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19 April 2023: Lab/In Vitro Research  

Effects of the Dibenzofuran, Usnic Acid, on Inhibition of Ocular Biofilm Formation Due to Coagulase-Negative Staphylococci

Sertaç Argun Kıvanç ORCID logo1ACDEF*, Berna Akova ORCID logo1ACDEF, Merih Kıvanç ORCID logo2ABCDEF

DOI: 10.12659/MSM.940266

Med Sci Monit 2023; 29:e940266

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Abstract

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BACKGROUND: Coagulase-negative staphylococci (CoNS) are gram-positive, aerobic, commensal bacteria found on the skin and mucus membranes, including the conjunctiva. Usnic acid (UA) is a dibenzofuran derivative isolated from lichens. This study aimed to investigate the effects of usnic acid on inhibition of ocular biofilm formation due to CoNS.

MATERIAL AND METHODS: Nine Staphylococcus epidermidis isolates, 5 Staphylococcus hominis isolates, 2 Staphylococcus saprophyticus isolates, and 1 Staphylococcus capitis and Staphylococcus lentus isolates were taken as test bacteria. They were inoculated into brain heart infusion broth and incubated for 24 hours at 35°C and activated. Antibiotic susceptibility was investigated by Kirby-Bauer disc diffusion method. Biofilm production was determined using the microtiter plate method and optical densitometry was measured at 570 nm using an automated microplate reader. Anti-biofilm activity of UA was determined by microtitration method and biofilm removal percentage was calculated.

RESULTS: All tested bacteria were found as high biofilm-producer strains; they were generally resistant to methicillin, but susceptible to vancomycin. UA inhibited the biofilm formation of S. epidermidis isolates, ranging from 5.7% to 81.5%. It inhibited the biofilm formation of S. saprophyticus and S. lentus by 73.3% and 74.3%, respectively. There was no effect of UA on mature biofilms of S. epidermidis 17.7H, S. epidermidis 15.41, S. hominis 9.3, S. hominis 17.2H, S. saprophyticus, and S. lentus.

CONCLUSIONS: It was determined that UA exerted anti-biofilm activity on some CoNS isolated from the ocular surface. Anti-biofilm activity was found to be higher even in strains that did not show antibacterial activity.

Keywords: usnic acid, Biofilms, Eye, Eye Infections, Bacterial, Coagulase, Humans, Staphylococcal Infections, Anti-Bacterial Agents, Dibenzofurans, Microbial Sensitivity Tests

Background

Coagulase-negative staphylococci (CoNS) are a heterogeneous group, and the historical definition of this group is based on diagnostic procedures used to distinguish between Staphylococcus aureus and less or non-pathogenic staphylococci. Currently, CoNS are typically opportunistic microorganisms, representing one of the nosocomial pathogens that cause significant impacts on human health. The most important CoNS species are S. epidermidis and S. haemolyticus, and they are generally responsible for foreign body-associated infections. CoNS has less virulence than S. aureus, with a different disease spectrum. Therefore, host susceptibility becomes even more important in CoNS infections. CoNS infections are difficult to treat infections due to the many methicillin-resistant strains and the increasing number of isolates less susceptible to glycopeptides [1]. The healthy ocular surface is characterized by a stable and low-diversity microbiome. Bacteria on the ocular surface contribute to maintain homeostasis and modulate immune function. Genetic analyses have shown that compositional changes in the microbiota occur in ocular surface disorders such as trachoma, blepharitis, dry eye, age-related macular degeneration (AMD), and uveitis [2]. Commensal bacteria are members of the ocular surface microbiota that do not cause infection in normal conditions. For example, Staphylococcus, Corynebacterium, Streptococcus, and Propionibacterium spp. can act as commensals on the cornea and conjunctiva. Commensal bacteria on ocular surface are tolerated immunologically, while an immune response occurs against pathogens [3]. Staphylococcus spp. was found in the ocular surface microbiome of 3–73% of healthy people, according to sequencing methods in different studies [4–6]. In a study using the sequencing technique, the 2 most common isolates detected in the ocular microbiome of healthy individuals were Propionibacterium acnes (88%) and S. epidermidis (73%) [6]. CoNS were the most frequently isolated bacteria from conjunctival swabs and the most predominant isolate was S. epidermidis [7]. CoNS is currently associated with implants and foreign bodies. Colonization of patients’ skin and mucous membranes is the main source of endogenous infections caused by CoNS. Once implants are placed, they can be colonized by CoNS and cause failure of the surgery [1]. The colonization is related to the biofilm-forming ability of CoNS [8]. In the field of ophthalmology, it has been reported that bacteria form biofilms on many implants. On abiotic surfaces such as corneal sutures, intraocular lenses (IOL), contact lenses (CL), punctal plugs, suture materials, socket implants, glaucoma tubes, scleral buckles, orbit implants, and eyelid implants, it has been reported that CoNS, especially S. epidermidis, form biofilms [9–15]. Many microorganisms form a biofilm in an extracellular matrix on abiotic or biotic surfaces to survive in unfavorable conditions [16]. Many bacteria form biofilms, which make them resistant to antibiotics and other environmental stressors [17]. Cells in biofilm show a higher resistance to antibiotics and disinfectants than planktonic bacteria. Biofilms complicate the treatment of infections, as they provide protection for microbial cells, prevent the penetration of antimicrobials, and facilitate the uptake of elements necessary for cell survival, such as water, oxygen, and nutrients [18]. There is a need to develop new strategies other than antibiotics to prevent biofilm formation. For this purpose, various plants and microorganisms are tried to be used. Natural products, especially plant-derived compounds known as phytochemicals, have been shown to be effective even against drug-resistant bacteria due to their excellent broad-spectrum antibacterial profile [19–22], and usnic acid (UA) is one of the substances studied. It is a secondary metabolite of lichens such as Usnea, Evernia, Lecanora, Cladonia, and Parmelia [23]. Usnic acid [2,6-Diacetyl-7,9-dihydroxy-8,9b-dimethyl 1,3(2H,9bH)-dibenzofurandione] is a yellowish pigment. Raw extracts of usnic acid-rich lichens have been used to treat many health disorders. Usnic acid has been thought as a candidate for use as an antimicrobial, anti-inflammatory, antioxidant, antiprotozoal, antiviral, and larvicidal agent. It is also widely used as an expectorant, in antibiotic ointments, perfumery, cosmetics, sunscreens, and personal hygiene products such as toothpaste, mouthwash, shampoo, and deodorant [24–28]. Elo et al reported UA’s antibacterial effects against methicillin-resistant S. aureus and vancomycin-resistant enterococcus [29]. Francolini et al reported the anti-biofilm effect of the UA against S. aureus and found that it can adhere to the surface of the polymer, but it cannot form a mature biofilm [30].

To the best of our knowledge, investigation of UA in the field of ophthalmology is very limited. This study aimed to investigate the effects of UA on inhibition of ocular biofilm formation due to CoNS.

Material and Methods

TESTED BACTERIA AND DETERMINATION OF ANTIBIOTIC SUSCEPTIBILITY:

The tested bacteria were Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus lentus, Staphylococcus saprophyticus, and Staphylococcus capitis. Bacteria isolated and stocked from previous studies regarding ocular surface were obtained from Eskisehir Technical University Microbiology Laboratory. After the bacteria were removed from the stock, they were inoculated into brain heart infusion (BHI) broth and incubated for 24 hours at 35°C and activated, then inoculated into BHI agar and incubated at 35°C for 24 hours. They were used in the tests after examining the colony characteristics and then checking their purity microscopically by gram staining. Antibiotic resistance status of test bacteria was investigated by Kirby-Bauer disc diffusion method using Mueller-Hinton agar (MHA). Tests were applied in line with the recommendation of the Clinical and Laboratory Standards Institute (CLSI) [31]. Penicillin G (P10), gentamicin (CN 10), kanamycin (K30), erythromycin (E15), tetracycline (TE30), and methicillin (ME5) antibiotic discs were used in susceptibility tests. Minimum inhibitory concentration (MIC) values of vancomycin were determined by microdilution broth method according to CLSI standards. MIC values were interpreted as ≤4: sensitive or ≥32: resistant.

DETERMINATION OF BIOFILM FORMATION:

Biofilm production was determined using the microtiter plate method. CoNS strains were inoculated with 10 ml of tryptic soy broth (TSB) with 0.25% glucose and incubated at 37°C for 24 hours. The cultures were then diluted 1: 40 with TSB with 0.25% glucose. We transferred 200 μl of the diluted cultures into the wells of 96-well polystyrene microtiter plates. Plates were incubated at 37°C for 24 hours. After incubation, the plates were washed 3 times with sterile phosphate-buffered solution (PBS, pH 7.2). After the plates dried, the wells were treated with 200 μl of 96% methanol for 5 minutes and washed again with PBS. Then, 200 μl of 2% crystal violet was transferred to the wells and kept at room temperature for 5 minutes. Excess dye was washed off by placing the plate under running tap water and the plates were air-dried. We added 160 ml of 33% (v/v) glacial acetic acid to the wells. Optical densitometry (OD) was measured at 570 nm using an automated microplate reader. Medium without bacteria was used as a negative control [32]. The study was done in pairs in parallel. The strains were classified as non-adherent (0), weakly (+), moderately (++), or strongly (+++) adherent, referring to the ODs of bacterial films.

DETERMINATION OF MINIMUM INHIBITORY AND MINIMUM BACTERICIDAL CONCENTRATION OF UA:

The minimum inhibition concentration of UA for the test bacteria was determined by the microdilution method [31]. Double-layer serial dilutions of UA in Muller-Hilton broth (MHB) were prepared, then we transferred 100 μl of different dilutions to the wells, and 100 μl of the overnight culture adjusted to 0.5 McFarland was added to each well. Plates were incubated at 37°C for 24 hours. After incubation, the lowest concentration without growth was determined as the MIC value. The plates were inoculated on BHI agar from all concentrations without growth, starting from the lowest concentration at which no bacterial growth was observed. After incubation at 37°C for 18–24 hours, we checked for growth, and the lowest concentration without growth was recorded as the minimum bactericidal concentration (MBC). Experiments were performed 3 times. Growth was controlled with tetrazolium chloride (TCC).

INVESTIGATION THE EFFECT OF UA ON BIOFILM FORMATION:

Anti-biofilm activity of UA was determined by microtitration method in multi-well flat bottom polystyrene plates. To determine the effect of UA on biofilm formation, 100 μl of TSB prepared with UA as much as its MIC value was transferred to each well. We transferred 100 μl of the test bacteria culture prepared in TSB containing 0.25% glucose, diluted 1/40 from the 18-hour culture of test bacteria developed in TSB containing 0.25% glucose. TSB was used as control. The amount of biofilm was determined after the plates were incubated for 24 hours at 37°C. To determine the effect of UA on the mature biofilm, a 1/40 diluted bacterial culture was prepared from the overnight culture prepared in TSB containing 0.25% glucose. We poured 200 μl of this bacterial culture into the wells and incubated at 37°C for 24 hours. At the end of the incubation period, 100 μl was removed from the wells and transferred from the solution containing 100 μl UA (as MIC). Plates were incubated at 37°C for 24 hours. After incubation, the biofilm was determined. All tests were done 3 times [33,34].

DATA ANALYSIS:

Descriptive statistical analyses were performed. Biofilm inhibition percentage was calculated and given according to an equation used in a previous study [35]:

Results

BIOFILM FORMATION PROPERTIES AND ANTIBIOTIC SUSCEPTIBILITY OF TESTED BACTERIA:

Nine S. epidermidis isolates, 5 S. hominis isolates, 2 S. saprophyticus isolates, and 1 S. capitis and S. lentus isolates were taken as test bacteria. All tested bacteria formed high biofilm on polystyrene (Table 1). The susceptibility of CoNS isolates to antibiotics was different. Antibiotic susceptibility properties of the tested bacteria are given in Table 1. While S. epidermidis isolates were resistant to penicillin and erythromycin, they were determined to be susceptible to gentamicin. All isolates were resistant to penicillin except S. epidermidis17.7H and S. lentus. All test isolates were susceptible to tetracycline except for S. epidermidis 4.11 and S. saprophyticus 2. Test isolates were generally resistant to methicillin but susceptible to vancomycin.

ANTIBACTERIAL EFFECTS OF UA:

The antibacterial effect of UA varied according to the isolates. While 62.5 μg/ml inhibited 5 S. epidermidis isolates, it was not effective on 4 S. epidermidis isolates (Table 1). It was not found to be effective on most of S. hominis, S. capitis, and S. saprophyticus. The MIC value for S. lentus was found to be 31.2 μg/mL. The MIC and MBC values of the tested CoNS strains are shown in Table 1.

ANTI-BIOFILM EFFECTS OF UA:

Anti-biofilm effects of UA were evaluated against biofilm formation of tested strains before and after adding into the media. Biofilm inhibition percentage was between 5.7% and 81.5% when UA was tested against S. epidermidis biofilms before biofilm formation. However, It did not affect the biofilm formation of S. epidermidis 15.81 or S. epidermidis17.11. It inhibited the biofilm formation of S. saprophyticus 1 and S. lentus by 73.3% and 74.3%, respectively. Usnic acid inhibited S. capitis preformed biofilm by 20.0%, and S. saprophyticus 2 by 20.3%. While it was not effective on S. hominis 17.2 and S. hominis 9.3 isolates, it inhibited other S. hominis isolates at between 27.2% and 73.6%.

After the biofilm was formed, the inhibition effect of UA decreased in all strains (Figure 1, S. epidermidis 15.81, S. epidermidis17.11, S. hominis 17.2, and S. hominis 9.3 were excluded since they were not inhibited). UA could not inhibit biofilm against 11 out of 18 strains after biofilm formed. UA inhibited the formed biofilm of S. epidermidis 17.71 by 2.9%, S. epidermidis 22.21 by 24.2%, and S. epidermidis 11.21 by 29.5%, while it inhibited the biofilm of S. epidermidis 4.11 by 35.1% S. epidermidis 11.11H by 44.4%. No effect on mature biofilms were observed for S. epidermidis 17.7H, S. epidermidis 15.41, S. hominis 9.3, S. hominis 17.2H, S. saprophyticus, and S. lentus. Figure 1 shows the inhibitory effect of UA on coagulase-negative staphylococcal isolates before biofilm formation and after biofilm formation. None of the isolates were stimulated for biofilm formation by UA.

Discussion

Usnic acid has been shown to have antitumor, antiviral, and antimicrobial activities [36], and its antibacterial properties against many susceptible and multi-drug-resistant bacterial strains have been proven [37,38]. In the present study, the antimicrobial activity of UA varied according to the tested bacteria. Usnic acid did not show antibacterial activity against S. epidermidis17.11, S. epidermidis17.7H, S. epidermidis15.41, S. epidermidis4.11, S. capitis, S. hominis 17.2, S. hominis29.2, S.saprophyticus1, and S.saprophyticus2 isolates. The MIC value against other isolates was determined as 31.3–62.5 μg/ml. The effect of UA on biofilm formation of CoNS strains varied according to the isolates. While it did not affect the biofilm formation of S. hominis 17.2, S. epidermidis 15.81, S. epidermidis17.11, and S. hominis 9.3 isolates, UA inhibited biofilm formation at different rates in other isolates. Addition of UA after biofilm formation had less effect on the biofilm. Although UA strongly inhibited biofilm formation in S. epidermidis17.7H and S. epidermidis15.41 isolates, it had no effect on mature biofilm. Similarly, UA had no effect on mature biofilms of S. saprophyticus, S. lentus, S. hominis9.3, and S. hominis 17.2H. In previous studies, UA was shown to have antimicrobial activity against gram-positive bacteria and mycobacteria such as S. aureus, Enterococcus faecalis, and Enterococcus faecium [39–42]. Usnic acid has been reported to be effective against antibiotic-resistant gram-positive bacteria and methicillin-resistant S. aureus (MRSA) [29,39,43]. While the inhibitory activity of UA was weaker against antibiotic-sensitive S. aureus strains, it has been reported to be higher against MRSA strains [43–45]. Maciazg-Dorszynska et al [46] reported that it showed antimicrobial activity by causing rapid and strong inhibition of RNA and DNA synthesis in gram-positive bacteria. Researchers have reported that UA causes disruption of DNA replication and also indirectly contributes to the antimicrobial activity of UA by inhibiting protein synthesis. Gupta et al [47] suggested that the antibacterial activity of UA against MRSA occurs by disruption of the cell membrane. Due to its lipophilic nature, UA is thought to penetrate the bilayer cell membrane and cause proton leakage and disrupt the bacterial membrane [48,49]. It has been reported that UA shows high similarity with vancomycin, inhibitor of cell wall synthesis and rifampin, and inhibitor of RNA synthesis. It has been suggested that antimicrobial capacity may be related to the protonophoric activity of UA [49]. Another possible mechanism of UA activity against S. aureus is binding to allosteric sites on the protein surface of FabI (enoyl reductase enzyme), which affects enzyme activity [48]. Usnic acid was found to be active against S. aureus, with a MIC of 21 μg/ml [50]. Minimum inhibitory concentration values of UA against different clinical isolates of S. aureus range from 8 to 50 μg/ml [33,34,43,46,47]. In a recent study, UA incorporated poly(ɛ-caprolactone) and decellularized extracellular matrix nanofibrous scaffold was found effective against S. aureus, S. epidermidis, Streptococcus mutans, Cutibactrium acnes, and fungal pathogens, but the sources of the tested bacteria were commercial kits. The authors also reported that it has anti-biofilm effect against Klebsiella pneumoniae and Pseudomonas aeruginosa, but they did not check anti-biofilm activity against CoNS [51]. Francolini et al [30] stated that UA has an inhibitory effect on S. aureus biofilm. Researchers have reported that UA can control the biofilm formation of S. aureus, mainly by killing the attached cells. They also suggested that it may be due to the effect of UA on signaling pathways. Sun et al [52] reported that it was effective on mature biofilm of MRSA. Pompilio et al [33] found UA to be effective against biofilm formation of MRSA and methicillin-susceptible S. aureus strains. However, they also found that UA was significantly active against preformed biofilms. In another study, Pompilio et al [34] reported that the antibacterial activity of UA was associated with damaged peptidoglycan synthesis, whereas the anti-biofilm effect was primarily due to impaired binding to host matrix proteins and decreased expression of lipase and thermonuclease. Although UA did not show antibacterial activity against S. epidermidis17.7H, S. epidermidis 15.41, S. epidermidis 4.11, S. saprophyticus1, S. saprophyticus 2, and S. hominis 29.2 isolates, it significantly inhibited biofilm formation of these isolates. It had also been reported that UA can affect the morphology of the P. aeruginosa biofilm without inhibiting bacterial growth, probably due to interference with bacterial signaling pathways [30]. In another recent study, glass coated with graphene loaded with UA was investigated for anti-biofilm effect against S. aureus and S. epidermidis; the authors used commercial kits for tested bacteria. According to their results, the graphene-coated material itself had no anti-biofilm effects, but graphen (loaded with UA)-coated material had an effect on biofilm formation of S. aureus and S. epidermidis. However, in that study the authors only studied S. aureus and S. epidermidis [53].

There are few studies on the antibacterial and anti-biofilm effect of UA on CoNS, and to best of our knowledge, the effects of UA on ocular surface microbiota have not been studied previously. It was determined that while UA was antibacterial on some CoNS, it was not effective on some strains, and anti-biofilm activity was found to be higher. Although UA did not show antibacterial activity in some strains, it was shown to have anti-biofilm activity. However, liver toxicity and contact allergy have been reported in some studies [54], which greatly reduces its potential as an anti-biofilm agent. There are studies showing that it is beneficial as a controlled-release drug to prevent local toxicity [51,53,55]. It has been demonstrated that ocular microorganisms, including S. aureus and S. epidermidis, which in the past were susceptible to antibiotics, have now become resistant [56–58]. According to the ESCRS Endophthalmitis Study, CoNS are one of the leading groups that causes endophthalmitis [59,60]. Similar to our findings, Bharathi et al [56] found that gram-positive cocci obtained from infections such as endophthalmitis, corneal infections, blepharitis, conjunctivitis, keratitis, and dacryocystitis were susceptible to vancomycin. The antibiotic susceptibilities of the test microorganisms in this study were different. Apart from endophthalmitis, treatment of ocular surface infections is mostly achieved with topical drops, thus avoiding systemic toxicity, especially with slow-release drugs. Cocchietto et al reported that usnic acid and its salts are used topically in skin and nail diseases with success [61]. Therefore, the effect of UA against ocular surface bacteria warrants further investigation. More studies are needed to determine the efficacy and safety of UA.

Our study has some limitations. First, this was an in vitro study, and in vivo studies are needed to assess the effects of UA on eyes and on infected ocular surface. To avoid the toxic effects of UA, studies should be performed with controlled-release UA products. Another limitation is that scanning electron microscopy and confocal laser microscopy were not used to visualize the biofilm formation.

Conclusions

In this study, it was determined that UA was effective at different rates in terms of antibacterial and anti-biofilm effects against coagulase-negative staphylococci obtained from the ocular surface. However, anti-biofilm effects were found to be limited after mature biofilm was formed in vivo in medium. Further studies should focus on inhibition of the formed biofilm. Considering that drugs are used as topical drops in the field of eye diseases, and with the development of slow-release drugs with technological developments, topical use of UA may be possible without systemic and local toxicities. Our findings suggest that UA needs to be investigated in further studies for its antibacterial and anti-biofilm effects on the ocular surface. We think that UA, which our study shows has an effect on ocular surface bacteria, may be a molecule that can be used for antibacterial and anti-biofilm purposes. We believe that the changes in antibiotic resistance in recent years necessitate the search for such alternative molecules. Also, UA should be studied both in vivo and in vitro against other ocular pathogens such as P. aeruginosa and methicillin-resistant S. aureus. Development of different slow-release UA systems will be the topic of future studies.

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750