Probiotic Application of Bacteriocin-Producing S. Epidermidis in A Cellulosic Pad to Treat Some Skin Infections

The aim of this study was to evaluate the possibility of using Staphylococcus epidermidis cells as a probiotic to treat some skin infections. For this purpose, S. epidermidis Y73, which is an active bacteriocin producer and non-biofilm forming isolate, was selected among 134 skin isolates through primary and secondary screening. Tryptic soya broth was selected as the best medium to support bacteriocin production, while the optimal pH and temperature for S. epidermidis Y73 growth were 7 and 37°C, respectively, which were invested in the formula preparation. Furthermore, the possibility of using this isolate as a probiotic was investigated by preparing 4 potential cellulosic pads with 4 different formulae which were all subjected to an in vitro trial to select the one which is superior to the others in terms of supporting bacteriocin production and cells viability. The shelf life of the pad was estimated and the results showed that the cells remained vital until the 20 th week. The selected pad formula was used to treat artificially induced wounds on rabbit skin. The wounds were infected with Staphylococcus aureus, Kocuria rosea and Pseudomonas. aeruginosa. The symptoms in both control and treated animals were recorded and, based on the results; the healing process with the presence of the S. epidermidis Y73 pad was significantly faster compared with that for the control. This research will serve as a base for future studies on using vital cells of S. epidermidis as probiotics and, hence, make a contribution to the current literature on using live cells to treat bacterial skin infections.


Introduction
Probiotics are living microorganisms having an array of beneficial effects in humans, such as reducing inflammation, speeding the wound healing process, and strengthening the immune system [1]. This is usually achieved through different mechanisms, including the production of inhibitory substances such as bacteriocins or acids, blockage of pathogen adhesion, antioxidant activity, and nutrient competition [2]. Probiotics have been vastly marketed and consumed, mostly as functional foods or dietary supplements. They exert their action through enhancement of the gut barrier, epithelial repair, and modulation of the immune response [1].
There are numerous microorganisms used as probiotics, such as Bifidobacterium and Lactobacillus which are most commonly used. However, some strains of E. coli, Bacillus, and Saccharomyces cerevisiae are also used [3]. The antibacterial activity of probiotics acts against different pathogenic bacteria through multifunctional ways, mainly by secreting antimicrobial substances, such as organic acids, bacteriocins, H 2 O 2 , lactic acid and others.
Human skin is populated by billions of numerous bacteria and due to their constant contact with the environment; microbes have easy colonization ability to various areas of the body. There are many microorganisms associated with skin infections, such as atopic dermatitis, eczema, acne and burns. These microorganisms include, for example, Propionibacterium acnes, Staphylococcus aureus, Pseudomonas aeruginosa, and Corynebacterium [4]. Experimental studies showed that probiotics exert specific influences in the dermatology by helping the prevention and treatment of skin infections, including bacterial infections, psoriasis, dermatitis, the external signs of aging, acne, rosacea and yeast infections [5].
A wound is a tissue damage usually caused by membrane laceration that generally results in skin damage. Most chronic wounds contain more than one bacterial species which result in a synergetic effect causing non-virulent bacterial species becoming virulent and evoking host damage [6]. In the literature, investigations of the bacterial diversity in chronic wounds showed that Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia are the most common in chronic wounds [6]. In addition, bacteria in chronic wounds were observed to develop biofilms which contribute to a delay in healing. Madsen et al. [7] reported that chronic wounds are colonized by various bacterial populations, which contribute directly and indirectly to chronic phenotypes of wounds. Colonizing bacteria such as Staphylococcus epidermidis and Corynebacterium are normally present on the skin and usually prevent pathogenic bacteria colonization by immune orientation and competition on resources as well as bacteriocin production, which are all criteria that are present in the selected bacterial strains. The aim of the current study was to utilize a safe and bacteriocin-producing isolate of S. epidermidis as a probiotic against some common skin pathogenic bacteria via introducing it in a suitable pharmaceutical formula.

Collection of S. epidermidis isolates
Skin samples (swabs) were collected from healthy individuals in order to seek a potential bacteriocin producing normal microbiota of Staphylococcus epidermidis. First, the swaps were placed in tubes containing 5 ml of brain heart infusion broth as an enrichment medium. After 24 hrs of incubation at 37 o C, serial dilutions were prepared in normal saline and a swab from the dilution of 10 -5 was applied on mannitol salt agar. Then, after 24 hrs of incubation at 37 o C, the potential candidates for being S. epidermidis which were fitting the morphological criteria were selected and sub cultured on MS agar 3311 to ensure purity. These isolates were identified through biochemical tests and cultural characteristics as described by Bergey's manual and using VITEK system.

Screening of S. epidermidis isolates for bacteriocin production Primary screening
Well diffusion assay was used to evaluate the production of bacteriocin, as follows [8]: Universal tubes containing 5ml of broth were inoculated with 2% of an overnight culture of the isolate containing approximately 1×10 8 cells/ml and then incubated for 24 hrs at 37°C. After incubation, the cell-free supernatant (CFS) was collected and assayed for the presence of bacteriocin using agar well diffusion assay, as follows: 100 μl of an overnight growth culture of the indicator bacteria (4 different isolates of S. aureus and 2 isolates of Pseudomonas aeruginosa) containing approximately 1×10 8 cells /ml was mixed with 25 mL of a sterile Mueller Hinton agar and then poured into sterile Petri dishes. Wells of 6 mm diameter were cut and 100 μl aliquots of the filtered CFS were dispensed in each well. Then, plates were incubated for 24 hrs at 37°C [9,10].

Secondary screening (Bacteriocin activity assay)
Bacteriocin activity was determined for the selected isolates from the primary screening by using the critical dilution assay which is similar to the minimum inhibitory concentration technique (MIC) for antibiotic assessment [11]. In addition, the biofilm detection for S. epidermidis isolates was integrated in the screening by using the microtiter plate assay [12,13,14].

Determination of best production medium for S. epidermidis
Different nutritional media (nutrient broth, brain heart infusion broth, tryptic soy broth, modified brain heart infusion broth, mueller hinton broth and Thomas medium) [15] were tested in order to select the medium that supports maximum bacteriocin production by the selected S. epidermidis isolate [16].

Characterization of S. epidermidis as a probiotic Antibiotic susceptibility testing
Antibiotic susceptibility was conducted using the disc diffusion method [17] on a Mueller-Hinton agar which was previously seeded with the selected S. epidermidis Y37. Determination of optimal pH and temperature for S. epidermidis Y37 growth Six universal tubes containing 5 ml of tryptic soy broth with pH values ranged from 4 to 9 were inoculated with the same cell concentration of the selected isolate (10 8 cell/ml) and then incubated for 24hr at 37ºC for 24 hrs (the tubes were weighed empty before the procedure ). Afterwards, the tubes were centrifuged at 10000 rpm for 10 min, the cell free supernatant was discarded, and the tubes were re-weighed with the biomass within. The difference in weight was marked as the biomass [18]. The same procedure was followed to determine the optimal temperature via incubation at different temperatures: 25, 30,35,37,40 and 45°C.

Biofilm formation assay
The Biofilm detection for S. epidermidis isolates was achieved by using the microtiter plate assay based on the method described earlier [12,13]

Probiotic preparation Pad design
Four pads of thick cellulosic filter paper with1cm width and 3 cm length were prepared. Four potential formulae were used as follows [19] 1. 150 µl TSB containing S. epidermidis at a concentration of 1×10 8 cells/ml. 2. 100µl TSB containing S. epidermidis at a concentration of 1×10 8 cells/ml plus 50 µl petroleum jelly, 3. 100µl TSB containing S. epidermidis at a concentration of 1×10 8 cells/ml plus 50 µl glycerol, 4. 100µl TSB containing S. epidermidis at a concentration of 1×10 8 cells/ml plus 25 µl petroleum jelly and 25 µl glycerol. Then, all pads were placed on MSA which was previously seeded with1×10 4 cells/ml of S. aurous as an indicator in order to investigate the antagonistic capacity of the tested pads. The superiority of the selected pad was determined based on beneficial criteria obtained in terms of competition capacity and shelf life.

Estimation of expiration date of the probiotic pad
Expiration date of the pad was estimated according to the strategy proposed by Capen et al. [20]. A twenty-weeks trial was conducted to estimate the shelf life of the pad and investigate how long the

Hussein and Luti
Iraqi Journal of Science, 2020, Vol. 61, No. 8, pp: 1932-1943 3311 cells will remain vital in the selected formula. This was achieved by preparing 20 pads in sterile conditions with the selected formula. The tubes were then sealed with parafilm and preserved in sterile tubes at 4 о C; this point was considered as the zero time. Along the 20 weeks, every seven days, one pad was picked out from the batch and placed on MSA plate which was then incubated for 24hr at 37 о C to cheek the vitality and purity of S. epidermidis cells.

In vivo application of the probiotic pad
Six local rabbits were used in this experiment, which were divided into 3 groups each with 2 animals; one was the treatment group and the other was considered as a control. Each animal was specified for one indicator pathogenic bacteria. Animals were shaved one day prior to the infection using scissors and razor at the shoulder site. The animals were housed in small cages (one animal per cage) and allowed for food and water. Incision was made with 2 to 2.5 cm in length using a lancet in the shoulder. Then, in each rabbit group, the rabbits were marked as 1, 2 and 3 which were infected with S. aureus, P. aeruginosa, or K. rosa respectively. The infection was achieved via introducing the pathogens into the wound using a swab at a concentration of 1×10 4 . The S. epidermidis-containing pad was fixed on the wound of the treated group of animals using wound patch (pad 3). It was placed at the zero time after introducing the inoculum and wrapped with bandage for extra fixation. Every 24 hrs, the old pad was removed and replaced with a new one. The trial was meant to last until full recovery was retrieved and this was accomplished within 7 days.

Results and Discussion
In this study, 134 S. epidermidis isolates from skin samples were obtained from healthy individuals. The morphological and biochemical examinations of S. epidermidis isolates were achieved based on Bergey's manual of systematic bacteriology [21]; catalase, blood hemolysis and coagulase tests are useful to identify S. epidermidis and, therefore, these tests were performed for all isolates (data not shown). Thereafter, S. epidermidis isolates were subjected to a screening process in order to select the higher bacteriocin-producing isolate that can be used in this study. The screening was achieved in TSB medium and for more reliability all isolates were cultivated under the same conditions in terms of inoculum size, cell number, pH and incubation period. Four isolates of S. aureus and 2 isolates of Pseudomonades aeruginosa were used as indicator strains in order to determine the antibacterial activity of S. epidermidis isolates. These indicators were chosen on the bases that all of them are important skin pathogenic bacteria. The primary screening (fig 1) was conducted using the well diffusion method to discriminate the isolates possessing the capacity of producing bacteriocin. Based on the results, 22 isolates were selected which were then subjected to a secondary screening program. Since S. epidermidis is not a text book probiotic bacteria, the secondary screening needs to be modified from the traditional ones. Therefore, in addition to the bacteriocin activity determination, the biofilm forming capacity of the isolates was integrated in this step for the value it has as a virulence factor [22]. Tissue culture plate method (TCP) was used to evaluate the biofilm forming ability and the results from the previous two factors are listed in Table-   By combining the data from both primary and secondary screening, including the wide range of activity against indicators and high bacteriocin activity as well as being non-biofilm producer, it was logical to select the isolate Y73 as the elected candidate among the other isolates. Next, six media were used in this experiment in order to investigate the best medium that can support maximum bacteriocin production. As can be seen in Figure-2, TSB was the best medium to support bacteriocin production with an activity of 40 AU/ml. Therefore, this medium was used in the next experiments as well as in the preparation of the pad containing S. epidermidis Y73 cells as a probiotic.

Characterization of S. epidermidis Y 73 isolate as a probiotic
Since S. epidermidis is rarely used as a probiotic [19], it was of upmost necessity to take the antibiotic sensitivity profile of the isolate S. epidermidis Y73 into consideration, especially with the increasing prevalence of multidrug resistant (MDR) infections worldwide. In addition, this was followed as an insurance policy in case of any manifestation. In this study, the chosen antibiotics were selected following the same guideline as the physicians use in treating bacterial skin infections [23]. Disc diffusion method was used in this study to determine the antibiotic susceptibility, which is already used in several studies [24].
According to the results, S. epidermidis Y73 was sensitive for 13 tested antibiotics: Erythromycin, Cefotaxime, Cephalothin, Amikacin, Clindamycin, Ampicillin, Mxifloxacin, Trimethoprim, Trimethoprim-sulfamethoxazole, Azithromycin, Ciprofloxacin, Norfloxacin, and Ofloxacin. Whereas, it was only resistant to Ceftazidim and Colistin sulfate. Therefore, it can be said that S. epidermidis Y73 is multidrug sensitive, unlike the results obtained by Lazaris et al [25] who isolated a novel multiresistance cfr plasmids in linezolid-resistant methicillin-resistant S. epidermidis. The other MRSE lacked cfr, but showed linezolid resistance-associated mutations. Taking into account that linezolid is often the drug of the last resort to treat infections caused by Gram-positive cocci; this strain could be very dangerous in case it has established an infection. Another study [26] reported a rifampicin resistant S. epidermidis isolate. Based on the previous data and with an overview concept, it is clear that S. epidermidis y73 is a safe isolate to be used in terms of antibiotic profile. In addition, the optimal pH and temperature for S. epidermidis Y73 growth were investigated. In fact, a relatively wide range of pH was used to mimic the pH variation in human skin and to cover all the potential possibilities, since the normal pH value of the stratum corneum is 4.1-6. In addition, physiological gaps that include axillae, groin, toe, and anus exhibit pH values between 6.1 and 7.4. Therefore, using such a range is justified by the natural variety of the human skin pH [27]. As can be seen in Figure-3, a maximum S. epidermidis Y73 growth of 11.3 mg/ml was obtained at pH 7. However, the biomass was slightly decreased at pH 8 and 6 reaching to approximately 10 mg/ml. On the other hand, several temperatures were investigated as a potential optimal condition and the results are shown in Figure-3. The optimal temperature for S. epidermidis Y73 growth was 37°C in which the biomass gained was 16.9 mg/ml. Based on these results, it can be said that this temperature is perfect since it is approximately equal to the human skin temperature. The above mentioned data of temperature, pH, and media were invested in further steps through the study to achieve the formula used in the application. S. epidermidis exists in healthy human skin as a commensal inhabitant and may become an important pathogen mainly if it has the ability of forming biofilms. Recently, increased resistance traits were suggested to be acquired in biofilm environments. Thus, both antibiotic resistant and biofilm formation are usually aligned. In a clinical study conducted by Sahal et al. [28], 65% of S. epidermidis were resistant to all b-lactam antibiotics (Penicillin, Oxacillin, Amoxicilin / Clavulonic acid), and 60% of all S. epidermidis strains were found as multidrug resistant. However, the results of strong biofilm forming S. epidermidis showed that 80% of them were b-lactam resistant whereas 100% of them were multi drug resistant. As mentioned earlier in Table-1, S. epidermidis Y73 was non-biofilm producer based on the results obtained in the secondary screening. Consequently, by combining the data obtained through the study, S. epidermidis Y73, as a multidrug sensitive, nonbiofilm forming, and strong bacteriocin producer isolated from normal healthy skin, is the elite candidate to be selected.

Probiotic application of S. epidermidis Y73
The use of living bacterial cells as probiotics was attended in several studies especially for bacteria like Lactobacillus, while for S. epidermidis only few attempts were performed. Cleland et al. [29] conducted a study in which S. epidermidis cells were introduced as cells without any immobilization to treat artificially induced rhinosinusitis infection with S. aureus. In that study, the ratio between the probiotic microorganism and the infection causing bacteria was 2 to 1; this is quite similar to the conclusion achieved in the current study. In addition, the use of S. epidermidis as a probiotic was fruitful in the mentioned study as it has eliminated S. aureus infection as well as prevented its ability to form biofilm in the site of colonization. On the other hand, Yang et al [19] also used S. epidermidis as a skin probiotic patch against Cutibacterium acnes, but in this scenario the cells were encapsulated with full immobilization. S. epidermidis mediated the fermentation of glycerol to produce antibacterial substances against Cutibacterium and the final result was a successful attempt to treat acne using that probiotic. In this study and since the goal is to treat skin infection, finding a middle ground was attempted through introducing the probiotic cells to the infected area with partial immobilization on a cellulose paper pad. This was associated with making an advantage of the nature of S. epidermidis as non-motile bacteria so that the cells will be restricted to the edges of the pad, as shown in the Figure-4. As mentioned earlier, 4 pad formulae were tested. The presence of petroleum jelly in both concentrations used in the formulae 2 and 4 acted as some sort of impediment to S. epidermidis Y73, which may be due to its thick consistency. Whereas, the use of only S. epidermidis Y73 cells in TSB was efficient and the probiotic cells swept the S. aureus 3. However, since the final design requires the probiotic cells to remain vital for a long period, glycerol was added to serve as another strength factor since S. epidermidis usually utilizes glycerol as a carbon source to produce succinic acid which has anti-bacterial properties [30]. This will certainly give the formula number 3 a very good multifactorial core structure, which led to its selection as the best candidate formula to prepare the pad containing S. epidermidis Y 73 cells.

Estimation of expiration date of the pad
Several expiration date estimation methods were proposed by Capen et al. [20], among which the original one inspired our work and was applied in the current study; a method in which the vitality of

Hussein and Luti
Iraqi Journal of Science, 2020, Vol. 61, No. 8, pp: 1932-1943 3313 cells was estimated and measured along a 20-week trial. The pads were all prepared at zero time and stored in sealed sterile tubes at 4°C; a single pad was extracted each week and placed on MSA to investigate the bacterial viability. The bacterial growth was represented by the yellow color around the pad as well as the bacterial growth at the edges. In addition, the purity of growth was examined and the same bases of diagnosis were followed. A slight reduction in the growth was noted after the 9 th week, but in general the cells remained vital until the 20 th week. It was found that S. epidermidis Y73 growth was restricted to the borderline of the pad. The appearance of the yellow color, which was due to the presence of glycerol in the pad formula, serves two purposes; (I) an indicator for the fermentation of carbon source that reflects the viability of S. epidermidis Y73 cells and (II) prolonging the shelf life of the pad.

In vivo application of S. epidermidis Y73 pad
The selected pad formula was used to treat artificially induced wounds on lab animals' skin. The wounds were infected with three clinical pathogenic isolates of S. aureus, Kocuria rosea and P. aeruginosa. In all cases, the same cut (approximately 2.5 cm) was conducted on the skin of the animals, and the excess blood was removed to avoid the direct contact between the inoculum (10 4 cells per 100 microliter) and the immune product found in the blood. . The three indicator species in this experiment were selected as common bacteria causing skin infections and the healing process was compared between the control and treatment groups.

Case 1: Staphylococcus aureus
S. aureus is responsible for most skin and soft tissue infections. Half of patients treated for primary skin infections experience recurrence within six months, despite sufficient antibiotic sensitivities and interventions to prevent infection [31]. S. aureus inhabits the skin in 20-30% of the population and induces 80-90% of all skin and soft tissue infections in humans worldwide [32,33,34]. The major infections fall into one of the following categories (I) Localized skin infections, (II) Deep, localized infections, and (III) Scalded skin syndrome.
Based on the results, it was clear that, with an overview perspective, the healing process with the presence of the S. epidermidis Y73 pad was faster, as shown in Figure-5. By the time of day 6, there was no trace to the wound in the treated animals, unlike the control. In addition, the pruritic rigid edges of the wound were noticed in the control, while in the presence of the pad the edges were smooth and the animal almost neglected the site of wound. Moreover, tenuous erythema was appeared in days 1 to 4 in the control, unlike the treated wound in which no redness was observed. K. rosea was used in this study due to its newly rising titer as a potential pathogen worldwide [35]. Therefore, having sufficient data will aid to participate in dealing with any problem that may be caused by this Genus. The results of this experiment showed that the control rabbit infected with K. rosea suffered from high fever and severe diarrhea. Oncel and his colleagues recently reported a human infant with prolonged history of diarrhea with the blood culture growth containing Kocuria spp. [36]. Interestingly, the previous symptoms were not found in the treated animal. Moreover, it was obvious that the healing process was significantly improved in the treated wound Figure-6.
Kocuria spp has been reported to be normal human skin and oral cavity flora and is generally considered to be a laboratory pollutant, while overlooked when isolated in the clinical specimens which leads to undermining its pathogenic potential. Back in 1974, kocuria was first reported as a cause of urinary tract infection and was first named as Micrococcus kristinae [37]. Reports of Kocuria species infection gained prominence in the late twentieth century and displayed a growing trend, which indicates its pathogenic potential. Kocuria-isolated infections include infections of the urinary tract, cholecystitis, catheter-associated bacteremia, dacryocystitis, canaliculitis, keratitis, native valve endocarditis, peritonitis, mediastinitis, descending necrotizing, meningitis, and brain abscess [38,39]. Based on the previously mentioned information, this bacterium has the actual potential to become a serious pathogen which should not be underestimated.

Case 3: Pseudomonas aeruginosa
The case of this bacterium was the most promising in terms of wound healing. After day 6, there was almost no trace for the wound, as shown in Figure-7. Furthermore, the animal totally neglecting the wound, which is a sign of being non itchy. In addition, mild erythema was noticed in days 1 and 2 in the control, unlike the treated animal. Similar to Kocuria rosea case, the control suffered from fever and relatively decreased normal range of movement when compared to the other cases. P. aeruginosa is also known to be a major skin opportunistic pathogen as it has the required characteristics such as biofilm formation and antibiotic resistance. Such infections are often characterized by vigorous pathogen development at the infection site and increasing antibiotic resistance. The opportunistic pathogen P. aeruginosa is accountable for a wide variety of infections in immunocompromised hosts [40]. Among the most important of these infections are those localized to soft tissues, containing chronic and burn wounds [40,41].

Conclusions
Nowadays, there is a global orientation towards untraditional methods of treating bacterial infections. The main trigger to this wave of orientation is the rising incidence of multidrug resistant bacterial infections. One of the examples on these previously uncommon methods is the use of benzoyl peroxide (BPO) instead of antibiotics to treat acne, with the key difference is that BPO does not induce antibiotic resistance [42]. There is an increasing interest in using probiotics in both sides of doctors and end users. Thus, the results achieved in the current study, which proves the efficiency of using vital cells of the isolate S. epidermidis Y73 as a probiotic, may hopefully participate in completing the image about such topic.