2.1 Antibacterial activity
TTO is a potential anti-bacterial[7, 17] and anti-fungal product[18, 19], but there were few studies about the anti-MRSA activity of terpinen-4-ol, a main ingredient of TTO. Our research focused on the antibacterial activity of terpinen-4-ol against MRSA. The MIC and MBC values for terpinen-4-ol against S. aureus were 0.08%~0.32% (Table 2), which is similar to Loughlin`s result (0.25%)[20]. In Table 1, we could see the MBC value was two times the MIC value, which leads us to know that terpinen-4-ol exhibited bactericidal properties against MRSA and other strains in this study [21]. In addition, a time-kill curve of MRSA exposed to terpinen-4-ol was generated by performing plate counts (Fig. 1 A). We found that 0.32% terpinen-4-ol atkilled all the bacteria within two hours and that terpinen-4-ol at 0.16% or 0.08% could obviously inhibit the growth of MRSA, which also showed terpinen-4-ol exhibited bactericidal properties against MRSA.
Table 1 Antimicrobial activities of terpinen-4ol against 13 S. aureus strains.
Bacteria
|
MIC(v/v)
|
MBC(v/v)
|
ATCC 43300
|
0.16%
|
0.32%
|
S-1
|
0.08%
|
0.16%
|
S-2
|
0.08%
|
0.16%
|
S-3
|
0.16%
|
0.32%
|
S-4
|
0.08%
|
0.16%
|
S-5
|
0.32%
|
0.32%
|
S-6
|
0.08%
|
0.16%
|
S-7
|
0.16%
|
0.16%
|
S-8
|
0.08%
|
0.16%
|
S-9
|
0.16%
|
0.16%
|
S-10
|
0.32%
|
0.32%
|
S-11
|
0.16%
|
0.32%
|
S-12
|
0.16%
|
0.32%
|
2.2 Antibiofilm activity
The antibiofilm activity of terpinen-4-ol was also assessed by the crystal violet staining. As shown in Fig. 1 B, there was almost no MRSA biofilm formation under treatment with terpinene-4-ol at concentrations of 0.64% and 0.32%. Terpinene-4-ol at 0.16% also had a significant effect on the formation of MRSA biofilm, and the inhibition rates at 24 h and 48 h were 48.09%±0.97% (P < 0.01) and 31.20%±2.13% (P < 0.01). It can be seen that higher concentrations of terpinen-4-ol effectively inhibited the formation of biofilm.
In this study, terpinen-4-ol behaved its inhibition in MRSA biofilm formation. It also confirmed other authors' results that terpinen-4-ol could destroy the biofilm of many oral pathogens such as, Streptococcus mutans, and Lactobacillus acidophilus[22] Porphyromonas gingivalis, Fusobacterium nucleatum[23]. We could see that terpinen-4-ol could inhibit biofilm formation of MRSA in a concentration-dependent manner. With the decrease of concentration of terpinen-4-ol, the inhibition rates gradually down.
The destruction of biofilms by terpinen-4-ol was also evaluated. As shown in Fig. 1 C, 0.16% terpinene-4-ol achieved a clearance rate of 93.90%±3.33% (P < 0.01) for MRSA mature biofilm. As the concentration of terpinen-4-ol decreased, the clearance rate gradually decreased. The viability of the preformed MRSA biofilm exposed to terpinen-4-ol was also verified by CLSM analysis. As shown in Fig. 2, as the concentration of terpinen-4-ol increased, the amount of red fluorescence (indicating dead bacteria) increased while that of green fluorescence (indicating live bacteria) decreased.
Terpinen-4-ol showed its activity in clearing MRSA biofilm. Generally, it is more difficult to remove mature biofilm than to inhibit its formation. Thus, terpinen-4-ol could consider to be a potential antibiofilm agent, for it can not only inhibit the formation of biofilms, but also destroy mature biofilm. Therefore, it is necessary to further study the anti-biofilm activity of terpinene-4-ol and explain its mechanism of action.
2.3 Antibiofilm mechanism
2.3.1 Transcriptomics
To understand the changes in MRSA due to treatment with terpinen-4-ol, we measured the transcriptome of MRSA exposed to terpinen-4-ol and screened 304 DEGs as defined by FC > 2 and Q < 0.05. Among these DEGs, 159 genes were downregulated and 145 genes were upregulated in terpinen-4-ol-treated relative to control (Fig. 3). The RNA-seq data were submitted to Gene Expression Omnibus (GEO) under accession number GSE157638.
KEGG pathway analysis was used to obtain insights into the biological functions of the DEGs (Fig. 4). Compared with the control group, there were some pathways different, including valine, leucine, and isoleucine (branched chain amino acids, BCAAs) biosynthesis. nitrogen metabolism, ABC transporters, 2-oxocarboxylic acid metabolism and etc., were significantly enriched (P < 0.05) in terpinen-4-ol-treated group. Some of those pathways related to the formation of MRSA biofilm. For example, BCAAs, synthesized by 2-oxocarboxylic acid, regulate the biosynthesis of bacterial amino acids[24] and nucleotide metabolism[25] by combining with CodY, and they can also regulate the biofilm of S.aureus[26-28]. There were 9 genes related to 2-oxocarboxylic acid and BCAAs have been down in MRSA treated with terpinen-4-ol, which showed us the BCAA metabolic pathway of MRSA may be affected by terpinen-4-ol.
Furthermore, there were other pathways that warrant our attention, such as purine metabolism, quorum sensing (QS), and β-Lactam resistance. Most of these pathways are related to the synthesis of nucleic acids or ATP, which are important to biofilm formation of MRSA. We then used metabolomics to verify the results of the transcriptomics analysis.
3.3.2 Metabolomics
Twelve sets of data from the six biological replicates were analyzed to determine the changes in metabolites that occur in MRSA following treatment with terpinen-4-ol. and 215 differential metabolites were screened based on FC > 1.5 or FC < 0.67, P < 0.05, and VIP value >1.0. Among these differential metabolites, 125 were upregulated and 90 were downregulated in terpinen-4-ol-treated MRSA relative to control MRSA (Fig. 5). 18 enriched pathways (Fig. 6) were identified by KEGG enrichment analysis, including caffeine metabolism (P < 0.05). Caffeine can be converted to purine, and we found that many intermediate products of purine metabolism and the contents of its constituents have decreased, including xanthine and xanthosin, which are intermediate products of purine metabolism, guanosine, and adenosine, which are components of purine metabolism, 2'-deoxyadenosine, and deoxyadenosine, which are components of DNA.
In addition, there were also some metabolites related to pyrimidine metabolism have decreased. The content of these substances is closely related to DNA synthesis, and the reduction of their content may lead to a reduction in the amount of bacterial DNA. Thus, we conjectured that terpinen-4-ol achieves its anti-MRSA activity by inhibiting the synthesis of DNA.
2.3.3 eDNA Quantification
eDNA an important component of the EPS released by the lysis of some bacteria during the formation of the biofilm[29], and it plays a vital role in all stages of biofilm formation, including initial bacterial adhesion, aggregation, microcolony formation, and determining the structure of biofilm[30]. The amount of eDNA in the medium of the biofilm was measured by spectrophotometer and reported as the eDNA per relative biomass to account for the number of bacteria in the biofilm. The production of eDNA from biofilm exposed to terpinen-4-ol was significantly decreased compared with that of the control (Fig. 1 D). In particular, the eDNA content of the biofilm treated with 0.16% terpinen-4-ol was decreased relative to the control content by 61.88% ± 0.58%. The decrease in the content of eDNA reduces the adhesion ability between bacteria, leading to the destruction of the integrity of its biofilm. Thus, we inferred terpinen-4-ol inhibited biofilm formation of MRSA by inhibiting its release of eDNA.
2.3.4 q-RT-PCR
To verify our results, we also performed qRT-PCR verification of some of the differential genes, which play important roles in DNA synthesis (Table 2). In terpinen-4-ol-treated MRSA relative to control MRSA, the expression of deoD was upregulated by 12.378±0.541 times, and the expression of pyrB was downregulated by -3.049±0.147 times. Those results were largely consistent with the transcriptome results that terpinen-4-ol inhibit purine and pyrimidine metabolism.
Table 2 Selected MRSA genes that displayed altered expression after terpinene-4-ol treatment of biofilm as determined by RNA-seq and real-time RT–PCR.
Gene
|
Description
|
Fold Change ± SD
|
|
|
qRT-PCR
|
RNA-seq
|
carB
|
carbamoyl phosphate synthase large subunit
|
-2.054±0.304
|
-6.36
|
arcC
|
carbamate kinase
|
+2.371±0.051
|
3.38
|
deoD
|
purine nucleoside phosphorylase
|
+12.378±0.541
|
24.723
|
pyrF
|
orotidine 5'-phosphate decarboxylase
|
-1.297±0.697
|
-5.07
|
pyrB
|
aspartate carbamoyltransferase catalytic subunit
|
-3.049±0.147
|
-5.45
|
2.3.5 Conjoint analysis
The differential genes identified from the transcriptomics analysis and the differential metabolites identified from metabolomics analysis were represented in the KEGG pathway database (Fig. 7). After the enriched pathways were identified for the differential genes and metabolites separately, it was found that the differential genes and metabolites were mainly involved in the synthesis of MRSA nucleic acid. In the transcriptome results, 16 genes related to nucleic acid synthesis were altered, including nrdF, arcC, deoD, pyrF, pyrB and carB. In the metabolome results, the contents of 11 metabolites related to nucleic acid synthesis, including xanthine, 2'-deoxyadenosine, inosine, cytosine, thymidine, and deoxyadenosine, were significantly reduced in terpinen-4-ol-treated MRSA relative to control MRSA. Based on some of the differential genes and metabolites, we constructed a diagram of the pathways through which terpinen-4-ol affects the synthesis of MRSA nucleic acid. It can be seen that terpinen-4-ol significantly affects the salvage synthesis pathway of MRSA DNA and RNA.
The biosynthesis of pyrimidine nucleotides can affect biofilm by influencing the expression of the genes csgDEFG[31]. The interplay between the rescue synthesis pathway of nucleotides and Curli amyloid fibers' generation may be closely related to cellulose and other EPS responses to environmental pressure[32-34]. pyrB and carB are the key enzymes of the nucleotide rescue pathway, being very important for the rescue synthesis of nucleotides. In our research, the expression of these two genes was inhibited by terpinen-4-ol treatment. Furthermore, the levels of four nucleotides, the raw material for UMP synthesis, declined significantly after terpinen-4-ol treatment, indicating that the salvage synthesis of pyrimidine nucleotides was inhibited by terpinen-4-ol. The inhibition of pyrimidine nucleotides might block the production of modified nucleotides acting as signal molecules for biofilm formation, such as c-di-GMP[35], which is an important signal molecule in biofilm regulation. In addition, the inhibition of pyrimidine nucleotides biosynthesis might lead to the inhibition of eDNA synthesis, which could lead to the destruction of biofilm. The observed decrease in eDNA production in terpinene-4-ol-treated MRSA relative to control MRSA supports this possibility.
ATP, which is composed of adenine, ribose and 3 molecules of phosphate[36], is the most direct source of energy in living organisms. The decrease of inosine in terpinene-4-ol-treated MRSA affected the synthesis of ATP in MRSA, resulting in the inhibition of energy metabolism and the membrane transport system to varying degrees. In the analyses of the transcriptome and metabolome, we found many ATP-related genes and small-molecule metabolites affected to varying extents by terpinen-4-ol treatment, especially the ABC transporter and genes related to energy metabolism. The ABC transporter is the main transporter of the MRSA efflux pump[37, 38] and is closely related to the formation of MRSA biofilm[39] and the quorum sensing system. A reduction in efflux pump activity can inhibit the formation of bacterial biofilm. The ABC transporter is also involved in one of the main mechanisms of MRSA drug resistance. A study has shown that terpinen-4-ol and β-lactam antibiotics applied in combination have a significant synergistic effect against MRSA[9]. In addition, TTO can inhibit the growth of Botrytis cinerea by inhibiting its energy metabolism[40]. Therefore, we speculate that terpinen-4-ol can inhibit the growth of MRSA by inhibiting its ATP synthesis.