A new treatment for eliminating surface bacteria using blue light and carvacrol, a phenol derived from edible oils, creates a photoxic, bacteria-specific reaction that does not contribute to antibiotic resistance.
The technique, developed by researchers from Harvard Medical School and Shanghai Jiao Tong University School of Medicine, is described in Science Translational Medicine.
Oral and topical antibiotics have been in wide use since the 1940s, when they saved the lives and limbs of thousands of soldiers and injured civilians in WWII. It is difficult to overstate how many lives have been saved or improved through antibiotic pharmacology, but the efficacy of many standby treatments is now rapidly declining as bacteria mutates to withstand our common treatments.
“Multidrug-resistant (MDR) bacteria have become one of the biggest threats to the public healthcare of our time and we are fast running out of treatment options because fewer and fewer antibiotics are available to treat these infections. The infections are associated with high mortality, leading to approximately 35,000 deaths each year in the United States alone,” says Dr. Mei X. Wu, Associate Professor at Harvard Medical School and corresponding author of the research.
But loss of life is not the only factor contributing to damages from antibacterial resistance. “An estimated cost of antibiotic resistance is in excess of 55 billion dollars annually when loss of productivity is included in the estimate,” Dr. Wu adds.
While multiple methods for treating internal infection are in development, this two-step treatment is perfectly suited for effective, easy treatment of surface wound infections. Dr. Wu describes the process: “We combine carvacrol, an ingredient of edible oils, with blue light to safely and quickly kill multiple MDR bacterial pathogens quickly without incurring any resistance. … This modality may be an alternative for patients, especially, diabetic patients with skin wound infections which cannot be treated effectively with antibiotics.” Such wounds are difficult to treat and cause considerable discomfort and secondary infections.
Carvacrol is a phenol compound long used as a preservative in food production; it is derived from thyme and oregano essential oils, occurs naturally in many edible plants, and is safe for topical and internal use.
The therapy entails treating a surface wound with carvacrol and then applying blue light to create a bacteria-specific phototoxic reaction. The treatment “may be used at home conveniently as carvacrol is edible and blue light has been safely used in clinics for treatment of acne and neonatal jaundice,” explains Dr. Wu.
The study focused on Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus, two prevalent pathogenic bacteria that are notoriously difficult to treat with currently approved methods. Additionally, mice infected with lethal Pseudomonas aeruginosa were saved using the new strategy.
From the research: “Mechanistic studies revealed that carvacrol was photocatalytically oxidized into a series of photoreactive substrates that underwent photolysis or additional photosensitization reactions in response to the same blue light, forming two autoxidation cycles that interacted with each other resulting in robust generation of cytotoxic reactive oxygen species.”
With one application, researchers found that substantial or complete eradication of biofilm bacteria with few or no adverse reactions in the patient. After 20 repeat applications, the researchers found no antibacterial resistance. Neither carvacrol nor blue light alone produced comparable results.
“A study with a large animal like swine is needed to verify the efficacy prior to clinical study,” Dr. Wu explains, in order to obtain FDA approval. If the treatment is found effective, it will be relatively low cost and easy enough to self-administer at home.
Skin wound infection is a widespread problem in both civilian and military healthcare settings. Skin wounds are particularly prone to bacterial infections because the wounds provide an ideal medium for bacterial proliferation and a portal of entry into the bloodstream and are direct exposure to the “dirty” environment.
The infections can be readily treated with a variety of antibiotics if the bacteria involved are susceptible. However, there are only extremely limited or no treatment options when antibiotic resistant strains are involved in the wound infections, which occurs at a worrying speed.
If the wound infection cannot be eliminated in a timely fashion, the infection would alter cellular metabolisms and induce persistent inflammation systemically that can predispose the patients to various complications and life-threatening sepsis (Fitzwater et al., 2003; Wang et al., 2018).
For instance, burn wound infection outbreaks caused by multidrug-resistant (MDR) organisms emerged as a serious problem early in the course of Iraq military operations despite that the United States military has provided rapid and highly effective care for wounded soldiers (Scott et al., 2007; Vento et al., 2013).
As a matter of fact, skin infections caused by MDR bacteria are the most common cause of morbidity and mortality in patients infected with MDR microbes and represent almost 61% of deaths of this infected population (Gomez et al., 2009).
Extensive uses of broad spectrum antibiotics are the single most important factor in evolution of bacterial resistance (Hampton, 2013). A number of studies have shown that the most frequently identified MDR strains of bacteria in nosocomial infections and on the battlefield are Gram-negative bacteria Acinetobacter baumannii and Pseudomonas aeruginosa, and Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) (Scott et al., 2007; Calhoun et al., 2008; Li et al., 2014; Levin-Reisman et al., 2017).
In addition, bacterial biofilms formed by MDR bacteria are the major obstacles in treatment of burn wounds (Bloemsma et al., 2008; Jiang et al., 2017). Bacteria within biofilms can be as much as 1,000 times more resistant to antibiotics and are responsible for recurrent antibiotic-resistant infections elsewhere in the body upon dissemination from the site of the biofilm (Ceri et al., 1999; Caraher et al., 2007).
Currently, the only effective treatments available to fight these infections are older drugs like colistin, which are highly toxic and detrimental to the overall health of the patients (Crane et al., 2009). There is a pressing need for the development of non-antibiotic approaches to combat MDR microbes.
Essential oils (EOs) are a mixture of volatile constituents produced by aromatic plant/herbs. There are about 3,000 well-recognized EOs, of which 300 are generally recognized as safe (GRAS) to humans by the United States Food and Drug Administration (U.S. FDA) and have broad applications in food preservation, additives, and favors, perfume, cosmetic industries, antiseptic oral solutions, toothpastes, cleaner, and air fresheners for centuries (Pandey et al., 2017; Sakkas and Papadopoulou, 2017).
These natural products are of particular interest as “green” antimicrobial agents because of their low-cost, biocompatibility, potential antibiofilm properties, and friendly to eukaryote cells and environment (Burt, 2004; Nostro et al., 2007; Kavanaugh and Ribbeck, 2012).
Among these safe EOs, oregano oil has been shown to have a variety of activities such as antioxidant (Yan et al., 2016), anti-inflammatory (Ocana-Fuentes et al., 2010; Shen et al., 2010), anti-fungal (Akgul and Kivanc, 1988; Soylu et al., 2007), and anti-allergic (Benito et al., 1996). Its antimicrobial effect has been demonstrated in vitro cell culture, food systems studies (Lopez-Reyes et al., 2010; Soylu et al., 2010; Munhuweyi et al., 2017), and in vivo systemic infections (Manohar et al., 2001; Preuss et al., 2005).
In the present study, we investigate effectiveness of oregano oil in inactivation of MDR bacteria isolated from combat casualties in vitro and bioluminescent strains of P. aeruginosa (PA01) and MRSA (USA300) in mouse burn wounds. Our study showed that oregano oil effectively inactivated various pathogenic bacteria and their biofilms irrespective of their antibiotic susceptibility. The study is the first in vivo attempt on the use of oregano oil for the treatment of burn wounds infected with clinically important MDR bacteria.
TEM and SEM Illustrated Ultrastructural Damages of Bacteria
Transmission electron microscopy showed ultrastructural damages of A. baumannii AF0005 (Figure Figure2B2B) and P. aeruginosa IQ0042 cells (Figure Figure2D2D) after exposure to oregano oil for 1 h at 0.16 mg/ml and 0.56 mg/ml, respectively.
The cell wall and membrane damages were apparent in A. baumannii AF0005 and P. aeruginosa IQ0042 cells with a severe leakage of intracellular substances resulting in cell membrane shrinking and separating from cell wall (Figures 2B,D, arrows). Moreover, cytoplasmic vacuoles in A. baumannii AF0005 (Figure Figure2B2B, asterisk) and many stainless-vesicles in P. aeruginosa IQ0042 (Figure Figure2D2D, asterisk) were observed.
Intracellular structural discontinuation such as dissociation between cell wall and membrane was also seen in A. baumannii AF0005 (Figure Figure2B2B, oval). In comparison, untreated A. baumannii AF0005 (Figure Figure2A2A) and P. aeruginosa IQ0042 cells (Figure Figure2C2C) had intact, clear cell wall and membrane and dense and homogeneous cytoplasm.
However, we did not find any significant differences in the ultrastructure between the control and oregano oil-treated MRSA USA300 by TEM (data not shown), which probably hints at different responses of MRSA from A. baumannii or P. aeruginosa. As with biofilms, dense and thick bacterial biofilm was observed on the dentin surface, comprised of numerous layers of densely concentrated cocci in 24-h-old P. aeruginosa IQ0042 (Figure Figure2E2E) and MRSA IQ0064 (Figure Figure2G2G) biofilms.
The biofilms of P. aeruginosa IQ0042 (Figure Figure2E2E) and MRSA IQ0064 (Figure Figure2G2G) were treated with oregano oil at 1 mg/ml and 0.4 mg/ml for 1 h, respectively. The cells decohered in the extracellular polymeric matrix, dead bacteria were readily seen all over, and biofilms were destroyed completely in oregano oil-treated samples (Figures 2F,H, arrows). There were only a few bacteria scantly growing on the dentin surface owing to intensive cell death (Figures 2F,H).
In seeking non-antibiotic microbicides, we have screened dozens of EOs from Chinese indigenous aromatic plants/spices because EOs have been long recognized as one of the most promising natural products for safe microbicides in folk medicines (Lu et al., 2013a,b,c). The selection was initially based on their antiseptic applications in the food industry and in agricultures after an extensive database search.
Among the dozen EOs tested, about one third showed significant antibacterial activities against clinically and agricultural important microbes (Lu et al., 2013a,b,c). Oregano oil stood out as one of the best ones in terms of safety and efficacy. We thus detailed the bactericidal activity of oregano oil against 11 MDR clinical isolates of P. aeruginosa, A. baumannii and MRSA as well as two bioluminescent strains of P. aeruginosa PA01 and MRSA USA300 in the current study.
Oregano oil effectively killed all the bacterial strains tested, with the MICs ranging from 0.08 to 0.64 mg/ml and at an order of sensitivity of A. baumannii > MRSA > P. aeruginosa (Table Table11). The finding is in agreement with previous studies demonstrating that oregano oil and its main component carvacrol had a higher MIC against P. aeruginosa compared to other species, such as S. spp. (Nostro et al., 2007), Chromobacterium violaceum, Salmonella typhimurium, and S. aureus (Burt et al., 2014). Similar to the clinical isolates, oregano oil also inactivated standard strains of A. baumannii ATCC 19606 (Rosato et al., 2010), P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 (Bouhdid et al., 2009), with the MICs 0.15 mg/mL, 1 mg/mL, and 0.33 mg/mL, respectively, which are comparable to our investigation.
Previous studies suggested that Gram-negative bacteria appeared to be more resistant than Gram-positive bacteria in response to EO (Tepe et al., 2005; Longaray Delamare et al., 2007; Gilles et al., 2010). This relative resistance of Gram-negative over Gram-positive bacteria may be ascribed to their cell wall structure and outer membrane arrangement. The outer membrane of Gram-negative bacteria is rich in lipopolysaccharide molecules, relatively impermeable to lipophilic compounds, thereby presenting a barrier to penetration of EO antimicrobial substances (Gao et al., 2011).
It may be also associated with the enzymes in the periplasmic space, which are capable of breaking down the antimicrobial substances upon their entrance of the cells (Nikaido, 1996). However, our studies disagreed with these observations and found Gram-negative A. baumannii was more sensitive to oregano oil than Gram-positive MRSA.
This different outcome suggests that antibacterial activity of oregano oil may not depend on the type of Gram reaction in contrast to other EOs, a possibility that is supported by the studies of Gao et al. (2011). In their studies, Gram-negative bacteria Klebsiella pneumoniae was the most sensitive bacteria whereas the Gram-positive bacteria Listeria monocytogenes was the most resistant strain to the Sphallerocarpus gracilis seed EO (Gao et al., 2011).
The possibility that the cell wall and membrane were primary targets of oregano oil was supported by TEM imaging of the ultrastructure of the bacteria. We found damages of the cell wall and membranes, occurrent with cytoplasmic vacuoles, stainless-vesicles, and disruption and discontinuation of the intracellular structures in a large number of bacterial cells after oregano oil treatment (Figures 2B,E).
This finding is consistent with an association of the antibacterial activity of oregano oil/carvacrol with disturbance of membrane embedded proteins and disruption of lipids, RNA synthesis, ATPase activity, and efflux pump previously demonstrated (Simoes et al., 2009; Tapia-Rodriguez et al., 2017). Moreover, oregano oil may cause an imbalance in intracellular osmotic pressure owing to a leakage of cytoplasmic contents following cell wall and membrane damages, and formation of cytoplasmic vacuoles, eventually inducing cell necrosis, although more investigations are required to conclude the mechanism in detail.
Biofilms are sessile organizations of bacterial cells with a strong adherence to surfaces. Biofilm-associated microbial cells are well protected by an extracellular matrix that comprises exopolysaccharides, proteins and DNA and is poorly permeable (Donlan, 2002; Husain et al., 2015).
Systemic antibiotics administered to treat bacterial infections frequently fails at least in part due to the poor permeability of biofilms. Interestingly, oregano oil was capable of biofilm-killing at least at an early stage (24-h-old biofilms) as efficiently as planktonic cells. Biofilms of A. baumannii, P. aeruginosa, and MRSA were eliminated by oregano oil at a concentration of 0.3, 1.0, or 0.4 mg/ml, respectively, similar to the corresponding MICs attained in planktonic cells.
The similarity can be extended to the order of sensitivity with P. aeruginosa biofilms more resistant than MRSA biofilms than A. baumannii biofilms (Figure Figure11 and Table Table11). This may be attributed to the superior permeability and lipid solubility of oregano oil to bacterial cell membrane and wall (Magi et al., 2015; Khan et al., 2017). Likewise, the effectiveness of oregano oil to inactivate S. aureus, S. epidermidis, and P. aeruginosa biofilms was also found at similar MICs as those against planktonic cells (Nostro et al., 2007; dos Santos Rodrigues et al., 2017; Tapia-Rodriguez et al., 2017).
SEM observations confirmed the physical damage and considerable morphological alteration in the P. aeruginosa IQ0042 (Figures 2E,F) and MRSA IQ0064 (Figures 2G,H) biofilms following oregano oil treatment. These observations raise an intriguing possibility that EO may have advantages over water soluble antibiotics in treatment of biofilms because bacteria living in the biofilms are well known to be more resistant to antibiotics (up to 1,000 times) than their planktonic counterparts, in part owing to poor permeability of biofilms to the antibiotics (Ceri et al., 1999; Caraher et al., 2007).
One concern of using oregano oil as an alternative for the treatment of infections in clinics will be whether MDR bacteria can develop resistance to oregano oil. Although this remains largely unaddressed to date, our results suggest that resistance may not be readily developed because 20 passages in the presence of sublethal concentrations of oregano oil did not alter their susceptibility to the oil (Figure Figure3A3A).
Moreover, oregano oil has been used in food prevention and other antiseptic application for centuries and no resistance has been reported so far. It is commonly believed that EOs act at multiple sites within bacterial cells (cell membrane, cell wall, structural proteins, enzymes, nucleic acids, unsaturated lipids, etc.) and would be less likely to induce the development of resistance (Burt, 2004; Simoes et al., 2009; Tapia-Rodriguez et al., 2017). On the contrary, the MIC of conventional antibiotics could gradually increase with a treatment length due to their single action to inactivate the bacteria (Baym et al., 2016; Levin-Reisman et al., 2017).
The bactericidal activity of oregano oil was corroborated in mouse burn models using model bioluminescent strains of Gram-negative P. aeruginosa PA01 and Gram-positive MRSA USA300. When applied at 24 h after bacterial inoculation forming early stage biofilms, oregano oil effectively reduced the bacterial burden by 25-folds for PA01 and 49-folds for USA300, respectively, in comparison to untreated wounds. While efficiently inactivating bacteria, oregano oil exhibited no cytotoxicity or genotoxicity to the skin, in good agreement with its long record of safety. Moreover, oregano oil did not adversely affect human keratinocytes (Babili et al., 2011) and was safe when administered orally in mice (Manohar et al., 2001; Preuss et al., 2005; Feng et al., 2017).
In summary, we reported here the effectiveness of oregano oil against a panel of MDR bacteria isolated from combating casualties and demonstrated for the first time efficacy of oregano oil for the treatment of burn infections in mice. The study serves as an initial effort in the pursuit of a novel therapeutic option for wound infections, especially those caused by MDR bacteria.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182053/
More information: Min Lu et al. Bacteria-specific phototoxic reactions triggered by blue light and phytochemical carvacrol, Science Translational Medicine (2021). DOI: 10.1126/scitranslmed.aba3571