Universal Journal of Microbiology Research 5(3): 40-48, 2017 http://www.hrpub.org DOI: 10.13189/ujmr.2017.050302 Production of Bacteriocin Like Substances as Antipathogenic Metabolites by Staphylococcus warneri Isolated from Healthy Human Skin Reazul Karim*, Mohammad Nuruddin Mahmud, M. A. Hakim Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh Copyright©2017 by authors, all rights reserved. Authors agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Abstract Antibiotic resistance is a serious problem of Microbes that colonize the human body during birth or present world and development of viable alternative is urgent. shortly thereafter, remaining throughout life, are referred to The research work was designed to mitigate this problem. as normal flora [1]. A diverse microbial flora is associated Different types of bacterial colony were isolated from skin of with the skin and mucous membranes of every human being 30 healthy human and their antipathogenic activity was from shortly after birth until death [2]. Human skin is not a tested against 9 pathogens. The isolate showed activity particularly rich place for microbes to live. This is an against four pathogens- Klebsiella. pneumoniae subsp. environment that prevents the growth of many pneumoniae, Klebsiella. pneumoniae subsp. ozaenae, microorganisms, but a few have adapted to life on our skin Staphylococcus. aureus and Pseudomonas. aeruginosa was [3]. The effects of the normal flora are inferred by identified as Staphylococcus. warneri. Variation was found microbiologists from experimental comparisons in optimization of cultural conditions (incubation period, between "germ-free" animals (which are not colonized by incubation temperature and pH) for the most potent any microbes) and conventional animals (which are antipathogenic metabolites production. Staphylococcus. colonized with a typical normal flora). The overall beneficial warneri showed most potent antipathogenic activity at pH 6 effects of microbes are synthesize and excretion of vitamins, to pH 9, at an incubation period of 24h to 48h and at an prevention of colonization by pathogens, antagonize other incubation temperature of 37 . Antipathogenic metabolites bacteria, stimulation of the development of certain tissues, were then detected as bacteriocin like substances. Variation development of gut associated lymphoid tissue, production was observed with bacteriocin℃ activity against different of natural antibodies etc. [4]. Many mechanisms have been pathogens and was found most effective against Klebsiella. postulated by which Normal flora could produce pneumonia subsp. pneumonia and Klebsiella. pneumonia antipathogenic activity. In addition to their competitive subsp. ozaenae Samples containing bacteriocin like inhibition of the epithelial and mucosal adherence of substances showed heat stability up to 80 for 60 minutes pathogens and inhibition of epithelial invasion by pathogens, and up to pH 8 for Klebsiella. pneumoniae subsp. normal flora also show antipathogenic activity by producing pneumoniae and Klebsiella. Pneumoniae ℃sub sp. ozaenae. antimicrobial sub-stances or stimulating mucosal immunity. Papain treated cell-free supernatant did not show any Generally normal flora produces three types of antimicrobial bacteriocin activity, suggesting that the substances could be substances- H2O2, organic acid and Bacteriocin [5]. antimicrobial peptides. Solvent extraction of bacteriocin was Bacteriocins are proteins or short polypeptides, which are performed by using chloroform where maximum bacteriocin generally only toxic to bacteria that are closely related to the activity was found in interface layer rather than aqueous and producing strain [6]. Some researcher found that,[7]. organic layer. Staphylococcus. epidermidis, a major constituent of the normal microflora on healthy human skin protect skin by Keywords Antipathogenic Metabolites, Bacteriocin, producing antimicrobial peptide Another research work [8] Antimicrobial Peptide, Normal Microflora on bacteriocin- like inhibitory substance (BLIS) which is produced by the Streptococcal normal flora of the nasopharynx were found active against pathogens Streptococcus pneumoniae, Streptococcus pyogenes, 1 . Introduction Haemophilus. influenzae and Moraxella. Catarrhalis. In our recent study, we have tried to investigate the ability of Universal Journal of Microbiology Research 5(3): 40-48, 2017 41 normal skin flora to produce antipathogenic metabolites and hours. After the growth of the pathogens, the plates were also tried to reveal the fact that whether the metabolites observed for any zone of inhibition. A clear zone of were bacteriocin like substances or not. inhibition appeared against only those organisms which were sensitive to the metabolites produced by the isolates. Those pathogens and respective isolates were selected for 2. Material and Methods secondary screening. Samples collection, isolation and purification Secondary screening for antimicrobial metabolite production by the isolates Swab samples were collected from the skin of upper arm, under arm, forearm, leg and forehead of 30 healthy human Primarily selected isolates were subjected to the secondary persons free from skin or any other infection during sampling screening procedures to ensure their antimicrobial activity time and with a history of not taking antibiotics or any other against the respective pathogen. For this purpose, each antimicrobial drugs in the previous six months. For the normal flora was inoculated in 10ml sterile nutrient broth and collection of normal skin flora, the person who met selection incubated at 37 for 24 hours. After incubation, culture criteria was first taken to laboratory. With the help of sterile broth was centrifuged at 12000 rpm for 15 minutes at 4 and moisten cotton bud sample was taken from forearm, upper then filtered through℃ membrane filter. After filtering off the arm, under arm, forehead and leg of the selected healthy biomass through .45µ cellulose nitrate filter paper,℃ the human persons. The swab stick was then soaked in 10ml antimicrobial activity of the crude filtrate obtained was sterile peptone water from which 1 ml sample was used for tested against pathogens using disc diffusion [11] and well pour plate method [9] by using Mannitol salt agar medium diffusion method [12]. The average clear zone greater than10 and then incubated at 37 for 24 to 48 hours. Following mm was considered positive results [13]. incubation, different types of colonies based on differences in size, shape, color and ℃other colony characteristics, were selected and transferred to nutrient agar slant and were 3. Identification further purified by repeated streaking on nutrient agar plates. Bacterial isolate (normal flora) which showed positive result on primary screening and secondary screening against Test pathogens maximum pathogens were subjected to biochemical tests and For our current study, some type strains of skin and results were compared with standard description given in “Bergey’s Manual of Determinative Bacteriology”, 8th ed. intestinal pathogens were collected from Department of th Microbiology, University of Chittagong, ICDDR, B and [14] and 9 ed. [15]. The tests include Gram-staining, Spore Chittagong Maa-Shishu O General Hospital (CMSOGH) to staining Acid-fast staining, starch hydrolysis, Voges observe the inhibitory activity of the normal skin flora Proskauer (V-P) Test, Production of hydrogen sulphide, against these pathogens. The used bacterial test organisms Gelatin liquefaction test, Nitrate reduction test, Indole test were Klebsiella. pneumoniae subsp. ozaenae (CMSOGH, Deep glucose agar test, Catalase reaction Methyl-red test, urinary tract infection), Klebsiella. pneumoniae subsp. Fermentation test, Urease test Motility test, Oxidase test Cultural and physiological studies were also done. A rapid pneumoniae (CMSOGH, urinary tract infection), Salmonella. TM typhi (AEI14296), Escherichia. coli (ATCC 25922), bacterial identification test kit BBL Crystal Identification Pseudomonas. aeruginosa (CRL, ICDDR,B), Systems Gram-Positive Is Kit [16] was also used to identify Staphylococcus. aureus (ATCC 6538). Streptococcus. species of bacteria. pyogenes (CMSOGH , pus) Bacillus. subtilis (BTTC17) Optimization of cultural conditions for antipathogenic Primary screening for antimicrobial activity of the metabolite production isolates Different environmental and cultural conditions may The isolated bacteria (normal flora) were subjected to influence the antipathogenic metabolite production ability of primary screening procedures to find out the ability of the the selected isolates. Considering this matter, in our present normal flora to produce antipathogenic metabolites by streak study, an attempt was made to optimize the cultural plate method [10]. The isolates (normal flora) were streaked conditions that favor maximum antipathogenic metabolite over the surface of petridish containing pre-poured solidified production by isolated bacterial strain. To determine the Mueller Hinton media across one side of the plate & effect of incubation period on antimicrobial metabolite incubated at 37 for growth and diffusion of antimicrobial production, 4 pieces 250 ml conical flask was labeled as 24h, metabolites in the growth medium. Then cultures of the test 48h, 72h, 96h and 50 ml sterilized nutrient broth was taken pathogens were℃ streaked right angles to