans to form a thicker biofilm and to have better protection against external bactericidal substances [6‐8].
Copper is indispensable for maintaining the normal physiological functions of the human body. Its physiological concentration in saliva ranges from 0.2 to 7.05 mg L−1 and high concentrations of copper ions can be used as bacteriostats [9]. Werner’s research had shown that copper ions could significantly inhibit the growth of S. mutans [10]. Furthermore, the abilities of S. mutans to form biofilms and develop genetic competence were impaired under copper stress [11]. It has also been reported that a higher concentration of copper ions in the oral cavity may reduce the incidence of dental caries [5]. Considering the antibacterial properties of copper ions, it has been used by scientists in various oral materials to prevent the occurrence and development of oral diseases such as caries and periodontal diseases [12, 13]. But the antibacterial mechanism is not fully understood.
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This study aimed to investigate the inhibitory effect of copper ions on the Streptococcus mutans mono biofilm and S. mutans–V. parvula dual biofilm, compare the inhibitory ability difference of copper ions on these two kinds of biofilms, and explore the mechanism of the inhibitory effects of copper ions on the S. mutans–V. parvula dual biofilm.
In this study, copper ions were found to have a better inhibitory effect on S. mutans–V. parvula dual biofilm than on S. mutans mono biofilm. The result of RNA-seq showed that copper ions may exhibit bacteria inhibitory effects by activating S. mutans reactive nitrogen species (RNS). But the mechanism still needs further clarification.
Material and methods
Bacterial strains and growth conditions
S. mutans strain UA159, S. gordonii strain DL 1, S. sanguis strain SK 36, and V. parvula PK1910 were used in this study. Biofilms were formed using a method similar to that of S.S. Garcia et al. [9]. In brief, S. mutans/S. gordonii/S. sanguis cultured overnight was diluted into fresh THB and grown to the exponential phase. Cultures were diluted at a proper ratio of 1:100 into 10 ml of pre-warmed THB containing 1% sucrose as the carbohydrate source to form the S. mutans/S. gordonii/ S. sanguis mono biofilm [14]. V. parvula cultured overnight was diluted into fresh THL (THB + 0.1% Sodium Lactate) and grown to the exponential phase. Cultures were diluted at a proper ratio of 1:20 for V. parvula and 1:100 for S. mutans/S. gordonii/S. sanguis into 10 ml prewarmed THL + 1% sucrose to form the S. mutans/S. gordonii/S. sanguis–V. parvula dual biofilm. Biofilms were grown statically for 18 h in 96-well polystyrene plates with three repeats.
Different concentrations of metal ions
Metal ions at different concentrations were added to the S. mutans–V. parvula dual biofilm. Iron (FeSO4) and magnesium (MgCl2, MgSO4) were added to the culture at concentrations of 100 µM, 500 µM, 1000 µM, and 2000 µM respectively. For zinc ions (ZnSO4), concentrations of 10 µM, 50 µM, 100 µM, 250 µM, 500 µM, 750 µM, 1000 µM, 2000 µM and 3000 µM were respectively added. As for copper ions (CuSO4), concentrations of 10 µM, 50 µM, 100 µM, 250 µM, 500 µM, 750 µM, and 1000 µM were respectively added.
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Biomass measurement of biofilms
To measure biomass, loosely adhered bacteria cells were gently washed off and the biofilms were stained with 0.1% crystal violet for 15 min and solubilized in 30% acetic acid. Biomass was quantified using OD562.
Transcriptome analysis by RNA-seq
Mono- and dual-species cultures were prepared similarly to those before. Biofilms were grown statically for 18 h in 6-well polystyrene plates overnight, and loosely adhered bacteria cells were gently washed off and the biofilms were scratched and harvested by centrifugation and frozen at − 80 °C until use. RNA was isolated by TRIzol® Reagent, and rRNA was removed. RNA-seq was performed by Illumina Hiseq4000.
QRT-PCR
To validate the RNA-Seq data, quantitative real-time PCR (qRT-PCR) was used to measure the changes in the expression of selected mRNA. The first cDNA was synthesized from 1 μg of purified RNA by the Bio-Rad iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, United States). And quantitative amplification condition was made with Bio-Rad iTaq Universal SYBR Green Supermix and Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc.). To determine the relative amount of cDNA molecules, standard curves were used for each primer (Additional file 1: Table S1). Meanwhile, relative expression was calculated by normalizing the validated reference gene gyrA transcripts [15, 16]. The publication of qRT-PCR experiments (MIQE) guidelines was followed to control the quality of the data as well as analyze the information [17].
Three separated approaches were used to calculate fold changes and significant differences in gene expression between growth conditions: DEseq, edgeR, and limma [18‐20], as implemented in the R/Bioconductor package metaseqR [21]. We assigned genes GO terms using Blast2GO v.2.5.0 [22]. And Fisher’s exact tests were used to assess the relative enrichment of GO terms [23]. The test was performed using the Gossip statistical package. According to previous research, the false discovery rate (FDR) of 0.05 was used to correct for multiple hypothesis testing [24].
A KEGG pathway impact analysis was performed by the software package Pathway-Express as implemented in the R/Bioconductor package ROntoTools [25]. Also, the FDR procedure of 0.05 was used to correct for multiple hypothesis testing [24].
Statistics
The biomass difference between co-cultured and mono-cultured biofilms without adding copper ions was analyzed through two independent samples t-test and elsewhere t-test. The ratio of “OD562 value without copper ions’ addition minus OD562 value with copper ions’ addition at different concentrations” to “OD562 value without copper ions’ addition” was taken as the inhibition rate of biofilms to represent the inhibitory effect on biofilms. The difference in the inhibition rate of biofilms of copper ions at different concentrations between co-cultured and mono-cultured biofilms was analyzed through two independent samples t-test. The difference in gene expression before and after adding copper ions was analyzed through paired sample t-test. A P value < 0.05 was considered significant.
Results
Copper ions had a unique inhibitory effect on S. mutans–V. parvula dual biofilm
Co-culture of S. mutans and V. parvula formed a significantly better biofilm than S. mutans mono biofilm when no copper ions were added (P < 0.05). And at the copper ions concentrations of 250 µM or below, the growth of S. mutans–V. parvula dual biofilm was significantly better than the S. mutans mono biofilm for the biofilm biomass was statistically greater than that of S. mutans mono biofilm (P < 0.05). While the inhibitory effect of copper ions on the dual biofilm was significantly better than that on the S. mutans mono biofilm when the concentration of copper ions reached 100 µM (P < 0.05) (Fig. 1A).
Fig. 1
Inhibitory effect of copper ions, magnesium ions, zinc ions, and ferrous ions on biofilm. A Copper ions had a unique inhibition on S. mutans–V. parvula dual biofilm (P < 0.05). B, C When no copper ions were added, there was a statistically larger biomass of S. sanguis–V. parvula dual biofilm than S. sanguis mono biofilm (P < 0.05), while the biomass of S. gordonii–V. parvula dual biofilm and S. gordonii mono biofilm had no statistical differences (P > 0.05). And copper ions showed a decreased inhibitory effect on these two dual biofilms than on the two mono biofilms as the concentration of copper ions increased (P < 0.05). D–G Magnesium ions, zinc ions, and ferrous ions were added, and these trace elements showed no specific inhibitory effect on S. mutans–V. parvula dual biofilm (P > 0.05)
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Copper ions’ inhibition effect on S. gordonii–V. parvula dual biofilm and S. sanguis–V. parvula dual biofilm
When no copper ions were added, there was a statistically larger biomass of S. sanguis-V. parvula dual biofilm than S. sanguis mono biofilm (P < 0.05), while the biomass of S. gordonii-V. parvula dual biofilm and S. gordonii mono biofilm had no statistical differences (P > 0.05). And copper ions showed decreased inhibitory effect on these two dual biofilms than on the two mono biofilms as the concentration of copper ions increased (P < 0.05) (Fig. 1B, C).
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The inhibitory effect of common trace elements on S. mutans–V. parvula dual biofilm
Common trace elements such as iron, magnesium, and zinc were added, and these trace elements showed no specific inhibitory effect on S. mutans–V. parvula dual biofilm (P > 0.05) (Fig. 1D–G).
RNA-seq
A total of 29.6, 38.7, 33.3 and 26.2 million reads were obtained from the S. mutans mono biofilm, S. mutans–V. parvula dual biofilm, S. mutans–V. parvula dual biofilm under 250 μM copper ions, S. mutans–V. parvula dual biofilm under 500 μM copper ions using sequencing respectively. RNA-seq results of the S. mutans–V. parvula dual biofilm revealed 179 genes up-regulated compared to S. mutans mono biofilm, involving multi biological processes, such as the biological process, cellular components, and molecular functions (Additional file 1: Table S2).
RNA-seq results of the S. mutans–V. parvula dual biofilm with 250 μM copper ions revealed that 723 genes changed compared to which without copper ions’ addition, involving multi biological processes, such as the biological process, cellular components, and molecular functions (Additional file 1: Table S3).
RNA-seq results of the S. mutans–V. parvula dual biofilm with 500 μM copper ions revealed that 34 genes changed compared to which with 250 μM copper ions, involving multi biological processes, such as the biological process, cellular components, and molecular functions (Additional file 1: Table S4).
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Differences in gene expression before and after adding copper ions
Copper transport system copY, copA, copZ, and nitrate/nitrite metabolic pathway: SMU_651c, SMU_652c, SMU_653c.
CopY, copA, copZ and SMU_651c, SMU_652c, SMU_653c showed no significant difference between S. mutans mono biofilm group and S. mutans–V. parvula dual biofilm group when no copper ions were added (P > 0.05) (Table 1).
Table 1
The gene expression difference between S. mutans mono biofilm and S. mutans–V. parvula dual biofilm before copper ions’ addition
Seq_id
sm_count
sm_vp_count
sm_fpkm
sm_vp_fpkm
log2FC (sm_vp/sm)
P value
FDR
Significant
Regulate
SMU_651c
674.443
842.485
74.9465
93.1269
0.31
9.49E−01
9.98E−01
NS
Up
SMU_652c
347.107
311.042
58.6953
52.1764
− 0.17
5.14E−01
8.37E−01
NS
Down
SMU_653c
360.289
228.988
42.2153
26.6818
− 0.66
1.68E−01
5.20E−01
NS
Down
CopZ
547.024
675.515
4736.76
4960.58
0.07
9.68E−01
9.98E−01
NS
Up
CopA
81,090.2
71,135.1
3641.4
3187.54
− 0.19
4.77E−01
8.19E−01
NS
Down
CopY
8326.18
10,783.4
3291.1
4183.63
0.35
8.83E−01
9.90E−01
NS
Up
NS P > 0.05
CopA, copZ, SMU_651c, SMU_652c, and SMU_653c were significantly up-regulated in the S. mutans–V. parvula dual biofilm group with 250 µM copper ions (while copY was up-regulated, although with no significant difference) (P < 0.05) (Table 2). Consistent with the previous result, the dual biofilm was significantly inhibited when copper ions’ concentration reached 250 µM.
Table 2
The gene expression difference of S. mutans–V. parvula dual biofilm before and after adding 250 µM copper ions
seq_id
sm_vp_count
sm_vp250_count
sm_vp_fpkm
sm_vp250_fpkm
log2FC (sm_vp200/sm_vp)
P value
FDR
Significant
Regulate
SMU_651c
842.485
20,727
93.1269
2287.36
4.62
2.04E−09
7.61E−08
*
Up
SMU_652c
311.042
14,031.6
52.1764
2347.88
5.49
7.53E−12
8.79E−10
*
Up
SMU_653c
228.988
13,989.2
26.6818
1627.2
5.93
4.25E−13
8.82E−11
*
Up
CopZ
675.515
2415.5
4960.58
17,156.7
1.79
9.56E−03
2.80E−02
*
Up
CopA
71,135.1
288,381
3187.54
12,913.6
2.02
4.26E−03
1.48E−02
*
Up
CopY
10,783.4
25,701.5
4183.63
9914.07
1.24
7.83E−02
1.51E−01
NS
Up
NS P > 0.05; *P < 0.05
After adding 500 µM of copper ions, copY, copA, and copZ showed no significant difference (P > 0.05), while SMU_651c, SMU_652c, SMU_653c showed a significant decrease (P < 0.05), which may be caused by massive cell death (Table 3).
Table 3
The gene expression difference between S. mutans–V. parvula dual biofilm under the copper ions’ concentration of 250 µM and 500 µM
seq_id
sm_vp250_count
sm_vp500_count
sm_vp250_fpkm
sm_vp500_fpkm
log2FC (sm_vp500/sm_vp200)
P value
FDR
Significant
Regulate
SMU_651c
20,727
3937.95
2287.36
434.257
− 2.4
5.58E−04
3.47E−02
*
Down
SMU_652c
14,031.6
2164.41
2347.88
361.763
− 2.7
1.21E−04
9.00E−03
*
Down
SMU_653c
13,989.2
2660.31
1627.2
309.201
− 2.4
5.63E−04
3.47E−02
*
Down
CopZ
2415.5
1508.67
17,156.7
10,408.6
− 0.72
3.21E−01
1.00E+00
NS
Down
CopA
288,381
237,674
12,913.6
10,639.8
− 0.28
6.98E−01
1.00E+00
NS
Down
CopY
25,701.5
12,145.2
9914.07
4672.76
− 1.09
1.10E−01
8.96E−01
NS
Down
NS P > 0.05; *P < 0.05
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QRT-PCR
QRT-PCR results showed that SMU_651c, SMU_652c, and SMU_653c were significantly up-regulated after adding copper ions (P < 0.05, Fig. 2A). CopA were up-regulated after adding copper ions in both S. mutans mono biofilm and S. mutans–V. parvula dual biofilm (P < 0.05). CopY and copZ were up-regulated in the presence of 250 µM copper ions in S. mutans mono biofilm (P < 0.05, Fig. 2B).
Fig. 2
QRT-PCR results. ASMU_651c, SMU_652c, and SMU_653c were significantly up-regulated after adding copper ions (P < 0.05). BCopA was up-regulated after adding copper ions in both S. mutans mono biofilm and S. mutans–V. parvula dual biofilm (P < 0.05). CopY and copZ were up-regulated in the presence of 250 µM copper ions in S. mutans mono biofilm (P < 0.05)
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Discussion
The mitis and sanguinis groups, including S. oralis, S. mitis, S. gordonii, and S. sanguinis, are the primary colonizers of the tooth surface and are commonly considered as commensals [26]. In particular, communities collected from dentin carious lesions contained notorious acidogenic and aciduric species, including S. mutans, Scardovia wiggsiae, Parascardovia denticolens, and Lactobacillus salivarius. In contrast, S. sanguinis, Neisseria species, and Leptotrichia species were found associated with samples collected from healthy sites [27]. In our study, we found that in mono biofilm, copper ions had an inhibitory effect on S. sanguis and S. gordonii while no significant inhibitory effect on S.mutans. Considering the antagonistic effects between these two bacteria and S. mutans, it seemed that copper ions could help S. mutans compete with the others. But in the complex oral biofilm, the final results require further investigation. We then investigate the copper ions’ effect on dual-biofilm of S. mutans and V. parvula.
A lot of studies have shown that S. mutans and V. parvula are symbiotic. Mashima et al. found that the S. mutans and V. parvula could form a better biofilm compared to the mono biofilm, and the survival rates of S. mutans and V. parvula were both increased in the dual biofilm [28]. A study by Kara [29] showed that dual-species biofilms of S. mutans and V. parvula were less susceptible to antimicrobials, such as chlorhexidine, hydrogen peroxide, erythromycin, and zinc chloride than single-species biofilms of the same microorganisms. Qi’s study showed that V. parvula could help S. mutans to outcompete S. gordonii by improving S. mutans carbohydrate utilization and H2O2 resistance [30]. This study also showed that S. mutans and V. parvula could symbiosis to form a better biofilm of larger biomass, consistent with previous studies. RNA-seq results showed that in the dual biofilm, 179 gene expressions up-regulated involving in the biological process, cellular components, and molecular functions, suggesting that these processes may be related to thickening dual-biofilm to help improve S. mutans’ ability of carbohydrate utilization and H2O2 resistance. But further studies are still needed to explain the mechanisms.
In this study, copper ions were found to show better inhibitive effect on S. mutans–V. parvula dual biofilm than on S. mutans mono biofilm. No similar inhibition was found in the other dual biofilm groups such as S. gordonii-V. parvula and S. sanguis-V. parvula dual biofilms. Other common metal ions such as Fe2+, Mg2+, and Zn2+ were also tested and no such inhibition effect was found, which meant copper ions’ inhibition on S. mutans–V. parvula dual biofilm was unique. The RNA-seq results showed that the S. mutans copper-binding chaperone copYAZ expression increased with the increase of copper ions’ concentration, and at the same time a group of genes that were predicted to express a group of membrane transporters-ABC transporters (ATP-binding cassette transporters) SMU_651c, SMU_652c and SMU_653 were also significantly increased, which was confirmed by qRT-PCR. The function of SMU_651c, SMU_652c, and SMU_653c were predicted to be NO/nitrate /nitrite metabolism. Therefore, we speculated that the inhibitory effect of copper ions on S. mutans–V. parvula may be related to NO/nitrate/nitrite metabolism.
ABC transporters are a group of transporters that widely exist in bacteria, archaea, and eukaryotes, and their function involved the transmembrane transport of ions, carbohydrates, lipids, and proteins. The newly discovered ABC transporters SMU_651c, SMU_652c, and SMU_653c found in this sequencing were predicted to function as nitrite/nitrate transporter-related proteins (Table 4).
Table 4
Function of ABC transporter SMU_651c, SMU_652c, SMU_653c
Gene ID
Function
SMU_651c
1028079
ABC transporter substrate-binding protein
SMU_652c
1029588
Nitrate ABC transporter, ATP-binding protein
SMU_653c
1028071
Nitrate transport protein, ABC transporter permease
The following ways implicated in the interaction of copper ions and S. mutans have been described in the literature:
(1)
Copper ions activate S. mutans ROS, causing oxidative stress and leading to cell death [11].
(2)
Copper ions inhibit the expression of biofilm-forming related gene, GTF genes (gtfB, gtfC, gtfD) and GBP genes (gbpB, gbpC) of S. mutans [11].
(3)
Copper ions irreversibly suppress the activity of S. mutans F-ATPase, affecting the glycolysis ability of bacteria in an acidic environment, resulting in cell death [31].
Since copper ions showed a specific inhibition on S. mutans–V. parvula dual biofilm, there might be some other mechanisms for this, combing with the RNA-seq data, we believed that excessive copper ions may activate reactive nitrogen species (RNS), leading to intracellular nitrate/nitrite metabolic disorders.
Since copper ions’ activation to RNS in S. mutans has not been reported, we reviewed the literature to confirm the reasonableness of the speculations.
Excessive copper ions activate RNS in eukaryotes to bring cell damage
It has been widely studied that excessive copper ions release a series of radical ions by redox reaction, and activate inducible nitric oxide synthase (iNOS) to release a large amount of NO, leading to nitrative stress in cells and causing cell death [32]. For example, Cuzzocrea found excessive iNOS expression in a large number of tissues including arteries, liver, and lungs of mice when injected excessive amount of copper ions, which led to excessive nitrotyrosine appearing in tissues, and activated nitrification stress and mediated cell damage [33]. Reddy et al. treated astrocytes with copper ions and found that copper ions mediated intracellular RNS and ROS, resulting in cell damage [34].
The literature on the activation of RNS by copper ions in prokaryotes was rare
Karreraetal’s study suggested that copper ions exerted the effects as an antibacterial agent in the innate immune system via interaction with reactive nitrogen species [35]. The presence of copper ions in the bacterial cytoplasm could potentiate nitrative stress by causing the uncontrolled release of NO from S-nitroso thiols, which may become toxic to the bacterial cells in the presence or absence of additional host-derived NO.
If it is true that copper ions specifically inhibit S. mutans–V. parvula dual biofilm by activating RNS, then in S. mutans–V. parvula co-culture, there should be more NO than in S. mutans monoculture, which is possible.
V. parvula may transfer nitrate into nitrite (reaction (1)), the existence of S. mutans furtherly reduces the pH value of the dual biofilm, leading to reaction (2) to produce nitrite, the nitrite forms NO through reaction (3) and (4):
Based on the above reactions, we speculated that the co-culture of V. parvula and S. mutans may generate exogenous NO, along with endogenous NO activated by copper ions to cause specific killing of dual biofilm cells. The S. mutans cultured alone were unable to begin reaction (1) due to the lack of V. parvula, so the inhibition of copper ions on S. mutans–V. parvula dual biofilm was better than on S. mutans mono biofilm.
Due to the positive inhibitory effect of copper ions on S. mutans–V. parvula dual biofilm, copper ions could be considered more in the development of oral antimicrobial agents and could be added to multi kinds of dental materials for caries prevention, treatment, and daily oral hygiene maintenance.
Conclusions
In this study, we found that:
1.
S. mutans and V. parvula were symbiotic, and they could form a better dual biofilm of larger biomass than S. mutans mono biofilm. RNA-seq results suggested multi gene expression increases involving biological processes, cellular components, and molecular functions.
2.
Copper ions had a unique and better inhibitory effect on S. mutans–V. parvula dual biofilm than on S. mutans mono biofilm when copper ions concentration reached 100 µM, and copper ions showed decreased inhibitory effect on S. gordonii-V. parvula dual biofilm and S. sanguis-V.parvula dual biofilm than on the two mono biofilms as the concentration of copper ions increased. And common trace elements such as iron, magnesium, and zinc showed no specific inhibitory effect on S. mutans–V. parvula dual biofilm. The RNA-seq results showed that the expression of S. mutans copper transporter copYAZ genes significantly increased by adding copper ions at the concentration of 250 µM or higher in S. mutans–V. parvula dual biofilm and a newly discovered nitrate/nitrite related ABC transporter (SMU_651c, SMU_652c, SMU_653c) expression showed a significant upregulation. Copper ions may inhibit S. mutans–V. parvula dual biofilm by activating S. mutans RNS.
Further investigation into the antibacterial mechanism of copper ions on S. mutans–V. parvula dual biofilm still needs to be carried out and the result may lead to the identification of novel virulence-regulatory pathways.
Acknowledgements
Not applicable.
Declarations
Ethics approval and consent to participate
This study was approved by the ethical review committee of Sichuan Provincial People’s Hospital.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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