Antimicrobial Activity of Partially Purified Bacteriocin from Lactobacillus Reuteri Against Pathogenic Bacteria and Fungi
Batool Abd Al Ameer Baqer*
| Maysoon Kh. Abbas | Shahrazad Najem Abdu-Allah | Masara F. Jasim
© 2024 The authors. This article is published by IIETA and is licensed under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).
OPEN ACCESS
Reuterin is an antimicrobial factor produced by Lactobacillus reuteri, a lactic acid bacterium involved in food fermentation and found in various ecological niches such as the intestinal gut. This is significant as the consumption of contaminated food leads to large economic losses in the nutrition industry. The aim of this study is to evaluate and estimate the antimicrobial activity of bacteriocin called reuterin produced by Lactobacillus reuteri isolated from dairy products against some pathogenic bacteria and fungi in Baghdad hospitals. Twenty-four clinical isolates were collected from patients suffering from urinary tract infections, including 20 (83.3%) urine isolates and 4 (16.6%) blood isolates. All Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans isolates were identified using the Vitek 2E compact system. The sensitivity of all bacterial isolates to 12 antibiotics was tested. On the other hand, 3 isolates of Lactobacillus reuteri were isolated from fermented dairy products, all isolates were undergone to the cultural, microscopically, biochemical test and API 50 CHL for the identification of species. Six of these antibiotics belonged to the cephalosporin group. The results showed that P. aeruginosa was 100% resistant to cefotaxime, ceftriaxone, and ceftazidime, but exhibited only 12% resistance to imipenem. There was a significant difference in their resistance levels, with S. aureus isolates showing high resistance to cefotaxime (91%) and ceftriaxone (70%), but a lower resistance to imipenem (12%). The results showed that reuterin at a bacteriocin concentration of 30% saturation produced the highest inhibition zones—21.3, 21.4, and 23.2 mm—against P. aeruginosa, S. aureus, and C. albicans, respectively. When bacteria were cultured under appropriate conditions on MRS medium, crude reuterin extract (bacteriocin) showed a good inhibitory effect against at levels (stock solution to 10-6) that all pathogenic isolates loss the Protease production, while little and normal activities were observed at concentrations (10-7 to 10-10). The result of our research was that P. aeruginosa 3 had the highest activity (19 mm) by examining the diameter of the lysis zone on skim milk agar medium, but the P.2 and C.9 were (12 mm in diameter). Bacteriocin (reuterin) demonstrates the greatest inhibition zone against pathogenic bacteria at concentrations (10-7 to 10-10).
antimicrobial, bacteriocin, reuterin, Lactobacillus reuteri, P. aeruginosa, Staph. Aureus, C. albicans, protease
Microorganisms, particularly lactic acid bacteria (LAB), play a key role in food fermentation and have been used in this process for centuries [1, 2].
There is increasing interest in LAB as probiotics due to rising antibiotic resistance [3]. The antibacterial effects of LAB inhibit the growth of potential pathogens in various ways, including reducing pH, secreting bactericidal proteins, and impeding bacterial adhesion to epithelial cells [4]. LAB was a group of gram-positive, non-spore-forming bacteria, including the genera Streptococcus, Lactobacillus, Lactococcus, Pediococcus, and Leuconostoc. These cocci or bacilli all produce lactic acid as the end product during carbohydrate fermentation [5]. These organisms create many compounds like organic acids, diacetyl, H2O2, acetaldehyde and bacteriocin through lactic fermentation [6]. Many studies have attempted to clarify the mechanisms behind reuterin's antibacterial influence, which has proven difficult to elucidate. This is due to the highly reactive aldehyde group in reuterin, which can convert into various compounds in aqueous solutions [7, 8].
Bacteriocins are peptides manufactured by ribosomes with antimicrobial properties, produced by bacteria such as LAB (Lactococcus, Lactobacillus, Pediococcus spp., Enterococcus, and Leuconostoc) that are of particular interest due to their potential use as bio-preservatives in the food industry [9, 10]. Bacteriocin is an antimicrobial factor generated by Lactobacillus reuteri, a food fermentative lactic acid bacterium found in different of environmental like intestinal gut [11]. Factors affecting the activity of bacteriocins in various food systems are constitution (proteins and lipids), hydrolysis of enzymes, synthesis process, physical properties, like pH, antimicrobial activity, and partial description of bacteriocin-like inhibitory material produced by Lactobacillus spp. Therefore, the successes of using bacteriocins in the food synthesis to control various pathogens are necessary to prove the antimicrobial activity of bacteriocins through laboratory studies and in food systems [12].
Lactobacillus reuteri is a heterofermentative lactic acid bacteria that belongs to the autochthonous microbiota of humans and animals [13]. Reuterin is an antimicrobial agent 0utput Lactobacillus reuteri, and has been suggested as a method of umpire, in part, the health benefits of probiotics impute to these bacteria. For all 20 years of investigation, the device of work by which reuterin apply its antibacterial effects has carry on shifty. Reuterin is a potent antimicrobial agent active opposed to Gram-positive and Gram-negative bacteria, beside yeasts, molds and protozoa [14].
Reuterin has been postulated that reuterin plays a major role in the probiotic action of L. reuteri. Reuterin is effective against enteric pathogens, yeasts, fungi, protozoa, bacteria and viruses [15, 16].
This study aims to isolate and identify Lactobacillus reuteri from dairy products and extract bacteriocins (Reuterin), assessing the antimicrobial activity of these bacteriocins against pathogenic microorganisms.
2.1 Collection of samples
2.1.1 Isolates of pathogenic bacteria of all Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans
Twenty-four clinical isolates were collected from patients suffering of urinary tract infections from Baghdad teaching laboratories in medical city hospitals in Baghdad city.
2.1.2 Isolates of Lactobacillus reuteri
Lactobacillus reuteri isolated from fermented dairy products, all isolates were undergone to the cultural, microscopically, biochemical test and API 50 CHL.
2.2 Culture media
Nutrient agar medium was used for the growth and conservation of pathogenic, such as Staph. aureus and P. aeruginosa, while Pseudomonas agar medium used as selective for P. aeruginosa and Mannitol salt agar medium was to select for Staph. aureus growth [17, 18].
Selective media was used for growth and conservation of Candida albicans [19].
Lactobacillus reuteri was grown in MRS broth and the medium was used to preserve the bacteria and to specify the growth density of the probiotics under study and their production of bacteriocins.
SMA medium was prepared with a composition of 2% skim milk (Lactona) and 2% agar, then bacteria were inoculated and incubated at 37℃ for 24 to 48 h. After the incubation period, it was possible to observe the indication that the growing bacteria were proteobacteria by the good growth of the isolates and the presence of a clear zone around the colony. This activity was tested before and after adding the reuterin extract to the N. agar medium at concentrations (10-1 to 10-10) [16, 18].
2.3 Solutions
2.3.1 Standard McFarland solution
McFarland solution was made rendering to (MacFaddin, 2000).
2.3.2 Physiological saline solution
It was prepared by dissolving 8.5g of NaCl in 1000 ml of distilled water, then sterilized by autoclave, and then kept at 4℃ until use.
2.3.3 KH2PO4 buffer 20mM, pH 7.0
It is prepared as following:
1. Liquefied KH2PO4 0.34gm / 100 ml distilled water.
2. Liquefy 0.27gm of KH2PO4 in 100 ml of distilled water.
Next 61 ml of KH2PO4 and 39 ml of KH2PO4 solutions were mixed and pH was adjusted to 7.0 and executed to 200 ml.
2.3.4 Ammonium per sulfate solution (NH4)2SO4
It is freshly prepared by dissolving 750g of (NH4)2SO4 to liter of distilled water in a container or beaker. Simply stir the solution at 25℃ - 30℃ with a magnetic stirrer for 15min. or until saturation. Pour out the clear supernatant solution. Carefully allow the undissolved solids to settle gently at the bottom of the container [19, 20]
2.4 Preparation of lactic acid bacteria (LAB) supernatant (Bacteriocin-like substance assay)
The bacteria were cultured in MRS broth at 37℃ for 24 hours. Subsequently, 10% of the subculture from 1000 ml of MRS broth was taken and incubated at 37℃ for 72 hours. After this, the efficacy of the crude extract of Lactobacillus reuteri as an antibacterial agent against pathogens was evaluated for 25 minutes at 4℃. The supernatant was collected, and its pH was adjusted to 6.5 using 5N NaOH, then sterilized through a cellulose acetate filter with a pore size of 0.2 µm [19].
To obtain the crude reuterin extract, the supernatant was placed in a water bath at 80℃ for 10 min.
2.5 Partial purification and concentration of a bacteriocin by ammonium sulfate precipitation
2.6 Bacterial antibiotics sensitivity test
The sensitivity of bacteria to some antibiotics was tested using the (1966) method. A bacterial suspension was prepared from the isolate to be tested by transferring (4-5) single, pure colonies, (24) hours old, growing on the nutrient agar medium using a microbial carrier, to (5) ml of the prepared functional saline solution. The turbidity of the suspension was compared with the turbidity of the prepared standard turbidity constant solution (McFarland (2.2.1)), which gives a number of cells approximately (108 × 1.5) CFU/ml. Using a clean, sterile cotton swab, a portion of the bacterial suspension was spread on the surface of plates containing Mueller-Hinton agar medium. Then, antibiotic discs were distributed on the surface of the agar at a rate of (6) discs for each plate by using sterile forceps, then plates incubated at 37℃ for 24 hr. Later that, results were observed and the inhibition zones around each disc were measured. By referring to standard tables specific to each type of antibiotic, it was determined whether the isolate was resistant or sensitive to antibiotics.
2.7 Evaluation of the efficacy of (Bacteriocin) reuterin extract of Lactobacillus reuteri as an antimicrobial opposite pathogenic bacteria
Three replicates were prepared for each isolate.
Figure 1. Positive of percentage of urine and blood infections
3.1 Identification of all Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans
Twenty-four clinical isolates were collected from patients suffering from urinary tract infections; these included 20 isolates (83.3%) from urine and 4 isolates (16.6%) from blood samples. All isolates of P. aeruginosa, S. aureus, and C. albicans were identified using microscopic and biochemical tests and confirmed with the Vitek 2E compact system. These results were illustrated in Table 1 and Figure 1.
Table 1. Number of positive bacterial cultures of samples and the percentage of urine and blood infections
|
Total Number of Bacterial Samples |
Positive Culture 24(100)% |
|
|
Number of Urine Samples |
Number of Blood Samples |
|
|
24 |
20 (83.3%) |
4 (16.6%) |
|
Pseudomonas aeruginosa |
6 |
1 |
|
Staphylococcus aureus |
6 |
2 |
|
Candida albicans |
8 |
1 |
3.2 Isolation and identification of all Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans
The bacterial isolates were cultured on nutrient agar and Pseudomonas agar, as shown in Figure 2, where they exhibited pyocyanin pigment colonies. This medium was generally used in the laboratory for the selective cultivation of P. aeruginosa, a Gram-negative bacterium.
Figure 2. P. aeruginosa produced of pyocyanin pigment on nutrient agar
Staphylococcus aureus isolates were cultured on mannitol salt agar medium which selective growth of Staphylococcus spp. Because of its constituents of sodium chloride (75.0 gm/L) is regarded as selective and differential medium for Staphylococci as shown in Figure 3 [18].
Few isolates could the ferment mannitol and composed a large golden colony surrounded by yellow zones and turned the medium color from pink to yellow, while others were non-mannitol-fermenter with white color was manifested on the mannitol agar agree with study [20].
Figure 3. S. aureus colonies on mannitol salt agar medium
Clinical Candida was cultured in aerobic and anaerobic blood culture bottles and grown in the automated blood culture system at 35℃. The aliquot was then removed and CHROM agar Candida was plated as a control and cultured on Sabouraud dextrose agar at the same time. No significant difference in colony form or color was observed between blood and control isolates in this agrees with study [20].
3.3 Antibiotic sensitivity tests
In the present study, standard disc diffusion test has been used for detection of susceptibility of pathogenic bacteria to antibiotics, as shown in Figures 4, 5 and 6.
Figure 4. C. albicans isolates growth on Muller Hinton agar
Figure 5. Sensitivity test using antimicrobial discs against P. aeruginosa cultured on Mueller Hinton agar
P. aeruginosa isolates appears a various resistance levels to antimicrobial as following: the isolates were resistant to Cefotaxime was (100%); Ceftriaxone (100%); Ceftazidime (100%); Cefepime (85%); Cefoxitin (63%); Ciprofloxacin (61%), while revealed lower resistance to Aztreonam (43%); Ampicillin (32%); Vancomycin (35%); Levofloxacin (47%); Rifampin (24%); imipenem was (12%) as shown in Figure 7.
S. aureus isolates revealed a various were relatively of resistant for than P. aeruginosa to Antimicrobial Cefotaxime was (91%); Ceftriaxone (70%); Ceftazidime (42%); Cefepime (26%); Cefoxitin (25%); Ciprofloxacin (41%), while Aztreonam (70%); Ampicillin (54%); Vancomycin (74%); Levofloxacin (22%); Rifampin (20%)); Imipenem was (15%) as shown in Figure 8, this does not agree with studies [6, 18].
Figure 6. Sensitivity test using antimicrobial discs against S. aureus cultured on Mueller Hinton agar
Figure 7. Results of susceptibility tests of P. aeruginosa against 12 antimicrobial agents according to (CLSI 2015)
Figure 8. Results of susceptibility tests of S. aureus against 12 antimicrobial agents according to (CLSI 2015)
3.4 Antibacterial activity of crud and partially purified reuterin (bacteriocin) from Lactobacillus reuteri on growth of some pathogenic bacteria
P. aeruginosa, Staph. aureus and C. albicans isolates were possessing high resistance to antibiotics and able to produce virulence factors.
The protease extracted from some pathogenic bacteria important a virulence factor that was especially for study. The results of this study showed that P. aeruginosa (3) were the most active (19 mm diameter) through measuring of the inhibition zone on skim milk medium (or by the well procedure), at the same time as the P. (2) and C. (9) were the lowest down protease activity (12 mm) in diameter as illustrated in Table 2.
Table 2. Protease activity produced of P. aeruginosa, S. aureus and C. albicans isolates detected measuring the diameter of lysis position later 18-24 hours at 37℃ of incubation
|
ACTIVITY Isolate No. |
PROTEASE Activity Diameter of Lysis Area in (mm) |
|
P. 1 |
14 |
|
P. 2 |
12 |
|
P. 3 |
19 |
|
P. 4 |
16 |
|
S. 5 |
14 |
|
S. 6 |
14 |
|
S. 7 |
16 |
|
S. 8 |
14 |
|
C. 9 |
12 |
|
C. 10 |
15 |
|
C. 11 |
14 |
|
C. 12 |
14 |
The reuterin extract, a bacteriocin, showed a good inhibitory effect on P. aeruginosa, Staph. aureus and C. albicans at concentrations ranging from the stock solution to 10-6. All isolates of these pathogens lacked protease enzyme activity, while few or normal activities were observed at dilutions ranging from 10-7 to 10-10, as shown in Table 3. This suggests that contact with other microbes in urine increases the production of reuterin, which inhibits bacterial growth by modifying thiol groups. This indicates that reuterin negatively affects multiple cell compounds, a finding that was consistent with study [7].
Table 3. Explain the activity of (Bacteriocin) levels on protease product by pathogenic bacteria
|
BACTERIOCIN Activity Levels |
PROTEASE Producing Activity Pathogenic |
|
Stock solution |
No-producing |
|
Con.10-1 |
No-producing |
|
Con. 10-2 |
No-producing |
|
Con. 10-3 |
No-producing |
|
Con. 10-4 |
No-producing |
|
Con. 10-5 |
No-producing |
|
Con. 10-6 |
No-producing |
|
Con. 10-7 |
Little (10mm) |
|
Con. 10-8 |
Normal (12mm) |
|
Con. 10-9 |
Normal (14mm) |
|
Con. 10-10 |
Normal (14mm) |
3.5 Evaluating the antibacterial activity of (Bacteriocin) reuterin extract of Lactobacillus reuteri against pathogenic bacteria
Lactic acid bacteria were used as antimicrobial agent. This comes down to its capability to secrete antibacterial materials that prevent the growth of pathogenics. LAB secretes anti-microbial composites (bacteriocins). This study shed light on a bacterium called reuterin, which is produced by Lactobacillus reuteri, and assesses its antibacterial activity against various bacterial and fungal pathogens found in hospitals. Roterin production (bacteriocin concentration (30%) saturation) showed the highest inhibition zone (21.3, 21.4 and 23.2 mm) opposed P. aeruginosa, Staph. aureus and C. albicans, respectively, at it’s grown anaerobically on MRS agar, pH 6.5 at 37℃ for one day. This study has permissible us to utilizes reuteri as an antimicrobial into reduce pathogenics. This agrees with study [7] that appears the reuterin has inhibitory activity against Gram (+) and Gram (-) bacteria. The inhibition zones were up measuring 23mm and 21mm in diameter contra Staph. aureus and E. coli, respectively.
Using the agar well diffusion assay (WDA), 0.1 ml of the suspension, at concentrations ranging from the stock solution to 10-10, was transferred to Mueller-Hinton agar and spread using the matting method. Wells with a diameter of 8 mm were created, and the plates were then left to dry.
Figure 9. Antibacterial activity of crude and partially purified reuterin (Bacteriocin) against P. aeruginosa
Figure 10. Antibacterial activity of crude and partially purified reuterin (Bacteriocin) against Candida albicans
Subsequently, 0.1 ml of reuterin extract (bacteriocin) was added to each well. After four hours, the plates were incubated under different conditions: anaerobically at 4℃ for lactic acid bacteria (LAB), and aerobically at 37℃ for other pathogens, for 18 to 24 hours. Three replicates were conducted for each isolate. The inhibition of growth was assessed by measuring the zone of inhibition surrounding each well, as illustrated in Figures 9 and 10.
There are supports our results through study [11] that reuterin showed in yogurt, a (fungistatic) effect at 1.4 mM of level but a (fungicidal) effect was geted at concentration of 7 mM. so, reuterin has a high possible as a nutrient protector, a specialty having to its biochemical characterizes and antimicrobial effectiveness es. in addition, reuterin illustrated a fungicidal effectiveness (killed 99.9% of all tested M.Os) at levels equal or less than 15.6 mM as indicated by MFC.
1. Lactobacilli can display antipathogenic bacterial activity through numerous mechanisms, as production of antimicrobial bioactive agents that can inhibit the growth of the pathogens.
2. Bacteriocins are antimicrobial compounds named reuterin which are produced by Lactobacillus reuteri have antimicrobial effectives against some hospitalized pathogens.
3. Bacteriocin (reuterin) demonstrates the greatest inhibition zone against pathogenic bacteria at concentrations (10-7 to 10-10). The results of our research were that P. aeruginosa 3 had the elevate effective (19 mm) by examining of the inhibition zone on skim milk agar medium, but the P. 2 and C. 9 were (12 mm in diameter).
4. The reuterin (Bacteriocin) is used as therapeutic agents.
The authors express their sincere gratitude to Mustansiriyah University, College of Science, Departments of Microbiology and Biology, for continued support of our research program.
[1] Christensen, I.B., Vedel, C., Clausen, M.L., Kjærulff, S., Agner, T., Nielsen, D.S. (2021). Targeted screening of lactic acid bacteria with antibacterial activity toward Staphylococcus aureus clonal complex type 1 associated with atopic dermatitis. Frontiers in Microbiology, 12: 733847. https://doi.org/10.3389/fmicb.2021.733847
[2] Kelly, M.S., Bunyavanich, S., Phipatanakul, W., Lai, P.S. (2022). The environmental microbiome, allergic disease, and asthma. The Journal of Allergy and Clinical Immunology: In Practice, 10(9): 2206-2217. https://doi.org/10.1016/j.jaip.2022.06.006
[3] Imtiaz, H., Imtiaz, K., Taara, N., Jabeen, S., Ismail, E., Ullah, M.S., Salman, M., Siddiqui, A., Sardar, S., Shireen, F. (2024). Antagonistic activity of lactobacillus and bifidobacterium isolated from yogurt against pathogenic enteric bacteria. Journal of Population Therapeutics and Clinical Pharmacology, 31(5): 544-551. https://doi.org/10.53555/jptcp.v31i5.5556
[4] Ahmadnejad, E., Dolatabadi, S. (2021). Isolation of Probiotic Lactobacilli from Indigenous Yogurt and Cheese and Their Antagonistic Roles Against Foodborne Pathogens. Shiraz E-Medical Journal, 22(5): e102313. https://doi.org/10.5812/semj.102313
[5] Hassanzadeh, S., Ebrahimi, S., Ganjloo, S., Jamehdar, S.A., Dolatabadi, S. (2021). Anti-pseudomonas aeruginosa activity of metal Schiff base complex and probiotics against planktonic-and biofilm-growing cells. Anti-Infective Agents, 19(2): 182-191. https://doi.org/10.2174/2211352518999200807152232
[6] Abbas, M.K., Abdu-Allah, S.N., Al Ameer Baqer, B.A. (2023). Study of the pathological antibacterial effects of tea extract and its role in reducing hypertension in pregnant women. Malaysian Journal of Microbiology, 19(1): 22. https://doi.org/10.21161/mjm.220008
[7] Niamah, A.K., Mohammed, A.A., Alhelf, N. A. (2023). Antibacterial activity and identification of produced reuterin from local Lactobacillus reuteri LBIQ1 isolate. Journal of microbiology, biotechnology and food sciences, 12(5): e4701-e4701. https://doi.org/10.55251/jmbfs.4701
[8] Darbandi, A., Asadi, A., Mahdizade Ari, M., Ohadi, E., Talebi, M., Zadeh, M.H., Emamie, A.D., Ghanavati, R., Kakanj, M. (2022). Bacteriocins: Properties and potential use as antimicrobials. Journal of Clinical Laboratory Analysis, 36(1): e24093. https://doi.org/10.1002/jcla.24093
[9] Varada, V.V., Kumar, S., Tyagi, N., Tyagi, A.K. (2022). Effects of compound lyophilized probiotics on selected faecal microbiota, immune response, and antioxidant status in newborn buffalo calves. Current Research in Biotechnology, 4: 493-502. https://doi.org/10.1016/j.crbiot.2022.10.003
[10] Soltani, S., Hammami, R., Cotter, P.D., Rebuffat, S., Said, L.B., Gaudreau, H., Bédard, F., Biron, E., Drider, D., Fliss, I. (2021). Bacteriocins as a new generation of antimicrobials: Toxicity aspects and regulations. FEMS Microbiology Reviews, 45(1): fuaa039. https://doi.org/10.1093/femsre/fuaa039
[11] Vimont, A., Fernandez, B., Ahmed, G., Fortin, H.P., Fliss, I. (2019). Quantitative antifungal activity of reuterin against food isolates of yeasts and moulds and its potential application in yogurt. International journal of food microbiology, 289, 182-188.
[12] Fotso Techeu, U.D., Kaktcham, P.M., Momo, H.K., Foko Kouam, E. M., Tchamani Piame, L., Ngouenam, R. J., Zambou Ngoufack, F. (2022). Isolation, characterization, and effect on biofilm formation of bacteriocin produced by Lactococcus lactis F01 isolated from Cyprinus carpio and application for biopreservation of fish sausage. BioMed Research International, 2022(1): 8437926. https://doi.org/10.1155/2022/8437926
[13] Alam, M.D., Islam, M., Ziaul, M.D., Tayab, M.D., Alam, K., Sahid, H., et al. (2022). Role of probiotic Lactobacillus reuteri in improving gut health and immunity in infants and toddlers: A review. International Journal of Nutrition Sciences, 7(2): 75-80. https://doi.org/10.30476/ijns.2022.94849.1182
[14] Maftei, N.M., Raileanu, C.R., Balta, A.A., Ambrose, L., Boev, M., Marin, D.B., Lisa, E.L. (2024). The Potential Impact of Probiotics on Human Health: An Update on Their Health-Promoting Properties. Microorganisms, 12(2): 234. https://doi.org/10.3390/microorganisms12020234
[15] Ju, J.H., Jeon, S.G., Lee, K.M., Heo, S.Y., Kim, M.S., Kim, C.H., Oh, B.R. (2021). The biocatalytic production of 3-hydroxypropionaldehyde and evaluation of its stability. Catalysts, 11(10): 1139. https://doi.org/10.3390/catal11101139
[16] Mazziotta, C., Tognon, M., Martini, F., Torreggiani, E., Rotondo, J.C. (2023). Probiotics mechanism of action on immune cells and beneficial effects on human health. Cells, 12(1): 184. https://doi.org/10.3390/cells12010184
[17] Missiakas, D.M., Schneewind, O. (2013). Growth and laboratory maintenance of Staphylococcus aureus. Current Protocols in Microbiology, 28(1): 9C-1. https://doi.org/10.1002/9780471729259.mc09c01s28
[18] Abbas, M.K., Abdul, F.R., Rasool, K.H. (2022). Immunological and Enzymatic study of Staphylococcus aureus Bacteria and fungi isolated from oral cavity. Research Journal of Pharmacy and Technology, 15(7): 3119-3124. https://doi.org/10.52711/0974-360X.2022.00522
[19] Eddleman, H. (1998). Making Bacteria Media from Potato. Indiana Biolab.
[20] Ghazaei, C. (2022). Study of the effect of bacteriocin-producing Bacillus subtilis strains on beta-lactamase-producing pathogenic bacteria. Journal of Clinical Research in Paramedical Sciences, 11(2): e130208. https://doi.org/10.5812/jcrps-130208
[21] Duong-Ly, K.C., Gabelli, S.B. (2014). Salting out of proteins using ammonium sulfate precipitation. In Methods in Enzymology, 541: 85-94. https://doi.org/10.1016/B978-0-12-420119-4.00007-0
[22] Hamdy, A.N., Abbas, M.K., Khudhair, M.A., Tektook, N.K. (2020). Interactions between Interleukin-6 and MDA in Women with Preeclampsia. Systematic Reviews in Pharmacy, 11(3): 752. https://doi.org/10.31838/srp.2020.3.104
