Antibacterial Activity of Live Cells of Lactobacillus Plantarum L40 as A Probiotic Against Pathogens Associated with Diabetic Foot Infections

The present study was designed to select a suitable isolate of Lactobacillus sp. in order to use it as a probiotic formula for treating diabetic foot ulcer disease in diabetic mellitus patients. In order to determine the inhibitory action, 240 isolates of Lactobacillus from different sources, were taken and exposed for screening tests to evaluate the (antagonism-activities) toward 4 multi-drug resistant foot ulcer pathogens which were chosen from 120 pathogenic isolates: Staphylococcus aureus, Klebsiella pneumonia, Proteus mirabilis, and P. aeruginosa . Twenty Lactobacillus isolates were selected from primary screening according to the capacity to restrain its expansion of four markers, that were exposed toward second screenings to detect their ability to produce a high amount of bacteriocin . Based on the results, the isolate Lactobacillus L40 was selected as the highest bacteriocin producing isolate which was further characterized as Lactobacillus plantarum Next, several tests were performed to study the properties of L. plantarum L40, in particular its biosafety and its suitability to be a successful probiotic. Results showed that L. plantarum was resistant to cephalosporin groups such as ceftriaxone and cefepime whereas, it was sensitive to amikacin and chloramphenicol. In addition, results revealed that this bacterium was strongly adherent and a good biofilm producer; had high auto-aggregation capability and a high ability to adhere with S. Staphylococcus aureus, Klebsiella pneumonia, Proteus mirabilis and P. aeruginosa . A gel formula containing live cells of L. plantarum L40 was prepared and based on the results, cells were successfully survived in this formula over 18 days. Therefore, it was selected to be utilized as a vector to store and transport viable cells of L. plantarum L40. This gel formula showed antibacterial activity by inhibiting Staphylococcus aureus, Klebsiella pneumonia, Proteus mirabilis, and P. aeruginosa growth. The results showed the possibility of using viable cells of bacteriocin-producing Lactobacillus plantarum as an effective probiotic to deal with some skin pathogens, and hence treat skin diseases such as diabetic foot ulcer disease.


Introduction
High rates of morbidity, death and healthcare costs have been linked to diabetic foot ulcers (DFU), a common consequence of diabetes.The International Diabetes Federation estimates that between 19% and 34% of patients with diabetes may develop DFU at some point during their lifespan [1].Diabetic foot ulcers have minimal practical significance.The development of diabetic foot ulcers has been linked to about a five% death risk during the first twelve months besides about a 42% mortality rate within 5 years, according to cohort research conducted in the United Kingdom.A study found that patients with diabetic foot ulcers had a 2.5-fold raised risk of death in comparison with those with diabetes but no foot lesions.They have a lot of patients to see every day [2].
With increasing resistance to the most antibiotics in use today, it becomes vital to develop and find new therapeutic strategies to combat microbial infections without affecting the patient.One of the suggested strategies, in this context, is to use bacteria from the resident human microbiome with probiotic characteristics to provide resistance against disease [3].
Treating diabetic foot ulcers accounts for nearly a 3rd of the entire price of diabetes care, which is valued at US$176 billion in direct healthcare expenses in 2012 [4].Despite the fact that there are defined guidelines for the organization of diabetic foot sores, therapy for these sores can be difficult.Wide range of new interventions to improve wound healing are being studied [5].finding new therapeutic protocols for diabetic foot ulcer confrontation are certainly important and urgent.In this context, several strategies were recently proposed such alasadi and Luti Iraqi Journal of Science, 2023, Vol. 64, No. 11, pp: 5624-5639 5626 as antimicrobial peptides, using probiotics, and even utilization of some bacteriophages as alternatives to currently available antibiotics [6].
The purpose of this study is to identify a locally isolated Lactobacillus sp. that is able to produce a bacteriocin with antimicrobial activity against several pathogenic bacterial isolates in diabetic foot ulcer samples.The study is particularly, will focus on the multidrug-resistant bacterial isolates that associated with the diabetic foot ulcers.In addition, this work will look into the viability of administering the Lactobacillus isolate topically(dermal probiotic) to treat DFU.

Sample collection (DFU-pathogens):
A total of 120 specimens were taken by swab from individuals who suffer from diabetic foot ulcers attending at the (Al-kindy teaching hospital, Special Center for Diabetes and Endocrinology glands and medical city /Baghdad) during the period January 2021 to April 2021 The primary methods used to identify these isolates were morphological features and standard biochemical assays.including: blood hemolysis, coagulase, catalase, oxidase and Gram stain.All isolates were further confirmed using VITEK system after it was primarily identified by culturing on MacConkey agar and Mannitol salt agar.

Lactobacillus collection
240 samples in total were collected; 80 mouths of humans (40 male, 40 female) of healthy individuals and 160 samples from the dairy product (50 samples from cow milk, 60 samples from yogurt, 50 samples from cheese); during the period from (12/January/ 2021 to 30/April/ 2021).After microaerophilic incubation at 37C for 48 hours in a candle jar, swabs were recultured in the agar of MRS like single colonies medium for another 48 hours.The obtained isolates were subjected to different morphological and biochemical tests to be identified presumably [3].

Primary screen
The following Agar plug diffusing technique was employed for the initial screening [7]: The Lactobacillus isolates were placed into the MRS agar media via streaks all across surface of the plate after overnight growth in MRS broth.They were then maintained at 37°C.over roughly 24 hours inside a candle container.Following incubation, plugs of every inoculum measuring 0.5 cm wide have been created using an aseptic cork borer and afterward positioned on Mueller-Hinton Petri dishes containing 200 ml l of bacterial isolate of overnight growth culture of the markers (Staphylococcus aureus, Klebsiella , P. mirabilis, Pseudomonas aeruginosa) having about one×10 8 cell by ml.The same procedures then were used for an overnight incubation of dishes.Plates were then incubated under the same conditions.A Zones surrounding the bacterial plags were measured to determine the antibacterial activity of each isolate.

Secondary screening
Inoculums of Lactobacillus isolates were prepared as follows: a few loop-full of pure isolate by culturing about 24 hrs.were taken and placed on MRS solid media placed in 10ml of MRS liquid media after that raised at (37°C) overnight.Furthermore, counted cell by spectrophotometer at 600 nm in order to reach the number of the cell of about 1 ×10 8 cell/ ml. the next step was cultivation method as follow: universal tubes containing 20 ml of MRS broth were inoculated with 2% of an overnight culture of the isolate reaching alasadi and Luti Iraqi Journal of Science, 2023, Vol. 64, No. 11, pp: 5624-5639 5627 approximately10 8 cells/ml.Then, tubes were incubated for 48 hours under anaerobic conditions at 37°C.Afterward incubation, the broth culture was centrifuged at 10000 rpm for 15 min to collect the cell-free supernatant (CFS) and bacteriocin activity was determined as described in the following section [8].

Bacteriocin activity determination
The significant diluting method, that is comparable to a lowest inhibitory concentration approach (MIC) for antibacterial evaluation, was used to evaluate the action of bacteriocins [8], [9].Such a procedure required making a serial of a double diluted sample of a cell-free supernatant. of Lactobacillus culture to be tested as follows: CFS was filtered with 0.22 μm Millipore filter paper under sterile conditions.To counteract overall impact that organic acids, a mixture of NaOH plus catalase was administered.H 2 O2 activity respectively.A series of Eppendorf tubes were prepared each containing 500 μl of sterile phosphate buffer ph7.A sterile micropipette was used to transfer 500μl of the Lactobacillus culture to the first Eppendorf tube and then mixed vigorously using a vortex mixer; this is the first two-fold dilution.Then, 500 μl from the first two-fold dilution was transferred to the second tube to carry out a second two-fold dilution.The series of two-fold dilutions were continued to make a series of two-fold dilutions.100 μl of each dilution was filtered and added to the wells of plates which were streaked by the selected indicator and after that, dishes had been kept at 37 degrees Celsius for 24 hours [10].The diameter of the inhibitory area that includes every well was then calculated.The level of bacteriocin activity was measured by the maximum dilution of an inhibitory circle.A bacteriocin efficacy is inversely related to a maximum dilution factor (DF) that results in an apparent inhibitory zone.The below equation was used to determine the number of bacteriocin activity units (AU), that were given as follows Eq.( 1): Asbitrary Unites AU/Ml = (1)

Characterization of the selected Lactobacillus isolates as a dermal probiotic
The selected Lactobacillus isolate was characterized as a prospective probiotic via investigation of some factors related to its growth such as

Antibiotic Susceptibility
This test was exanimate the ability of the selected isolate to resist some antibiotics including(Cefotaxime, Ceftriaxone and Vancomycin, Clindamycin, and Erythromycin) by using antibiotic disc diffusion method [11].

Detection of the Ability of Biofilm Formation
A lactobacillus strain were cultivated over 48 hours at 37°C inside MRS-broth containing 1 percentage glucose anaerobically.Then, A sterile tryptic soya broth was prepared.Inside a microtiter tray, 180 µl of TSB and 20 µl Lactobacillus (grown for 48 hours)were poured for every well.After incubating the well's growth at 37 C for 72 hours without shaking, it was mixed 10 times with a pipette.The liquid was taken out post incubation, then phosphate buffered was used to rinse every well three times.Two hundred microliters of about 1% crystal violet were added and left on the counter for 15 minutes.A phosphate buffer solution was used to wash the wells three times before they were allowed to dry in the air for half an hour.After 15 minutes, the supernatant was discarded and 200 ml of 96% ethanol was added The negative control was a sterile medium.The result was read using an ELISA reader at 630nm [12].

Auto-aggregation assay
After an overnight culture of Lactobacillus isolates, cells were harvested at 7000 rpm for 15 min, and then the collected cells were washed twice with phosphate buffer saline (pH 6.0) and the concentration was modified to 10 8 CFU/ml.4 ml of Lactobacillus cells suspension were mixed by vortex for 10 seconds.During 5hr, auto-aggregation of Lactobacillus was determined For ten seconds, a vortex was used to mix the solution at room temp, and an auto-aggregation of Lactobacillus was found to be caused by moving 100 µl of the suspension tube's top surface to a 3.9 ml tube containing PBS.At 600 nm, the absorbance was then calculated.The below equation Eq.( 2) was used to calculate the auto-aggregation %: [13].% Auto-aggregation = [ODi-ODf/ ODi] 100 (2) Where OD i represents the absorbance of the start time (t = zero) for auto-aggregation and ODf represents: absorbance in 1,2,3,4 and 5hr.

Co-aggregation assay
To examine the co-aggregation ability between the selected isolate Lactobacillus and the indicator S. aureus, P. aeruginosa, Klebsiella and Proteus mirabilis, 2 ml of each of both bacteria were mixed by vortex for 10 seconds.4 ml of each bacterial suspension was utilized as controls.Finally, the absorbance was measured at 600 nm during 5hr at room temperature [13], The percentage of co-aggregation was counted by using this equation Eq.( 3 Preparation of gel formula contained live cells of of the selected Lactobacillus Lactobacillus biomass preparation Lactobacillus Inoculums were prepared as follows: a few loopfuls of Lactobacillus growth from an overnight culture on MRS agar was inoculated in 10ml of MRS Broth, incubating it at 37°C, pH 6 for 24 hours.After being incubated, the final cell count was adjusted using a 0.5 McFarland Standard tube to a concentration of about 1×10 8 cell/ ml per milliliter.The media was then poured into the precipitant after the biomass had been obtained with a centrifuge at twelve thousand rpm for 15 minutes.As useful biomass, this sample solution was saved to be added to the recipe later.

Bacterial pathogens Preparation
Four pathogenic isolates were used: (S. aureus, P. aeruginosa, Klebsiella and Proteus mirabilis) isolated from diabetic foot ulcer.These indicators were prepared as follows: A chosen colony was transferred via a sterile wire loop into a tube containing 5ml of brain heart infusion broth, and the tube was placed in an incubator at 37°C for 24 hours.After that, the cell density was standardized in the McFarland tube to 1x10 8 cells/ml at 600 nm.Preparation of gel formula100gm [14]: Carbopol 941 was dissolved in D.W. using an aliquot weighing 4 gm, and the mixture was next vigorously stirred with a magnetic stirrer.Then, 0.1 grams of methyl paraben was applied.The mix was then allowed to remain over 24 hours.After that, a sample(Lactobacillus L40) solution in MRS medium was added and well combined.The formula's total volume was then finished to 100g.The ingredients with all components, including biomass, medium, and other formula substances, were blended for a whole hour.Triethanolamine was later added in sufficient quantities to create the desired texture for the gelling agent, and the pH was adjusted to pH 6.The formula was kept at 4°C in a well closed container.

Results & Discussion
As mentioned earlier, the present study aimed to investigate the antibacterial properties of a potential probiotic Lactobacillus sp.against pathogenic bacterial isolates collected from samples of diabetic foot infection.Therefore, to achieve such an aim, it was necessary to select diabetic foot ulcer isolates in order to use them as indicators in the selection of a suitable Lactobacillus isolate that can be used as a dermal probiotic in this study.For this purpose, one hundred and twenty subjects were included in this study, 60 (50%) males and 60(50%) females who were pre-diagnosed with diabetic mellitus disease and diabetic foot ulcer.The results demonstrated that Gram-negative bacteria were the most common bacteria isolated from diabetic foot, including Proteus mirabilis (8.3%), Klebsiella pneumoniae (10.8%),Pseudomonas aeruginosa (12.5%),E. coli (7.5%).Whereas, Gram-positive presented 25.82% for both S. aureus and Staph.epidemidis.These results come in align with some previous studies [15] that reported p, clindamycin, chloramphenicol, tetracycline and amoxicillin with clavulanic acid in order to select the multi-drug resistant isolates that can be used as indicators in this study.Four isolates were used as indicators: S. aureus, Klebsiella pneumonia, Proteus mirabilis and P. aeruginosa.[16].
Two hundred and forty samples were collected from different dairy product samples including raw milk, yogurt, cheese as well as from the human mouths of healthy individuals.Based, on results, the homemade yoghurt and raw cow milk were the best source for Lactobacillus.All isolates were subjected to a number of biochemical tests.As a total isolation process for the present study the following isolates were collected: 71 (29.58%) isolates were Lactobacillus sp., 68 (28.3%) isolates were cocci shape, whereas 56 (23.3%) samples had no growth and 45 (18.7 %) samples were contaminated.Morphological identification of Lactobacillus isolates was mainly achieved by investigating the appearance of colonies on the solid medium (MRS) as well as the microscopic examination.All colonies on MRS agar were white, rounded in shape and ranged in consistency from creamy white in color to glossy white and moist-mucoid colony appearance on the surface.the growth of Lactobacillus isolates appeared as a single colony on MRS agar after anaerobic incubation of 48 h at 37 ˚C.The microscopic field examination for all isolates was investigated.Under light microscope, Lactobacillus is a Gram-positive, rod-shaped (bacilli) and occurred singly, in pairs, or even in chains [17].
To choose the isolate with the highest bacteriocin production that may be employed in the current study's additional investigations, all isolates were put through a screening procedure.The screening procedure was carried out to see if Lacto strains might stop the multi-drug resistance bacteria that cause diabetic foot ulcers from growing.To emphasize the conflict among these isolates and the markers, the agar plugs diffusing technique was applied.It involves making an agar plug from the culture of Lactobacillus sp isolates to be tested for antimicrobial production and then deposit on the agar of another plate before inoculating with the indicator bacterium.Ordinarily, microbial cells secrete antimicrobial active compounds which diffuse in the agar medium from the plug.Now, this antimicrobial activity of the Lactobacillus is recognized by the appearance of an inhibition area round of agar plug.Results showed that several Lactobacillus samples had the ability to give the activity of bacteriocin alongside the four markers used with different inhibition zones.Consequently, the strategy used to select Lactobacillus isolates for the secondary screening were based on: a) The isolate is active against indicators with an inhibition zone of 15 mm and above.
b) The isolate is active against more than two indicators.
According to results, twenty Lactobacillus isolates had antagonistic activity against more than two indicators with an inhibition zone of 15 mm and above.,The rest of isolates had no or weak (less than 10 mm) or medium (less than 15 mm) inhibitory activity.Therefore, only twenty isolates were selected for further experiments in the secondary screening.
Research has demonstrated that this inhibitory activity of lactobacilli can be different in broth compared to solid medium because of better diffusion of the substance secreted by Lactobacillus [18].Therefore, in the second step of screening, the well diffusion method was used to detect the ability of Lactobacillus isolates to produce bacteriocin in liquid culture.The highest bacteriocin-producing isolate was detected based on its performance by forming an inhibition zone around the well in an agar plate containing cell-free supernatant.Results demonstrated that the activity was different (320,160,80 40, 20 AU/ml) of bacteriocin produced by lactobacillus isolates.According to the results, the isolate Lactobacillus L40 from yoghurt showed the best activity isolates to antibacterial activity of 320 AU/ml toward 4 indicators: (Staphylococcus aureus Pseudomonas.aeruginosa, Proteus.mirabilis, and Klebsiella pneumonia) as described in (Table and Figure 1).Thus, the isolate numbered L40 was selected to be employed in the work.Lactobacillus L40 was ten and subjected to a process of identification according to VITEK 2 system in order to determine its species.According to results, the selected Lactobacillus isolate showed positive results for 18 biochemical tests and negative results for the rest.These tests were performed to differentiate the species that belong to the genus Lactobacillus.The results revealed that the selected isolate L40 was Lactobacillus plantarum with a very good probability of 93%.The main target of this work was to use the isolate Lactobacillus plantarum L40 as probiotic, therefore, it was useful to take the antibiotic sensitivity profile of the isolate into consideration.For this purpose, nine different antimicrobial drugs were utilized and tested against Lactobacillus plantarum L40.Disc diffusion method was utilized in this study to identify the antibiotic susceptibility [19].Based on results, Lactobacillus plantarum was resistant to beta-lactam groups like ampicillin and penicillin whereas, that one showed sensitivity toward Amikacin and chloramphenicol.In addition, Lactobacillus plantarum L40 showed resistance to Cefotaxime, Ceftriaxone and Vancomycin, Clindamycin and Erythromycin.The result similarity [20] and [21] reported Lactobacillus sp were usually susceptible toward antibacterial that inhibit protein production like chloramphenicol and tetracycline, and strongly resistant to aminoglycoside like streptomycin and gentamicin.In general, to evaluate the biofilm formation, different methods were used including quantitative methods, for example, tissue culture plate (TCP) [22] which is considered as a standard method as well as qualitative methods using the Congo red agar (CRA) [23].and tube method (TM) [24].For the current research, TCP techniques were applied for Detection the Biofilm Formation Ability by Lactobacillus plantarum L40.The TCP method provides a numerical value based on OD determination and each value represents a certain phenotype result non-producer, weak, moderate and strong [25].Therefore, this method is a quantitative assay to determine the ability of an isolate to produce biofilm by calculating the degree of biofilm production.The result showed that OD of sample was 0.37 whereas, OD of control was 0.08, So this isolate is a strongly adherent and good biofilm producer.This result was agreed with [26] who confirmed the ability of L. plantarum to produce biofilm.In addition, many studies reported the ability of different species of Lactobacillus to produce biofilm [27], [28].
Biofilm-producing microorganisms have an advantage that is not found in planktonic ones such as protection and resistance to drugs (antibiotics), adhesion capability, mechanical properties, avoiding immune system (antibodies and phagocytes), protection from environmental stresses and cellular communication [29].Biofilm formation is a property that provides the ability to colonize probiotic cells and avoid the colonization of pathogens [30].Auto-aggregation is an important property of probiotics, Some Lactobacillus strains have the ability to inhibit adherence of pathogens either by forming a barrier via auto aggregation or by direct co-aggregation with the pathogens [31], [32].Therefore, this test is important to determine the possibility of using L. plantarum L40 free-cells equally prospective probiotics.As the results presented in Figure (2), Lactobacillus plantarum L40 had a high autoaggregation capability of approximately 87.2% after 5 hrs.This test clearly proves that this isolate has a good ability to adhere and inhibit adherence to pathogens.The results agreed with [28] who reported that Lactobacillus Nu14 cells had a high auto-aggregation capability of more than 86% after 5 hrs.Furthermore, Lactobacillus plantarum L40 had a high Co-aggregation capability of approximately 76.1% after 5 hrs. Figure (3).This test clearly proves that this isolate has a good ability to adhere to inhibit adherence to pathogens.The results agreed with [28] who reported the high ability of L. crispatus and L. parabuchneri to adhere with S. mutans which reached 71.66% after 5 hrs.The obtained result was also in agreement with study that reported the ability of Lactobacillus to co-aggregate with pathogens [33].According to the definition by the World Health Organization (WHO) and the American Food and Agricultural Organization (FAO), a probiotic is "a live organism which has a beneficial effect on human health when they take in enough amounts" [34].Antagonistic activity is a very important criterion for the selection of probiotics because they are natural antimicrobials for potentially harmful bacteria.Therefore, CFSs from Lactobacillus spp.isolated from dairy products and the mouth of healthy individuals were screened for their antagonistic activity against Gram-positive and Gram-negative diabetic foot ulcer-causing pathogens.In this context, different bacterial probiotic cells, in particular Lactobacillus sp., were utilized in formula to treat several diseases such as Lactobacillus plantarum cells in an emul formula that is used against some skin pathogens [35].In addition, [27] used Lactobacillus crispatus in an emul gel formula against some vaginal pathogens.This study was performed to select an acceptable formula which can be used to provide a suitable medium for Lactobacillus plantarum L40 to survive.

alasadi and Luti
Iraqi Journal of Science, 2023, Vol. 64, No. 11, pp: 5624-5639 5634 In fact, the challenge in this study was to select a useful formula which can act as a vector to store and transport viable cells of Lactobacillus plantarum L40 into the skin with an ulcer in diabetic patients.After several tests, formula containing lactobacillus plantarum L40 (live cells) was selected and optimized to choose the most useful one that can be used in this study.Lactobacillus plantarum L40 was used in this study against four MDR pathogenic bacteria that isolated from diabetic foot ulcer.The physiological characteristics of the created emul gel formula include a silky finish, a white appearance that is clear and homogenous, and no odor.Lactobacillus plantarum L40 biomass was successfully included in the formulation of the emul gel bases in this investigation.By daily culture on MRS agar following manufacture and homogeneity of the formula ingredients, the vitality of Lactobacillus plantarum L40 cells was monitored for three weeks.According to the findings, cells maintained their vitality for 18 days.A number of factors, including pH, homogeneity, spreadability, and skin irritancy, are often used to assess the manufactured formula emul gels [36].In vitro monitoring of pH and homogeneity made the formula's stability clear.Through the course of 18 days, the produced formula demonstrated homogeneity and stability at pH (5.9-6.5).Using a technique previously described by Mostafa et al. [37], we investigated the antibacterial efficacy of Lactobacillus plantarum L40 biomass within emulgel formulations against Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia, and Proteus mirabilis.
(the control is a formula without lactobacillus).The formula's efficiency was demonstrated by its ability to suppress the growth of four indicator bacteria when tested at doses of 10 8 CFU/ml and according to the following: 1. Viability of Lactobacillus plantarum L40 cells.2. Its ability to produce bacteriocin.3.There is no way for the bacteriocin to become trapped in the formula, and no way for any of the components of the formula to interfere with the bacteriocin's ability to inhibit the tested bacteria as it diffuses out into the surrounding environment (the tested media).
The results showed inhibition of bacterial growth to: Staph.aureus with 19mm in diameter with control formula of no inhibition zone Figure (4).While in Pseudomonas aeruginosa, L. plantarum L40 cells formula showed an inhibition zone of 16mm.Furthermore, L. plantarum L40 cells formula showed inhibition toward Klebsiella pneumonia with 18mm in diameter and 17mm in Proteus mirabilis).According to the results obtained in this study, Pseudomonas aeruginosa was the most causing pathogen for foot ulcer therefore, it was used as an indicator to test the formula.Moreover, the formula was tested for eighteen days in order to measure the availability of lactobacillus plantarum L40 to inhibit bacterial growth.The L40 cells formula showed increases in activity with 22mm in diameter against Pseudomonas aeruginosa and lack of its green color altering into yellow improving the Lactobacillus plantarum L40 availability and ability to produce compounds that affect or inhibit bacterial growth Figure(5Data confirmed that the recipe distributed extremely well, which is important since gel or emul gel's capacity to treat ailments often hinges on how well they spread.Emul gels also have perfect quality in dermal administration due to their exceptional spreading capacity.In contrast, is thus thought to be a crucial component of patient adherence to therapy [38].Based on results of the viable cell count method, Lactobacillus plantarum L40 successfully survived in this formula over 18 days as shown in Figure (6).The percentage of viable cells decreased after three weeks.Therefore, this formula was selected to use in this study which can be utilized as a vector to store and transport viable cells of Lactobacillus plantarum L40. the presence of glycerol in the formula may play an important role to store bacteria for long term at 4 o C, and hence, may participate in maintaining Lactobacillus parabuchnei Nu14 cells viable for long time [28].

Conclusions
In conclusion, the present study supports the idea of using vital cells as a dermal probiotic for the treatment of skin infections and as an alternative method to face the widespread multidrug resistance.The results showed the possibility of using viable cells of bacteriocinproducing Lactobacillus plantarum as an effective probiotic to deal with some skin pathogens, and hence treat some skin diseases related to diabetic foot ulcer patients.The results of this study validated the survivability of the Lactobacillus plantarum L40cells within the formulas and their capacity to generate and secrete bacteriocin without interacting with formula ingredients, demonstrating the effectiveness of the cells-containing formula.

Figure 2 :
Figure 2: Auto-aggregation assay after 5 hours of incubation

Figure 3 :
Figure 3: Co-aggregation assay after 5 hours of incubation

Figure 4 :
Figure 4: The activity of live cells of lactobacillus plantarum L40 formula against diabetic foot infections-causing pathogens compared with control (lack of lactobacillus bacteria).A: P. aeruginosa, B: Klebsiella, C: Proteus mirabilis, D: S. aureus.

Figure 5 :
Figure 5: Antibacterial activity of live Lactobacillus plantarum L40 cells formula against P. aeruginosa after 18 days

Figure 6 :
Figure 6: Monitoring of viability of Lactobacillus plantarum L40 in formula for three weeks

Table 1 :
Lactobacillus isolates Secondary screening for production of bacteriocin