Mechanism of Eucalyptus Volatile Oil Application for Preventing and Treating Pseudomonas Aeruginosa Infection in Vitro

Background The mechanism of eucalyptus volatile oil application on the prevention and treatment of burn infections caused by Pseudomonas aeruginosa (PA) was need to examined. Methods The effect of eucalyptus volatile oil on bacterial biolm was investigated using a PA biolm model. The expression of LasI mRNA in PA was detected by RT-PCR. Results The minimum inhibitory concentration test showed that the volatile oil of Eucalyptus urophylla in 20% concentration or more could exert an antibacterial effect. However, neither high nor low concentration of the eucalyptus volatile oil had a zone of inhibition. In scanning electron microscopy, the volatile oil group exhibited a signicant delay compared with the control group. The volatile oil group also had a signicantly lower LasI mRNA expression than the control group. Conclusion As a Chinese medicine, the volatile oil of E. urophylla can affect PA proliferation and biolm formation by interfering with the expression of LasI, thereby successfully preventing and treating burn infection caused by PA.

Conclusion As a Chinese medicine, the volatile oil of E. urophylla can affect PA proliferation and bio lm formation by interfering with the expression of LasI, thereby successfully preventing and treating burn infection caused by PA.

Background
Infection is one of the most common clinical complications of burn. It can cause septic shock and multiple organ dysfunction syndrome, which ultimately lead to death (1,2). Sepsis is a complex process and a clinical manifestation that occurs in severe infection cases with systemic in ammatory response syndrome (3). As pathogens invade the blood circulation system, they multiply and produce toxins, causing acute systemic infections. Synthesized by pathogenic bacteria, endotoxin stimulates in ammatory cells to produce a multitude of in ammatory mediators, inducing immune dysfunction.
Subsequently, immune cell function is inhibited, causing septic shock, multiple organ failure, and even death.
Pseudomonas aeruginosa (PA) is a common pathogen of burn (4), and bio lm formation is its most important survival strategy. In a mature PA bio lm, the extracellular polysaccharide ber or the polysaccharide-protein complex is entangled and interlaced into a network. In addition, a negatively charged alginate, which constitutes the external environment of bio lm, lls the gap, creating a strong diffuse barrier. This barrier hinders the penetration of antibiotics, especially positively charged ones(5, 6), greatly enhancing bacterial viability (7). To regulate the formation of toxic factors, PA can use the Quorum Sensing (QS) system named Las system (lasI/lasR) (8). LasI encodes the signal molecule 3-oxododecanoyl homoserine lactone (3-OXO-C12-homoserinelactone, 3-O-C12-HSL) and other signaling molecules that regulate transcriptional activators. LasR and rhlR gene defects can affect the ability of PA to form a bio lm in vitro, and the QS system plays an important role in the establishment and chronic development of PA lung infection (9,10).
Traditional Chinese medicine possesses unique advantages in preventing infection caused by a bacterial bio lm; it can also inhibit bacterial bio lm formation. This type of medicine has become the focus of further anti-infective drugs following antibiotics because of its wide source of drugs, affordability, and minimal side effects (11). Eucalyptus urophylla is a kind of Myrtaceae plant cultivated in Guangxi Province. Eucalyptus leaves have been widely used by the public. They are commonly applied to the skin to prevent mosquito bites. As con rmed by modern medicine, eucalyptus leaves contain gallic acid, phenols, mellow alcohol, and Eucalyptol. The tests of gallic acid in vitro extracted from E. urophylla leaves inhibited Staphylococcus aureus, pneumococcus, typhoid, paratyphoid bacillus, and PA; thus, eucalyptus leaves have good antibacterial and anti-in ammatory effects (12,13). Therefore, this study aimed to investigate the effect of E. urophylla volatile oil on patients with burn for preventing and treating concurrent PA infections.
Materials And Methods

Experimental materials and reagents
In this research, PA was obtained from clinical patients. RT-PCR-related extraction, reverse transcription, and ampli cation reagents were purchased from TAKARA (Japan). Eucalyptus leaves were collected from

Analysis methods
After extraction, the eucalyptus leaf oil was analyzed by GC-MS. To con rm each chromatographic peak, we searched the mass spectrum in NIST98 standard mass spectrum database (HPMSD ChemStation).

MIC test
Considering that the volatile oil of eucalyptus leaves is insoluble in water, cosolubilization was performed using the cosolvent Tween-80. The ratio of volatile oil and cosolvent is 5:1000. We extracted 10 μL of 0.5 MCF of PAO1 bacterial suspension and inoculated it into 1 mL of LB medium. Then, we added soluble volatile oil to con gure a gradient-mixed suspension with a drug concentration of 10% to 50% in volume. Next, we incubated it in a CO 2 incubator for 24 h and transferred to the blood plate to examine bacterial growth.

Protein ngerprint analysis
The protein ngerprint of PA in each group was tested and analyzed by mass spectrometry (Micyo ex LT/SH, BD, US) and FlexAnalysis (Bruck, US), respectively. For validation and speci cation issues, we used the standard MBT method, which is commonly employed for typical laboratory standard samples. MS/Parent mode: On. Initial laser power: 30%, and maximal laser power: 40%. Allow only: 80 satisfactory shots per raster spot. Matrix blaster: Initially, 10 shots were red, with a laser power of 40%. The sample was pasted to the sample holder, placed on the IB3 ion sputtering instrument, and subsequently observed under an electron microscope.

RT-PCR detection
After constructing the bio lm model, we gently washed the gastric tube with sterile PBS and sonicated the bacteria on the inner and outer surfaces of the gastric tube to obtain the bio lm. After enrichment, the sample RNA was extracted using the TAKARA genome extraction kit and then reversed. After 15 min of recording, the LasI expression (6 cases per group) was tested using an RT-PCR detector (Redstone SLAN-96P, Shanghai, China).

Statistical methods
For statistical analysis and picture processing, we used the SPSS 19.0 and GraphPad, respectively. Measurement data were analyzed by t test. Sample means were expressed as mean ± standard deviation. The test level was α = 0.05. Furthermore, P < 0.05 indicated a statistically signi cant difference.

Antibacterial effect of eucalyptus volatile oil
Eucalyptus volatile oil at 20% concentration or more can yield an ideal antibacterial effect (Figure 2a).
Drug diffusion experiments showed that various concentrations of eucalyptus volatile oil have no bacteriostatic ring ( Figure 2b); thus, the evaporative capacity of eucalyptus is poor, and its antibacterial effect can only be achieved by direct contact. Figure 3 illustrates the PA (blue curve) in the control group and PA (red curve) in the experimental group after intervention with medication (drug concentration: 10%). The mass-to-nucleus (m/z) was signi cantly different at 7594 and some other areas. Thus, eucalyptus volatile oil has an effect on various proteins of the bacteria, and at the same time, it interferes with the PA QS system. In future studies, we will further explore and specify these proteins. Most of these proteins are related to bacterial resistance. Figure 4 shows the surface bio lm morphology observed by SEM after the successful construction of the bacterial bio lm model. The control group formed a thick early bio lm. In the group treated with eucalyptus volatile oil, the bio lm morphology was relatively thin, indicating that the eucalyptus volatile oil has an inhibitory effect on bacteria and that bio lm formation was delayed.

Observation of PA after intervention with eucalyptus volatile oil
As depicted in Figure 5, the RT-PCR detection results showed that the LasI mRNA expression in the experimental group was (30.68 ± 0.087) after being converted by standard RT-PCR curve [Y = -3.346LOG (X) + 34.52, R = 0.998]. Meanwhile, the average value of the control group was (31.12 ± 0.063). Compared with that in the control group, the LasI mRNA expression in the experimental group was signi cantly reduced after the eucalyptus volatile oil intervention, and the difference was statistically signi cant (P < 0.05).

Discussion
Guangxi Province is one of the regions in China where Zhuang people live, with rich medical resources and culture. Thus, the research on Chinese medicine is the advantage and characteristic of this article.
Considering the numerous ethnic minorities in Guangxi, its diverse ethnic culture and unique local customs and activities are rare intangible cultural heritages in China; however, it also causes burden to the society. For instance, numerous people suffer from injuries and burns yearly caused by rework and recracker exposure, consuming huge medical resources for the treatment. Therefore, while protecting China's intangible cultural heritage, burn treatment is particularly important and necessary as well.
The bio lm-forming system can lead to high resistance of PA, suggesting as one of the important factors causing clinical intractable infection (14). Thus, people explore other means to treat PA infection, such as the development of new antibacterial drugs, alginate monoclonal antibody, gene regulation, and biomedical material improvement; however, these methods are expensive and have a narrow application range, resulting in limited clinical application(15). The QS system plays an important role in PA bio lm formation (16), and it is a mechanism of information transmission between bacteria. Cells cannot sense the presence of bacteria themselves but rather the concentration of signal molecules. Signal molecule concentration depends on the density of the cells. The QS system participates in regulating the expression of virulence factors, and interference with the QS signaling system may affect the regulation of the pathogenic factors of PA.
Our study results showed that the E. urophylla volatile oil has a lower MIC and better bactericidal effect and the growth of PA can be fully inhibited by at least 20% concentration. Furthermore, SEM results revealed that bacterial bio lm formation can be delayed after exposing the PA to the eucalyptus volatile oil. Bio lm formation is closely related to bacterial resistance (17)(18)(19)(20). Hence, destroying drug resistance is one of the mechanisms of the eucalyptus volatile oil that can be used in clinical bacterial treatment. In addition, the RT-PCR detection results demonstrated that the expression of LasI mRNA, which is a QS system-related gene, can be reduced by the eucalyptus volatile oil. The QS system regulates the secretion, virulence factors (e.g., elastin, exotoxin A, and pyocyanin), and bio lm formation of PA (21). Therefore, the eucalyptus volatile oil has a signi cant effect not only on bacteriostatic but also on the virulence-related system.
In summary, the volatile oil of E. urophylla has a high application value in the treatment of clinical infections caused by PA. Its main mechanism of action includes the following: directly inhibiting bacterial growth and reproduction, delaying bacterial bio lm formation to destroy its drug resistance, and reducing the expression of the LasI gene to reduce its virulence. However, the E. urophylla volatile oil also has its shortcomings. For example, its preparation process emits a pungent odor, and professional distillation and condensation recovery equipment is required for its extraction. Thus, this oil cannot be readily used.
In the current worldwide battle against the new coronavirus (2019-nCoV), Chinese medicine has become essential in patient treatment, rehabilitation, and daily infection prevention (22,23). The use of integrative medicine for 2019-nCoV treatment has exhibited a signi cant clinical e cacy in Wuhan, Hubei Province, and even the entire China (24). Therefore, we hope that the theory of traditional Chinese medicine be extended to the worldwide medical eld. In the future research, we will further work on its therapeutic mechanism but also improve the shortcomings in exploring and promoting its use value.

Conclusion
As a Chinese medicine, the volatile oil of E. urophylla can affect PA proliferation and bio lm formation by interfering with the expression of LasI, thereby successfully preventing and treating burn infection caused by PA.

Consent for publication
Not applicable.

Availability of supporting data
The datasets generated and/ or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests
We declare no competing interests.  Antibacterial assay of eucalyptus volatile oil Figure 2a An ideal antibacterial effect can be achieved by using more than 20% concentration of eucalyptus volatile oil. Figure 2b Subsequent drug diffusion experiments showed that regardless of the concentration of eucalyptus volatile oil, no bacteriostatic ring could be observed (Figure 2b), indicating that the evaporative capacity of eucalyptus is poor and its antibacterial effect can only be attained by direct contact. Scanning electron microscopy (SEM) observation. The control group had formed a thick early bio lm. In the group treated with eucalyptus volatile oil, the bio lm morphology is relatively thin, implying that the eucalyptus volatile oil inhibits bio lm formation.