is an opportunistic, gram-positive bacterium that is a major cause of nosocomial infections [1
]. S. aureus
can cause several different infections in humans and animals, ranging from pneumonia to sepsis and endocarditis [2
]. S. aureus
infections are treated with various antibiotics. However, the indiscriminate use of antibiotics has resulted in the emergence of antibiotic-resistant strains, including methicillin-resistant S. aureus
(MRSA), which is a serious public health hazard [3
]. Patients with community-acquired and nosocomial infections are frequent carriers of MRSA [4
The proportion of MRSA-attributable, hospital-acquired bacterial infections has been increasing every year, and MRSA is a serious threat to postoperative patients [5
]. Most patients with postoperative MRSA infections die due to the infection, rather than due to surgical complications, as the fatality rate of MRSA infections is high [6
]. Approximately 39–51% of the pathogens causing infections at surgical sites have been estimated as being resistant to conventional antibiotics in the United States [7
Previous studies have shown that MRSA can form biofilms on infected tissues and medical instruments, thus it is gradually becoming even more difficult to treat MRSA infections [9
]. Biofilms are formed by aggregates of bacteria that adhere to various surfaces; they are mainly composed of DNA, proteins and extracellular polysaccharides [12
]. Biofilm formation is primarily initiated by the adherence of planktonic cells [14
]. After biofilm maturation, some bacterial cells disperse, and these dormant cells are reconverted to planktonic cells [15
]. Compared with planktonic cells, biofilm-embedded, bacterial cells exhibit lower growth rates, more frequent cellular communication, and lower sensitivities to antibiotics, which makes them more difficult to eradicate [12
]. Bacteria in a biofilm are more resistant to antibiotics (up to 1000-fold), and can evade the immune system of the host. Thus, biofilm-associated infections can be persistent [17
] and even fatal [10
]. Previous studies have demonstrated that S. aureus
biofilms show a reduced permeability to vancomycin [18
]. Compared to typical bacterial infections, biofilm-associated infections are much more laborious to treat [19
]. Given the increasing incidence of multidrug resistance and our current knowledge of the tenacious nature of biofilms, it is necessary to develop novel strategies and drugs that target biofilm-associated MRSA infections.
Plant-derived compounds have been widely used to combat microbial infections because they are inexpensive and easy to extract [20
]. The traditional Chinese medicine, 2-isopropyl-5-methylphenol (IPMP), commonly known as thymol, is a monoterpene phenol isolated from plants. It is a component of the essential oils extracted from various plants in the Lamiaceae
, and Scrophulariaceae
families, and it exhibits anti-inflammatory, antioxidant and potent antimicrobial properties [22
]. In our previous studies [25
] we showed the potent effects of thymol against MRSA infections. Thymol inhibits bacterial growth by altering the membrane permeability and disturbing both protein synthesis and binary fission. At subinhibitory concentrations, thymol also reduces biofilm formation [25
]. Therefore, in this study, we first investigated the ability of thymol to inhibit biofilm formation and to eliminate mature biofilms in vitro. Second, we evaluated the effects of thymol on extracellular DNA (eDNA) release, polysaccharide intercellular adhesin (PIA) production, and the expression of biofilm-associated genes. Finally, but most importantly, we evaluated a combination of thymol and vancomycin for the treatment of MRSA biofilm infections in a mouse model.
MRSA is a “superbug” that has spread globally. The first drug of choice for treating MRSA infections is vancomycin. However, with the emergence of vancomycin-resistant S. aureus
(VRSA), MRSA has become an even more serious clinical issue [26
]. In addition, MRSA can form biofilms, and biofilm bacteria are markedly less susceptible to antibiotics [27
]. MRSA biofilms have become the greatest threat to critical care patients, especially patients in the intensive care unit (ICU), and patients with implanted medical devices or long-term wounds [28
Compared to conventional antibiotics, thymol has a higher MIC, similar to that of emodin [29
], baicalin [30
], and other compounds extracted from plants. However, even at low concentrations, thymol has prominent anti-biofilm activity. Here, we used sub-MICs of thymol (8, 16, 32 and 64 μg/mL), which were determined based on the results of susceptibility testing. These concentrations showed no significant (p
< 0.05) effect on the growth of MRSA, and thus applied less selective pressure. The median lethal dose (LD50
) of thymol when administered by intraperitoneal injection is 608 mg/kg; therefore, these concentrations are safe for use in humans.
This study evaluated the effects of thymol upon the formation MRSA biofilms and the clearance of pre-existing biofilms. The results showed that thymol exhibited remarkable inhibitory effects against MRSA biofilms. To explore the underlying mechanism of action, we investigated the effects of thymol on PIA production, eDNA release, and biofilm-regulated gene expression. The life cycle of a biofilm has four stages: bacterial attachment, formation of microcolonies, maturation of the microcolonies into a biofilm, and cell dispersal [12
]. PIA and eDNA are important components of MRSA biofilms [32
]. PIA plays a crucial role in adhesion and aggregation [33
]. It has been reported that although bacteria lacking PIA could adhere to biomaterials at the initial stage, at the later stage, they are unable to form a biofilm because cell-to-cell adhesion was greatly reduced. Similarly, eDNA is an essential component for biofilm formation, as it plays key roles in bacterial adhesion, aggregation, microcolony formation and biofilm architecture. The expression of PIA is regulated by the genes of the intercellular adhesive (ica
) operon (ica
ABCDR) and sar
]. The ica
A gene, which encodes acetylglucosamine transferase [35
], cannot be expressed in the absence of sar
A, because sarA binds to the promoter of the ica
]. Recent research has suggested that cid
A and sar
A also regulate biofilm formation and clearance through an ica
-independent pathway, especially in MRSA [39
]. Valle et al. [38
] reported that the sar
A mutation increased the expression of extracellular protease and nuclease, which reduced biofilm formation. Contrary to other findings, agr
did not affect the biofilm-forming ability of S. aureus
]. Therefore, we selected ica
A and sar
A for our study. We found that thymol reduced PIA synthesis and eDNA release, and RT-PCR showed that the transcript levels of these three genes were reduced in a dose-dependent manner after treatment with thymol. The changes in sar
A, which regulates biofilm formation through various pathways, were the most obvious. These findings suggest that thymol is effective against TCH1516 biofilms.
Even after adopting various preventive measures, biofilm-associated infections have become commonplace. Once a biofilm is formed on a tissue or on the surface of an implanted medical device, it is nearly impossible to eliminate using conventional doses of antibiotics [43
]. The situation is even more serious in the case of MRSA biofilm-associated infections. In such cases, combinatorial treatments are considered the first choice, as the dose of vancomycin, that is conventionally used to treat MRSA infections, should not be increased due to its toxicity. Thymol showed substantial inhibitory effects against biofilm formation and eliminated pre-existing biofilms. Therefore, clinical MRSA biofilm-associated infections might be cured with a combination of vancomycin and thymol. We extended our previous in vitro results by modeling a clinical MRSA biofilm-associated infection using an intraperitoneal foreign-body infection mouse model. We speculated that the biofilms on medical instruments may be damaged by thymol, and then vancomycin can penetrate the biofilms and kill the bacterial cells.
After treatment with either thymol or vancomycin alone, many bacteria were still adhered to the surface, and colony counting and SEM showed that the biofilms were not completely eliminated. However, after treatment with a combination of the two drugs, especially at a high dose of thymol, the destruction of the biofilm was obvious, and only a few bacteria still adhered. This result showed that thymol treatment alone could only eliminate some of the MRSA biofilm bacteria in vivo, and that thymol could improve the therapeutic effects of vancomycin on MRSA biofilm infections, in a combinatorial therapy.
Histopathological observation is a convenient way to diagnose disease and evaluate treatment efficacy. Thymol and vancomycin alleviated the pathological lesions associated with the biofilm-covered implants, which was consistent with the findings of other reports [46
]. The results showed that mice in the high-dose combination treatment group had a good prognosis after treatment.
WBCs are an important indicator of inflammatory responses. In this study, both vancomycin and thymol reduced the WBC counts compared to those in the PBS group. Vancomycin might reduce inflammation by killing planktonic bacteria. However, it is unlikely that thymol reduced inflammation by killing bacteria because its MIC and MBC were too high for the administered doses to reach an effective concentration. Thus, thymol might reduce the inflammation via its anti-inflammatory activity.
TNF-α and IL-6 are widely used as markers to study inflammatory responses. TNF-α is one of the earliest and most important mediators of inflammation. IL-6 induces a humoral response, more specifically B cell differentiation and antibody production, as well as a cell-mediated response, more specifically T cell activation, proliferation and differentiation, and it participates in the immune response [48
]. A previous study demonstrated that thymol inhibits TNF-α and IL-6 production via the nuclear factor-kappa B (NF-κB) signaling pathway in mice with an LPS-induced acute lung injury [46
]. However, in this study, TNF-α levels were not significantly (p
> 0.05) reduced in the thymol group, but were significantly (p
< 0.05) decreased in the high- and middle-dose combination treatment groups. This might be due to a decrease in immune stimulation, as most of the biofilms were eliminated. Contrary to the findings of a previous report [46
], the increase in IL-6 in the thymol group and the high- and middle-dose combination treatment groups might be due to the destruction of biofilms and the release of a higher number of bacteria, leading to the rapid cellular and humoral immune responses.
In summary, this study explored the ability of thymol to effectively inhibit and eliminate MRSA biofilms by reducing the synthesis of PIA and the release of eDNA in vitro. We confirmed that thymol could enhance the bactericidal activity of vancomycin by damaging biofilms. Thymol also improved the immune status of model mice, resulting in a good prognosis. As a monomeric drug with a well-defined chemical structure, thymol shows a potential for use as a novel drug against MRSA biofilm-associated infections. However, further studies on the pharmacokinetics, pharmacodynamics and toxicology of thymol must be performed before initiating any clinical trials. We will study more MRSA strains in the future to confirm whether these findings hold for all MRSA strains.