Pharmacognosy Magazine

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 16  |  Issue : 72  |  Page : 780--788

Terfezia claveryi and Terfezia boudieri extracts: An antimicrobial and molecular assay on clinical isolates associated with eye infections


Lorina Ineta Badger-Emeka1, Promise Madu Emeka2, Saif Aldossari3, Hany Ezzat Khalil4,  
1 Department of Biomedical Sciences, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia
2 Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia
3 Department of Ophthalmology, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia
4 Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia; Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia 61519, Egypt

Correspondence Address:
Lorina Ineta Badger-Emeka
Department of Biomedical Sciences, College of Medicine, King Faisal University, Al-Ahsa 31982
Saudi Arabia

Abstract

Background: Ocular infections are capable of spreading to different anatomical sites of the eyes and, if not appropriately treated, can lead to blindness. The emergence of difficult to treat microbial infections has led to the search of alternatives from natural sources. Objectives: The antimicrobial effects of Terfezia claveryi and Terfezia boudieri (T. boudieri) against bacteria isolates associated with eye infections and their molecular mechanism were investigated. Materials and Methods: Crude aqueous and methanolic extracts, including fractions of chloroform, petroleum, and ethyl acetate of T. claveryi and T. boudieri, were used for the investigation. Bacterial isolation and identification were carried out using basic microbiological and biochemical techniques. scanning electron microscopy.(SEM) and molecular docking were used to adduce possible antimicrobial mechanism of these extracts and their fractions. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus lugdunensis, Serratia odorifera, Serratia liquefaciens, Pseudomonas stutzeri, Pseudomonas oryzihabitans, Proteus mirabililis, Kocuria kristinae, Kocuria rosea, and Micrococcus luteus were isolated from patients with ocular infections. Results: Isolates were resistant to benzylpenicillin (78.0%), rifampicin (57.0%), tetracycline (56.0%), clindamycin (33.3%), and tigecycline (24.0%). Furthermore, the percentage resistance to gentamicin and ciprofloxacin was 13.0% each. All isolates were susceptible to extracts/fractions of T. claveryi and T. boudieri. Docking analysis showed binding with surface protein Sortase A of Staphylococcus aureus, indicating that stigmasterol, the active compound in both Terfezia species, interacted with valine amino acid 110. SEM imaging showed morphological alterations in treated isolated Staphylococcal species. Conclusion: Therefore, extracts of both Terfezia species have demonstrated the potential to possess antibacterial activity, which can be further exploited for clinical use.



How to cite this article:
Badger-Emeka LI, Emeka PM, Aldossari S, Khalil HE. Terfezia claveryi and Terfezia boudieri extracts: An antimicrobial and molecular assay on clinical isolates associated with eye infections.Phcog Mag 2020;16:780-788


How to cite this URL:
Badger-Emeka LI, Emeka PM, Aldossari S, Khalil HE. Terfezia claveryi and Terfezia boudieri extracts: An antimicrobial and molecular assay on clinical isolates associated with eye infections. Phcog Mag [serial online] 2020 [cited 2021 Apr 16 ];16:780-788
Available from: http://www.phcog.com/text.asp?2020/16/72/780/309306


Full Text



SUMMARY

Extracts and fractions of Terfezia claveryi and Terfezia boudieri exhibited significant antimicrobial activity against sensitive and resistant bacterial clinical eye isolates. Therefore is a potential source of future antibiotic.Molecular mechanism shows that it binds Sortase A protein on the surface of Staphylococcus aureus.

Abbreviations used: CoNS: Coagulase-negative staphylococci; SEM: Scanning electron microscope; ADT: Autodock tools; PBS: Phosphate-buffered saline; DMSO: Dimethyl sulfoxide; MICs: Minimum inhibitory concentrations; CAZ: Ceftazidime; FEB: Cefepime; AZM: Aztreonam; IMP: Imipenem; MINO: Minocycline; TGC: Tigecycline; PEN: Benzylpenicillin; ERY: Erythromycin; TET: Tetracycline; RIF: Rifampicin; CLI: Clindamycin; NLDO: Nasolacrimal duct obstruction; PUK: Peripheral ulcerative keratitis; WHO: World Health Organization Srt. A: Sortase A.

[INLINE:1]

 Introduction



Ocular infections such as keratitis, conjunctivitis, orbital cellulitis, endophthalmitis, among a wide range of other eye infections, can be attributed to bacteria.[1],[2],[3],[4] Particularly, 50%–70% of conjunctivitis cases are a result of bacterial infections.[5] Such infections might not remain localized but are capable of spreading to other anatomical sites of the eye[6] and consequently lead to corneal blindness or endophthalmitis if not timely treated.[7],[8],[9] Even when detected early, empirical management can either help in resolving the causative bacterial infection or failure. This is due to the global increase in difficult to treat bacterial infection, including those responsible for eye infections. Recent report[10] observed a growing resistance by Gram-negative bacteria associated with eye infections between 2013 and 2016 as compared to the observations for 2011 and 2013. These have been attributed to an increase in the use of antimicrobials like fluoroquinolones for both prevention and treatment.[11] Furthermore, earlier studies[12] have reported resistance to the fourth-generation fluoroquinolones; this growing resistance was particularly with concerns to moxifloxacin, gatifloxacin and imipenem, carbapenem. Therefore, there is a public health crisis arising as a result of difficult to treat bacterial infections to which the World Health Organization (WHO)[13] warned that the world could be entering an era like those preceding those of the discovery of antibiotics. The “golden era” of antibiotics resulted in the production and rise of antibiotics.[14] It is however of the view that the era ended due to misuse as well as the inability by researchers to keep up with the pace of the discovery of new drugs.[15] Antibiotics are not being produced as fast as they are needed, to meet with the rate at which antimicrobial resistance by bacterial isolates is evolving.

There is, therefore, a global urgency in the search for practicable alternative options. Thus, there is an increase in the search for, and the use of herbal medicinal remedies with about 25-50 % of current pharmaceuticals.[16] An increase in the discovery and use of herbal remedies could lead to interesting possibilities in combating antimicrobial resistance, as noted by researchers globally.[17] More so as these herbal remedies are less toxic when compared to conventional antibiotics.

Although the discovery of antibiotics was a defining moment in the medical management of microbial infections,[18] the advent globally of multi drug-resistant bacteria and the fact that the synthetic production of new antimicrobials has declined over the decades has led to a surge in the alternatives to antibiotics in herbal medicines. For ocular infections, there have been suggestions by researchers[19],[20],[21] for the use of dessert truffles as alternatives to currently used antibiotics.

Truffles are ectomycorrhizal fungi belonging to a family of complex hypogenous fungi containing species of which includes Terfezia claveryi and Terfezia boudieri.[22] Geographically, they are distributed in semi-arid and arid lands[23],[24] and have been employed in traditional/folk medicine in Arab communities for over two millennia with no know toxicity to its users. The Bedouins recommend the use of its water extracts for the treatment of common eye infections.[25]

The antimicrobial effects of Terfezia species have been reported previously, particularly the aqueous extracts.[26] These reports show that T. claveryi extracts is effective against clinical isolates of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.[27],[28] The documented efficacy of extracts of T. claveryi in curing trachoma disease and treatment of cornea infection in mice is based on its highlighted antibacterial bioactivity.[26],[29] In line with the need to search for alternative medications in place of existing antibiotics, the present investigation seeks to look into the clinical bacteria isolates associated with ocular infections, their susceptibility to commonly used antimicrobial, as well as the effect of extracts and fractions of T. claveryi and T. boudieri and to elucidate molecularly the mechanism of these effects. This is with a view of providing further insight into the use of extracts and their fractions as alternatives in the prevention or treatment of ocular infections.

 Materials and Methods



Fungal material

Desert truffles species such as T. claveryi and T. boudieri, which are usually available in March and April, were procured from a weekly local market in Al-Ahasa located in the Eastern Region of Saudi Arabia. The fresh truffles were dried in the shadows for 21 days while avoiding direct sunlight until constant dried weights were obtained. The resultant dried truffles were grounded into fine powder and stored in a dark, dry place inside containers at ambient temperature until use. Voucher specimens were deposited at the College of Clinical Pharmacy in the Department of Pharmaceutical Sciences, King Faisal University, Al-Ahsa, Saudi Arabia.

Preparation of extracts of Terfezia species and fractionation

One hundred grams of fine powder of T. claveryi and T. boudieri were weighed out, soaked separately in distilled water in 1:3 ratio, for 48 h at temperatures of 4°C. The resultant solution was homogenized after 48 h, filtered through a double layer of cotton. The filtrate was centrifuged at 3000 rpm for 10 min at room temperature, with the resulting supernatants collected and labeled as crude sample of aqueous extracts. These crude samples were further dried under reduced pressure with a rotary evaporator to give 6.2 g for T. claveryi and 5.1 g T. boudieri. These were represented as total dried crude aqueous extracts.

For the preparation of methanolic extract, 400.0 g of dried powdered truffles extracts of T. claveryi and T. boudieri were exhaustively extracted three times with methanol (MeOH) for 7 days using 5.0 L of 70.0% MeOH under room temperature. The resulting extracts were then separately filtered using Whatman filter paper and evaporated to dryness. The concentration of the extracts was with rotary evaporator at reduced pressure to give a dark reddish-yellow extract weighing about 20.9 g for T. claveryi and 18.3 g for T. boudieri and labeled as total MeOH extracts.

Furthermore, 17.0 and 15.0 g of T. claveryi and T. boudieri were each suspended in 200.0 ml deionized water in separating funnels and partitioned with petroleum ether (5.0 × 500.0 ml). The resulting petroleum ether fractions were evaporated to dryness using rotary evaporator. These extractions were dried to give 5.1 and 4.6 g for T. claveryi and T. boudieri, respectively and then stored in closed containers. The rest of the mother liquor was mixed with chloroform (4.0 × 500.0 ml) and the chloroform fraction obtained from this mixture was also evaporated to dryness to concentrate it by the use of rotary evaporator, which was then freeze dried. A weight of 2.3 and 1.9 g was obtained, respectively, for T. claveryi and T. boudieri. The preparation of ethyl acetate extracts is the same as the above-mentioned protocol to give 4.9 and 3.7 g for T. claveryi and T. boudieri, respectively. The remaining mother liquor fractions were also dried to give 4.1 and 3.5 g for T. claveryi and T. boudieri, respectively, stored in an airtight container and kept in the freezer for further use.[30],[31] From the ten extract fractions of crude aqueous extract, petroleum ether, chloroform fraction, ethyl acetate, MeOH extracts for T. claveryi and T. boudieri each, 20 mg/ml solutions were prepared and stored in the freezer for the evaluation of minimum inhibitory concentrations (MICs).

Minimum inhibitory concentrations determination

The MICs determination of T. claveryi and T. boudieri extracted fractions were done using the broth dilution method in accordance with a previously described.[32] All the extracted fractions were prepared to the highest possible concentration of 20.0 mg/ml (stock concentration) in 10.0% dimethyl sulfoxide (DMSO) solution. These were serially diluted to give concentrations of 15.0, 10.0, 5.0, and 2.5 mg/ml. A volume of 1.0 ml of the standardized microbial broth cultures were inoculated into the tubes containing the diluted extracts and labeled accordingly. The tubes were placed in CO2 incubator at 37°C and were observed for 24 h. They were examined for the presence or absence of bacterial growth. The least concentrations of the extracts which inhibited the growths of inoculums were considered as the MICs. Therefore, MICs were found to be 5.0 mg/ml for crude aqueous extract, MeOH extract, and chloroform fraction. However, ethyl acetate and petroleum ether fractions were 2.5 mg/ml, respectively.

Clinical ocular bacteria isolates

Specimens were from people with diabetes and hypertensive patients with glaucoma, bacterial and viral conjunctivitis, cataracts, which included those with mature and post-cataract surgical removal, nasolacrimal duct obstruction and peripheral ulcerative keratitis (PUK). Patient eye discharge was collected by the attending ophthalmologist using soft-tipped sterile cotton swabs under sterile conditions and was brought to the Microbiology department of the College of Medicine. Each swab was inoculated into the nutrient broth and incubated aerobically at 37°C for 24 hr. Overnight growth was plated on blood agar and MacConkey agar obtained from Oxoid, Hampshire, UK and was incubated aerobically for 24 h at 37°C.

Bacteria isolation and antimicrobial susceptibility test

Pure bacteria cultures were used for the identification using basic bacteriological and Biochemical techniques as recommended by Cheesbrough.[33] Confirmation of isolate identity was carried out using the VITEK 2 compact automated system (BioMerieux, Marcy L'Etoile, France) according to the manufacturer's guidelines, with GP and GN cards for Gram-positive and Gram-negative isolates, respectively. Susceptibility to antimicrobials and determination of MICs was carried out by the VITEK 2 compact automated system using antimicrobial susceptibility testing cards, against the following antibiotics: Ampicillin/Sulbactam (AMS); augmentin (20/10 μg); piperacillin/tazobactam (100/10 μg); ceftazidime (30 μg); cefepime (30 μg); aztreonam (AZM); ertapenem [10 μg]; imipenem (10 μg); meropenem (10 μg); amikacin (30 μg); gentamicin (10 μg); tobramycin (TOB); ciprofloxacin (30 μg); levofloxacin (5 μg); minocycline (30 μg); tigecycline (30 μg); moxifloxacin (MXF); oxacillin (OXA); trimethoprim/sulfamethoxazole (1.25/23.75 μg) benzylpenicillin (10.0 μg); erythromycin (15.0 μg), vancomycin (30 μg), and tetracycline (30 μg). Interpretation of results was according to the Clinical and Laboratory Standards Institute[34] recommendations.

Antimicrobial effect of Terfezia claveryi and Terfezia boudieri

Well diffusion antimicrobial susceptibility method[35],[36] was used for the determination of the antimicrobial effect of the desert truffles against clinical ocular infection isolates. Each bacteria isolate was inoculated and spread on Muller Hilton agar. Using 0.8 mm cork borer, wells were cut into the agar with extracts of both desert truffles introduced into each well. All plates were incubated aerobically at 37°C overnight after which zones of inhibition were measured using mm ruler. Experiment was carried out in three replicates.

Molecular analysis

Scanning electron microscopy

The SEM was used to determine the effect of extracts fractions of T. claveryi and T. boudieri on the cells of species of Staphylococcus using the previous method[37] with modifications. Staphylococcal species were cultured in Muller Hilton broth at 37°C in a shaking incubator for 6 h. Final adjustment of turbidity was according to McFarland 0.5 standards with obtained bacteria cell suspension as described.[37]

Treated bacteria cultures were incubated in a shaker incubator at 37°C for 24 h while untreated bacteria cells were used as controls. The resulting growth was centrifuged, prefixed overnight in 2.5% glutaraldehyde solution at 4°C. All samples were then rinsed with phosphate-buffered saline and post fixed as described[37] with 100% acetone applied at the last stage. A 20 nm thick layer was obtained by sputtering of gold. The SEM micrographs were obtained with SEM (JSM 6390 LA, JOEL) at 15 KV accelerating voltage.

Molecular docking analysis

In other to further investigate the molecular mechanism of extracts of T. claveryi and T. boudieri, whose main active component is Stigmasterol, hence it was docked with Staphylococcus aureus surface protein. Molecular docking was done using Autodock tools V.1.5.4 and Autodock V.4.2 program. The aim was to perform in-silico analysis of the interactions between stigmasterol, a ligand candidate and Sortase A (Srt-A), a Staphylococcus aureus surface protein. The Srt-A (PDB ID: 1TD2P) three-dimensional (3D) chemical structure was retrieved from Protein Data Bank, while that of stigmasterol (CID_5280794) was obtained from PubChem compound database. Q-site finder was used for the identification of active sites of targeted protein with docked ligand reflected as rigid bodies to the receptors. Predicted binding energy was used for the evaluation and sorting of results. This described method was adopted from Hanieh et al.[38]

Statistical analysis

Data are presented as mean ± standard deviation and Graphpad Prism 8.2.3 (San Diego, USA) was used for statistical analysis. Two-way analysis of variance was used to compare the statistical difference between and within zones of inhibitions produced by different extract fractions of both plants under study. Furthermore, Paired t-test was used to compare zones of inhibitions produced by the same extract fractions from the two plants. The significant difference was taken as P < 0.05.

 Results



Patient demography

Collected specimens were from males (57%) and females (43%) with ages that ranged between 1 and 80 years. The isolated bacteria were Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus lugdunensis, Serratia odorifera, S. liquefaciens, Pseudomonas stutzeri, Pseudomonas oryzihabitans, P. mirabililis, Kocuria kristinae, Kocuria rosea, Micrococcus luteus, as shown in [Table 1].{Table 1}

Antimicrobial susceptibility

The results on the antimicrobial susceptibility of isolated Gram-negative and Gram-positive bacteria are presented in [Table 2] and [Table 3], respectively. [Table 2] shows that in addition to being intrinsically resistant to tigecycline, all isolated strains of Proteus mirabilis were resistant to minocycline, ceftazidime (CAZ), cefepime (FEP), and aztreonam (AZM). Two of the strains were also resistant to imipenem. For the Gram-positive Staphylococcal eye infection isolates, there was a 100% resistance to the penicillins, as shown in [Table 3]. Furthermore, there was high antimicrobial resistance against the following conventional antibiotics: erythromycin, clindamycin, tetracycline, and rifampicin. One strain of S. epidermidis isolates tested positive to cefoxitin screen, while another strain also showed high antimicrobial resistant to different groups of antibiotics [Table 3]. For all the isolates, according to the antibiotic classification, the β–lactams, there was a 78% resistance to benzylpenicillin, 50% resistance to cefepime, 30% to imipenem, and 22% ampicillin/sulbactam and ceftazidime each [Figure 1]. For other classes, [Figure 2] shows antimicrobial resistance for the isolates as follows: rifampicin (57%), tetracycline (56%), clindamycin (33.3%), and tigecycline (24%). There was a 13% resistance to gentamicin and ciprofloxacin, while 6% of the isolates were resistant to levofloxacin and trimethoprim/sulfamethoxazole.{Table 2}{Table 3}{Figure 1}{Figure 2}

Zones of inhibition against extracts and fractions

The results on growth inhibitory effects of fractions of T. claveryi and T. boudieri are shown in [Table 4]. Both Gram-positive and Gram-negative bacteria eye isolates were inhibited by crude aqueous and methanolic extracts of both plants to varying degrees, more so with fractions of T. claveryi extracts. Crude aqueous extract of T. claveryi showed better zones of inhibition than that of T. boudieri at a very statistically significant with P value of 0.0013. Furthermore, the chloroform fraction of T. claveryi showed better inhibition against all isolates as compared to those of the same fraction of T. boudieri, with the difference in the mean zone of inhibition being statistically significant (P < 0.0001), as shown in [Table 4]. A similar pattern was exhibited by ethyl acetate fractions of both plants, with T. claveryi having a better zone of inhibition, which was statistically significant (P = 0.0001). Results in [Figure 3] showed that methanolic extracts observed zones of inhibition of bacteria growth was not statistically different when compared together. Of all plant fractions, those of petroleum ether were the least effective against the eye infection isolates, with observed differences between the two fractions being statistically not significant (P = 0.077).{Table 4}{Figure 3}

Comparison between antimicrobial susceptibility and fractions of extracts

The results in [Table 2] are the antibiogram of the isolates to conventional antibiotics employed in the treatment of Gram-negative infections. It shows encountered strains of P. mirabilis to be resistant to ceftazidime (CAZ), cefepime (FEB), aztreonam (AZM), imipenem (IMP), minocycline (MINO), and Tigecycline (TGC). For the Gram-positive Staphylococcal species, antibiogram results presented in [Table 3] showed resistance by different strains to benzylpenicillin (PEN), erythromycin (ERY), tetracycline (TET), rifampicin (RIF), and clindamycin (CLI). However, there was growth inhibition of this bacterium by extracts and extract fractions of both T. boudieri and T. claveryi as shown in [Table 4]. Extracts and fractions of T. claveryi showed better zones of inhibition than those of T. boudieri against the different isolated strains of P. mirabilis. The difference in growth inhibition by chloroform and ethyl acetate fractions were statistically significant (P < 0.05) as compared with other fractions of T. claveryi. Furthermore, with regard to Gram-positive Staphylococcal species, these isolates where found to be susceptible to extracts and fractions of T. boudieri and T. claveryi [Table 4], with T. claveryi exhibiting greater zones of inhibition. Results showed that both crude aqueous and methanol extracts displayed better antibacterial activity than other fractions, with methanol extract of T. claveryi exhibiting better inhibitory activity.

Results also showed that crude aqueous extract, methanol extract, and fractions of chloroform and ethyl acetate did significantly inhibit S. epidermidis more than other Staphylococcal species. Staphylococcus aureus was more sensitive to crude aqueous and methanol extracts, chloroform fraction, and ethyl acetate fraction isolated from T. claveryi.

Molecular analysis

The results of scanning electron microscopy and molecular docking used to determine the antimicrobial mechanism of action of T. boudieri and T. claveryi are presented in [Figure 4] and [Figure 5], respectively. Morphological alterations in treated Staphylococcal species by fractions of Terfezia species are shown in [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e, [Figure 4]f, [Figure 4]g, [Figure 4]h, [Figure 4]i, [Figure 4]j. In [Figure 4]a, untreated S. epidermidis is seen with defined smooth clustered margins as compared with those treated with aqueous extract of T. claveryi [Figure 4]b, which showed altered surface margins. A similar pattern is seen in the untreated [Figure 4]c and treated [Figure 4]d S. epidermidis with aqueous extract of T. claveryi. For the SEM micrograph of S. epidermidis untreated [Figure 4]e and treated [Figure 4]f with methanolic extract of T. claveryi shows the surface appearance of the bacteria was dramatically altered. This is similar to observations in the micrograph of S. epidermis with extracts of T. boudieri [Figure 4]g and [Figure 4]h. While the micrographs of MeOH extract of T. claveryi showed complete distortion of treated S. epidermidis [Figure 4]j as against those of the untreated [Figure 4]i.{Figure 4}{Figure 5}

Results in [Figure 5] are those of 3D docking analysis of Srt-A surface transport protein of S. epidermidis with stigmasterol, the main constituent of T. boudieri and T. claveryi. Stigmasterol is the active compound of T. boudieri and T. claveryi. Docking analysis of stigmasterol with surface protein Srt-A of Staphylococcus aureus showed this active compound interacted with valine amino acid 110. This interaction with the Srt-A domain has a binding energy of-6.69, with a ligand efficacy of 0.22. The binding energy reflects a high binding efficacy to the bacteria, possibly depicts its antimicrobial activity.

 Discussion



The clinical features of ocular bacterial infections in the present study were diverse and so was their susceptibility against tested antibiotics. Bacteria conjunctivitis was the most prevalent of the encountered eye infections caused by both Gram-positive and Gram-negative bacteria. Researchers had previously reported similar findings in different regions of the world.[39],[40],[41] Causative bacteria included Staphylococcus aureus, co-agulase negative Staphylococcus [CoNS], Gram-negative bacteria with species of Pseudomonas and strains of P. mirabilis among others. The listed bacteria are similar to those reported by other researchers.[10],[42] Differences in observed reports are in the antimicrobial susceptibility of the bacteria isolates against tested antibiotics.

In the present investigations, Staphylococci isolates were all (100%) resistant to Penicillins, observations that are similar to those of earlier researchers.[42],[43] Furthermore, high resistance against erythromycin, clindamycin, tetracycline, and rifampicin, all of which are the drug of choice in the treatment of ocular eye infections, were noted. This is a disturbing trend in antimicrobial susceptibility of bacteria associated with ocular infection but then reflects the global public health problem of the reduced susceptibility against antibiotic of choice by bacteria. Furthermore, troubling are the antimicrobial susceptibility of the various strains of P. mirabilis isolates with high resistance to minocycline, ceftazidime, cefepime, aztreonam, as well as some strains being resistant to imipenem. High antimicrobial resistance by bacteria associated with ocular infections, such as in this study, had also been reported by other researchers.[44],[45] This trend in bacteria susceptibility to antimicrobials highlights the challenges faced globally in the general management of bacterial infections, inclusive of those involved in ocular infections, as shown in this report. It is, however, worthy of note that irrespective of antimicrobial susceptibility by the isolates in the present investigation, the extracts and or fractions of both desert truffles in the present investigation inhibited the growth of Gram-positive and Gram-negative bacteria isolated from patients with eye infections. There were, however, differences as seen in the study between T. claveryi and T. boudieri with regard to the zones of inhibition of the bacteria isolates as well as differences in the type of extracted component within a plant species. The statistically significant better zones of inhibition obtained from the crude aqueous extract, chloroform, and ethyl acetate fractions of T. claveryi than those of T. boudieri suggest variations between species of desert truffles that could be due to differences in their chemical compositions and content. A similar observation[46] pointed out differences in the antimicrobial properties of a number of desert truffles species. This they had attributed to possible differences in their chemical composition as well as the treated microbial strains. This might explain why methanolic extracts of T. claveryi and T. boudieri inhibited the growth of bacteria isolates in this study with the exception of S. lugdunensis. The bacteria strain could be contributory factor more so as the comparison in differences in zones of inhibition of the methanolic extracts of T. claveryi and T. boudieri were not statistically significant, an observation that is similar to those of other researchers.[47],[48]

Besides the differences in plant species used in this study, there were observed variations in extract or fractions activity as encountered with both species of Terfezia in the present investigation. That petroleum ether fraction of both desert truffles was the least effective against the bacteria isolates suggest that this is might not be the most suitable for the inhibition of microbial growth, particularly in CoNS. These findings differ from earlier reports[29] where there was lack of growth inhibition by aqueous and petroleum extracts of T. claveryi against Gram-positive and Gram-negative bacteria. The report[29] suggested that this could be due to the chemical composition of the bacteria. Such differences could also be attributed to a number of other factors, such as of extraction methods that might affect the quantity and quality of antimicrobials present in them, a view that had also been expressed previously.[49]

Overall, it is, however, worth noting that there was growth inhibition of all the isolates in the present investigation by extracts and fractions of extracts of T. claveryi and T. boudieri, thus suggesting their suitability for the treatment of ocular infections. The molecular docking results and the SEM imaging gave an insight into the antimicrobial inhibitory mechanism of the desert truffles. The suggested mechanism is seen in the docking analysis by the interaction between stigmasterol that is one of the active compounds of Terfezia and Sortase A protein on the surface of Staphylococcus aureus.[50] This could be collaborated by the SEM analysis, where the bacterial cell wall showed partial or complete distortion when treated with Terfezia extracts.

 Conclusion



Our study shows that T. claveryi and T. boudieri significantly inhibited the growth of clinical bacteria isolates associated with ocular infections, even for those that were resistant to conventional antibiotics. SEM and molecular docking analysis also confirm their antimicrobial activity. Therefore, the use of extracts of T. claveryi and T. boudieri as herbal remedy for eye infections could hereby be affirmed and justified.

Ethical consideration

Permission for the research was given by the Deanship of Scientific Research, King Faisal University [Research number 186176].

Acknowledgements

The authors would like to acknowledge the Deanship of Scientific Research at King Faisal University for the financial support under Nasher Track (Grant No. 186176). The authors thank Dr. Hairul for his kind help in molecular analysis. We thank Mr. Hani Al-Rasasi and Ms. Haijer Al-Del for their technical assistance.

Financial support and sponsorship

This study was supported by a grant from the Deanship of Scientific Research, King Faisal University, Saudi Arabia support under Nasher Track (Grant No. 186176).

Conflicts of interest

There are no conflicts of interest.

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