Bacteriocin and its effect against skin pathogens
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This study investigates the extraction of bacteriocin from Lactobacillus isolated from dairy products and its inhibitory effects against various skin pathogens, including Candida albicans and Staphylococcus aureus, using the Kirby-Bauer disc diffusion method. Results indicate significant antagonistic activity of the extracted bacteriocin against these pathogens, suggesting potential therapeutic applications in combating skin infections. The findings highlight the importance of Lactobacillus and its bacteriocin in medical microbiology and the fight against drug-resistant infections.
Bacteriocin and its effect against skin pathogens
- 1. 1 Ravi and Thankavel International Journal of Microbiology and Mycology | IJMM | pISSN: 2309-4796 http://www.innspub.net Vol. 18, No. 2, p. 1-7, 2024 Bacteriocin and its effect against skin pathogens Aparna Ravi*1 , M. Thankavel2 1 Department of Microbiology, Nehru Arts and Science College, Coimbatore, India 2 Deen School of Life Sciences, Nehru Arts and Science College, Coimbatore, India Keywords: Bacteriocin, Inhibitory effect, Antagonistic activity, Skin pathogens, Kirby Bauer disc diffusion Publication date: February 22, 2024 Abstract This paper aims to examine the isolation of Lactobacillus from dairy products (milk, curd, and yogurt), extraction of Bacteriocin from it as well as to determine their inhibitory effect against few fungal skin pathogens and bacterial skin pathogens such as: Candida albicans, Aspergillus sp., Malassezia sp., Fusarium sp., and Penicillium sp., Staphylococcus aureus, Streptococcus pyogens, Klebsiella sp. The antagonistic activity of Lactobacillus sp. is mainly due to the bacteriocin present in it, therefore in this study, the bacteriocin is extracted and checked against the indicated microorganisms. Kirby Bauer Disc Diffussion method is used and zone of clearance is observed around the pathogenic species indicating that they shows some kind of antagonistic activities which are further measured and noted, which gives a clear picture about the degree of resistance of bacteriocin against the pathogenic microorganisms. * Corresponding Author: Aparna Ravi aparnaravimicrophd20@gmail.com Open Access RESEARCH PAPER
- 2. 2 Ravi and Thankavel Introduction Skin is an important organ that represents the first line of defense against the external environment. Some microorganisms are pathogenic but some are usually present on the skin does not causing damages but during adverse condition like immunosuppressant phases the organisms develop infections in the host (Hall and Dorsch, 2002). Some of such cases like; Primary cutaneous aspergillosis usually involves site of injury, at or near intravenous catheter sites, occlusive dressings, burns, or surgery (Walsh and Groll, 1999). Secondary infections infect the underlying structures or from wide spread blood borne seeding of the skin. Superficial infections including keratitis, otomycosis are commonly caused by Penicillium sp., Malassezia sp. are another example of normal skin flora colonizes as commensals and during adverse conditions develops diseases at the head, neck dermatitis, and malassezia folliculitis (Gupta and Kohli, 2004). Fusarium sp. Causes infections in patients in conditions like hematologic malignant or bone marrow transplant (Nucci and Anaissie, 2007). Candida nail infections occur in patients with chronic mucocutaneous candidiasis caused by Candida albicans, they invade the entire nail plates (Kirkpatrick, 2001). Staphylococcus aureus is a major cause of bacterial skin infections namely, abscesses in boils, furuncles, Cellulitis (Prendiville, 1989). Streptococcus pyogens are also bacterial species causing infections in the superficial keratin layer called impetigo, the superficial epidermis layer-erysipelas, the subcutaneous tissue layer-cellulitis, fascia called necrotizing fasciitis or in the muscle-myositis, myonecrosis (Stevens and Bryant, 2017). Klebsiella causes surgical wound infections, they usually enters through break in the skin and gradually leads to soft tissue infections (Paterson and Bonomo, 2005). Balanced skin is crucial for maintaining healthy skin functioning; but changes in the skin microbes are associated with skin diseases such as those caused by Candida albicans, Aspergillus, Malassezia furfur, Fusarium, Penicillium, Staphylococcus aureus, Streptococcus pyogens, and Klebsiella sp. Lactobacilli are probiotic belonging to the group of lactic acid bacteria; they are Gram-positive, non-sporulating, anaerobic or facultative anaerobic rods. They are commonly present in dairy products, soil, lakes, and the intestinal tract of humans and animals. They possess antagonistic activity against various pathogenic microorganisms (Salminen et al., 2004). In the present study, the Lactobacillus sp. from dairy products like milk, curd, and yogurt are isolated; then bacteriocin is extracted from it and checked their efficacy against fungal and bacterial pathogens using the technique of Kirby Bauer disc diffusion method (Aasen and Moretro, 2018). Bacteriocins are low molecular weight peptides secreted by the bacterial cells to kill sensitive cells present in the same ecosystem competing for food and other nutrients. Bacteriocins, along with their native antibacterial property, also exhibit additional antiviral and antifungal properties nowadays (Riley and Wertz, 2002). The dairy samples (milk, curd, yogurt) were collected in sterile containers from different places of Vandithavalam, Palakkad, Kerala. The samples were analyzed microbiologically, identified, and confirmed by biochemical tests. The study aimed to extract bacteriocin from Lactobacillus sp. and to see whether its effect against the mentioned microorganism. Materials and methods Collection of milk samples Raw milk samples of cow and goat were collected in a sterile container in the month of February 2022 at various places of Vandithavalam, Palakkad, Kerala for the isolation of Lactobacillus species and examined under aseptic conditions.
- 3. 3 Ravi and Thankavel Collection of curd samples Homemade curd samples and commercially available curd samples of two different brands were collected for the isolation of Lactobacillus sp. Collection of Yogurt samples Yogurt samples of two different commercial brands were collected for the isolation of Lactobacillus sp. Collection of microorganisms Candida albicans Swabs were taken from patients with toe nail and skin infections characterized by fluid discharge , Colour changes of the nail, Pain in the nail and toe. Malassezia Malassezia samples were collected by taking the Scrappings from various patients of head and neck dermatitis characterized by white patchy scales present in the affected areas with itching and dryness. Aspergillus Swabs were collected from patients with skin injury and burns. Fusarium Swabs were collected from skin lesions through aseptic techniques. Penicillium Swabs were taken from patients of skin lesions with swelling and redness in the skin with discolouration. Staphylococcus aureus Swabs were taken from the pimples of patients with granules and pus. Streptococcus pyogens Discharges from the wound is collected through sterile cotton swabs. Klebsiella Swabs were taken from patients with boils and wound with discoloration. Isolation and characterization of Lactobacillus from dairy samples 1.The milk, curd and yogurt samples were serially diluted in various test tubes. 2.MRS (de Man, Rogosa, and Sharpe agar- selective media for LB) agar plates were prepared. 3.Swabs from various dilutions from each of the samples of milk, curd and yogurt were swabbed onto MRS agar plates using sterile techniques and control plates were maintained. 4.All the inoculated plates of three different samples of different dilutions were incubated at 37 degree Celsius for exactly 24 hours and then plates were observed for growth. 5.Colonies formed in the plates were subjected for Staining and biochemical tests for the confirmation of the species of microorganisms present. Grams staining A clean grease free glass slide was taken, washed thoroughly, surface sterilised using ethanol and smear of single colony has been made on the slides and allowed to dry, the smear is heat fixed by passing the slide above flame of Bunsen burner for 2-3 times. The smear was cooled, flooded with the primary stain -crystal violet, then waited for one minute was washed it under running tap water. Then added the Mordant- Gram’s iodine, waited for one minute, decolorized it with 95% ethyl alcohol, washed under running tap water. Finally, added few drops of the counter stain-Safranin, waited for one minute and washed under running tap water, air dried and observed under oil immersion of 100X (Buchanan, 1982).
- 4. 4 Ravi and Thankavel Catalase test A clean glass slide was taken, washed and added a drop of hydrogen peroxide to the center of the slide, then fresh culture of test organism is mixed properly to it using a tooth pick or inoculation loop and observed for the presence of bubble formation (Schlegel, 1976). Oxidase test Oxidase disc was placed in a moisture free area and a small portion of test culture is spreaded on to it using a toothpick or inoculation loop, observed for the presence of colour change (Beveridge, 2001). Indole test Tryptophan broth was prepared, poured into test tubes, sterilized, cooled and inoculated with the test organisms, incubated at 37°C for 24 hours. After incubation few drops of Kovac’s reagent as an indicator was added to the test tubes and observed the colour change (Buchanan, 1982). Methyl red test MR-VP broth was prepared, poured into sterile test tubes and sterilized. Broth was inoculated with the test organisms and incubated at 37°C for 24 hours. After incubation Methyl red reagent was added and observed the colour change (Beveridge, 2001). Voges-Proskauer test MR-VP broth was prepared and poured into sterile test tubes and sterilized, inoculated with the test organism and incubated at 37°C for 24 hours. After incubation few drops of Barrit’s reagent A and Barrit’s reagent B, is added, Shaked well and observed the colour change (Buchanan, 1982). Citrate utilization test Simmon citrate agar medium was prepared and sterilised, poured into test tubes and slants were prepared by keeping the test tubes by sliding above a glass rod. Then streaked with the test organism and incubated at 37°C for 24 hours (Schlegel, 1976). Carbohydrate fermentation test Glucose, Fructose, Sucrose, Xylose, Lactose fermentations were checked. The test was performed by using 1% sugar in MRS broth. Media was prepared and poured into test tubes and Durham’s tube was inserted invertably into it. Test culture is inoculated and the tubes were incubated at 37°C for 24 hours. Phenol red was used as an indicator and observed the colour changes and formation of gas bubbles (Buchanan, 1982). Extraction of Bacteriocin Extraction of bacteriocins from Lactobacillus was done using chloroform extraction method (Beveridge, 2001). Isolation of microorganism Candida albicans Sabouraud Dextrose Agar (SDA) media was prepared and the Samples collected from patients were swabbed onto it. Plates were incubated at 37°C for 2-5 days and growth was observed (Buchanan, 1982). Malassezia Dandruff like flaky scrapings from patients were introduced to Dixon agar and SDA agar plates and incubated at 37°C for 3-5 days and observed growth (Buchanan, 1982).. Aspergillus, Penicillium and Fusarium Swabs taken from patients were introduced to SDA plates and incubated at 35°C for 2-5 days (Buchanan, 1982). Staphylococcus aureus Swabs from patients were inoculated into HiCrome Staph Selective Agar plates and incubated at 37°C for 24 hours (Buchanan, 1982).
- 5. 5 Ravi and Thankavel Streptococcus pyogens Swabs from patients were inoculated into Nutrient agar plates, incubated at 37°C for 24 hours (Buchanan, 1982). Klebsiella Swabs from patients were inoculated into MacConkey agar plates, incubated at 37°C for 24 hours (Buchanan, 1982). Checking the antagonistic activity Antimicrobial activity was tested by disc diffusion method (Beveridge, 2001). Candida albicans, Aspergillus, Malassezia, Fusarium and Penicillum, Staphylococcus aureus, Streptococcus pyogens and Klebsiella samples were swabbed onto separate Muller Hinton Agar plates, sterile discs coated with bacteriocins were placed on it. The plates were incubated and observed the zone of inhibition around the discs. The diameter of zone was measured. Results In the MRS agar plates, small to medium white colonies were observed. Gram staining showed purple rods indicating Gram-positive rod shaped bacteria (Fig. 1, Table 1). Colonies on MRS Agar Microscopic observation of LB Fig. 1. Colonies on MRS Agar and microscopic observation of LB Table 1. Morphological characteristics of Lactobacillus Characteristics LB -1 observations LB -2 Observations Colony morphology on Nutrient agar Creamy pale white colonies were seen Small white raised colonies were seen Colony morphology on MRS agar Pale white coloured colonies were seen Pale white coloured round colonies were seen Gram staining Purple coloured rods in chains were observed Purple coloured rods in chains were observed Table 2. Biochemical characteristics of Lactobacillus Name of tests Results (LB-1) Results (LB-2) Indole Positive Positive Methyl red Positive Positive Voges- proskauer negative negative Citrate utilization Positive Positive Oxidase Negative Negative Catalase Positive Positive Triple sugar iron agar Negative Negative Carbohydrate utilisation Glucose positive Lactose positive Sucrose positive Glucose positive Lactose positive Sucrose positive Table 3. Measurement of zone of inhibition against bacteriocin of Lactobacillus Organisms Diameter of zone (mm) Candica albicans 14mm Aspergillus sp. 15mm Malassezia furfur 14.5mm Fusarium sp. 13mm Penicillium 12mm Staphylococcus aureus 12.5mm Streptococcus pyogens 13mm Klebsiella 13mm Biochemical tests showing: oxidase- positive, catalase- positive, Indole -positive, MR -positive, Citrate- positive, lactose fermentation- positive indicated that the isolated microorganism is confirmed as the (LAB) Lactic acid bacteria- Lactobacillus sp. (Table 2). Bacitracin was extracted from the organism. Candida albicans, Aspergillus, Malassezia, Fusarium and Penicillum, Staphylococcus aureus, Streptococcus pyogens, Klebsiella swabbed plates were inoculated with the bacteriocin
- 6. 6 Ravi and Thankavel coated discs, zone of clearance was observed. The diameter of zone was measured and tabulated (Fig. 2, Table 3). Candida sp. Fusarium sp. Aspergillus sp. Malassezia sp. Penicillium sp. Streptococcus sp. S. aureus Klebsiella sp. Fig. 2. Zone of inhibition against bacteriocin of Lactobacillus Discussion One of the major challenges that microbiologists are facing nowadays is the multidrug resistance shown by various pathogenic species (Ventola, 2015). If the compound from a microorganism itself is efficient to control the growth and adverse effects of pathogens, it will be a revolutionary moment (Fernández, 2019). The use of probiotics is one example of such an application. Probiotics have been considered effective in maintaining gut health for many years, and now they are being explored for more possible applications (Hill et al., 2014). The probiotic Lactic acid bacteria, Lactobacillus, is isolated, and its virulent compound, Bacteriocin, is extracted, purified, and checked. It is confirmed that it has the ability to kill or suppress the growth of various fungi and bacteria (Cotter et al., 2005). Antagonistic activity of Bacteriocin of Lactobacillus sp isolated from dairy samples was subjected to the study and observations against fungal and bacterial pathogens, showing good degrees of inhibition (Mokoena, 2017). Considering this as a reference, recent studies are ongoing, expecting the formulation of new therapeutics from bacteriocins, which could be a broad-spectrum drug and a solution for the challenge of multidrug resistance shown by various pathogens (Perez et al., 2014). Conclusion A total of two raw milk samples from cow and two from goat, two curd samples (homemade, commercially available), two yogurt samples were collected, Morphological, physiological, microscopic observation, biochemical characterisations were made and confirmed as Lactobacillus species. The antagonistic activity of bacteriocin is checked. They are found to be sensitive against human pathogen Candida albicans, Aspergillus sp., Malassezia sp., Fusarium sp., Penicillium sp., Staphylococcus aureus, Streptococcus pyogens, Klebsiella sp. causing various skin infections in human beings. This has an important role in medical microbiology and human health. References Aasen IM, Moretro T. 2018. Extraction and Purification of Bacteriocins from Lactic Acid Bacteria. In Bacteriocins: Production, Applications and Safety (pp. 73-91). Springer. Beveridge TJ. 2001. Use of the Gram stain in microbiology. Biotechnic & Histochemistry 76(3), 111-118. Buchanan RE. 1982. Microbial staining methods. ASM Press.
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