COMPARATIVE EVALUATION OF PURIFICATION METHODS FOR PRODUCTION OF POLYPEPTIDE ANTIBIOTICS – “POLYMYXIN B” AND “CEREXIN A” FROM BACILLUS SPECIES

Pharma Admission

pharma admission

 

{ DOWNLOAD AS PDF }

ABOUT AUTHORS:
Pratyush Kumar Das1, Shilpa Das1, Debasish Sahoo2, Jikasmita Dalei2, V.Madhav Rao2, Sunakar Nayak2, Swadhin Palo3
1Centre of Biotechnology, Siksha O Anusandhan University, Bhubaneswar, Odisha, India.
2Nitza Biologicals (P.) Ltd.Chandra Towers, Near Fortune Honda Showroom, Neredmet 'X' Road, Secundrabad, Andhra Pradesh, India.
3Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha, India.
onlypratyush11@gmail.com

ABSTRACT:
Polymyxin B and Cerexin A are two polypeptide antibiotics, the first one discovered and incorporated quite earlier and the later one has still not been used in clinical trials for its high cytotoxic nature. Although Polymyxin was discovered very earlier but in the mid-way for some time it had lost its importance and was not used frequently due to its narrow spectra of action that only acts on gram negative microbes and because of its toxicity level. But with several new resistant gram negative microbes coming into the limelight responsible for causing many infections, Polymyxin B (the least toxic of all Polymyxins) has again been started to be used in pharmaceutical formulations and drugs. In this project, both Bacillus polymyxa and Bacillus cereus responsible for production of Polymyxin B and Cerexin A respectively were isolated from the rhizosphere of grass and cultured in the lab. They were confirmed by biochemical tests and then used to produce the corresponding antibiotics by submerged fermentation. The crude antibiotic thus obtained were purified by various methods like adsorption through activated charcoal, acetone precipitation, dialysis, Ion Exchange and Sephadex column chromatography and the results were compared to find the best possible way to purify the antibiotics keeping in mind that they show the maximum activity as possible on a lab scale. Further work on Cerexin A was not possible due to the unavailability of its standard solution. Work was carried out for quantitative estimation of purified and crude Polymyxin B by performing spectrophotometric assay against standard polymyxin.

REFERENCE ID: PHARMATUTOR-ART-2224

PharmaTutor (ISSN: 2347 - 7881)

Volume 2, Issue 8

Received On: 08/06/2014; Accepted On: 13/06/2014; Published On: 01/08/2014

How to cite this article: PK Das, S Das, D Sahoo, J Dalei, VM Rao, S Nayak, S Palo; Comparative Evaluation of Purification Methods for Production of Polypeptide Antibiotics – “Polymyxin B” and “Cerexin A” from Bacillus Species; PharmaTutor; 2014; 2(8); 188-200

INTRODUCTION:
An antibacterial is a compound or substance that kills or slows down the growth of bacteria. The term is often used synonymously with the term antibiotic(s); today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic(s) has come to denote a broader range of antimicrobial compounds, including antifungal and other compounds[1].

The term antibiotic was coined by Selman Waksman in 1942 to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. This definition excluded substances that kill bacteria, but are not produced by microorganisms (such as gastric juices and hydrogen peroxide). It also excluded synthetic antibacterial compounds such as the sulfonamides. Many antibacterial compounds are relatively small molecules with a molecular weight of less than 2000 atomic mass units[2].

Antibiotics are extremely important in medicine, but unfortunately bacteria are capable of developing resistance to them. Antibiotic-resistant bacteria are germs that are not killed by commonly used antibiotics. When bacteria are exposed to the same antibiotics over and over, the bacteria can change and are no longer affected by the drug. The problem of antibiotic resistance is worsened when antibiotics are used to treat disorders in which they have no efficacy (e.g. antibiotics are not effective against infections caused by viruses), and when they are used widely as prophylaxis rather than treatment. Resistance to antibiotics poses a serious and growing problem, because some infectious diseases are becoming more difficult to treat[3].

Resistant bacteria do not respond to the antibiotics and continue to cause infection. Some of these resistant bacteria can be treated with more powerful medicines, but there some infections that are difficult to cure even with new or experimental drugs[4].

In the last two decades, numerous oligopeptides have been isolated from sporogenic bacteria and fungi. These peptides are almost invariably characterized by the presence of macro cyclic structures, linkages other than cr-peptide bonds, amino acids that do not occur in protein, n-amino acids, and non-amino acid moieties. The formation of some of these peptides by microorganisms has been studied in considerable detail[5]. The biosynthesis of the actinomycins has been investigated in the laboratory of Katz[6] [7], that of bacitracin A by Bernlohr[8][9]and by Snoke[10] [11], that of gramicidm S by Winnick[12], that of valinomycin by MacDonald[13], and that of penicillin by several groups.

In all cases, the production of the peptide is related in a characteristic manner to the growth cycle of the microorganism, and it can proceed in the absence of protein synthesis.

Polypeptide antibiotics are a chemically diverse class of antibiotics containing non-protein polypeptide chains. Examples of this class include actinomycins, bacitracin, colistin, and Polymyxin B. Actinomycin-D has found use in cancer chemotherapy. Most other polypeptide antibiotics are too toxic for systemic administration, but can safely be administered topically to the skin as an antiseptic for shallow cuts and abrasions.

Bacillus polymyxaalso known as Bacillus aerosporousis a gram positive bacterium that is mostly found in the rhizosphere of grass. It is an endospore forming bacterium and the colonies have a flat surface with undulate margins. It is mainly responsible for production of PolymyxinB[14] – an antibiotic used to treat gram negative infections.

Bacillus cereus is a large, 1 x 3-4 µm, Gram-positive, rod-shaped, endospore forming, facultative aerobic bacterium[15]. It was first successfully isolated in 1969 from a case of fatal pneumonia in a male patient and was cultured from the blood and pleural fluid[16]. 16s rRNA comparison reveals Bacillus cereus to be most related to Bacillus anthracis, the cause of anthrax, and Bacillus thuringiensis, an insect pathogen used as pesticide[17]. Although they have similar characteristics, they are distinguishable as B. cereus is most motile, B. thuringiensis produces crystal toxins, and B. anthracis is non-hemolytic.

Polymyxin B is a polypeptide bactericidal antibiotic[18]. The Polymyxins were discovered in 1947 and introduced to the medical community in the 1950s. Colistin, also called Polymyxin E and its parenteral form, colistimethate, are related Polymyxins. Polymyxins can be administered orally, topically or parenterally, including intrathecally and intraperitoneally. However parenteral administration is primarily used in life threatening infections caused by Gram-negative bacilli or Pseudomonas species that are resistant to other drugs. Polymyxins exert their effect on the bacterial cell membrane by affecting phospholipids and interfering with membrane function and permeability, which results in cell death. Polymyxins are more effective against Gram-negative than Gram positive bacteria and are effective against all Gram-negative bacteria except Proteus species. These antibiotics act synergistically with potentiated sulfonamides, tetracyclines and certain other antimicrobials. Polymyxins also limit activity of endotoxins in body fluids and therefore, may be beneficial in therapy for endotoxemia.

Resistance to Polymyxins is uncommon and is exclusively chromosome dependent. The paucity of development of resistance is likely due to the drugs’ unique detergent action on the cell membrane. However, development of resistance to colistin in Pseudomonas aeruginosa is not uncommon with long term inhalation therapy for cystic fibrosis[18].

Review of Literature:
Microbes are a part and partial of our life. All the time we are surrounded by the microbes out of which some are beneficiary whereas some are harmful too. The microbes that are harmful cause many types of disease out of which some are very severe and life threatening. To tackle with these infections and disease our scientists and researchers have come out with many antibiotic compounds and drugs.

In this review the focus is entirely concentrated on two such polypeptide antibiotics viz. – “Polymyxin B” and “Cerexin A”. The Polymyxin were discovered in 1947 and introduced to the medical community in the 1950s. With large number of multi drug resistant gram negative bacteria emerging and lack of discovery of novel drugs to target them, it has become a significant public health issue. Hence the focus has now again shifted onto antibiotics like Polymyxin B which had once been ignored due to their higher toxicity level. Polymyxins also limit activity of endotoxins in body fluids and therefore, may be beneficial in therapy for endotoxemia. Many works have been carried out on Polymyxins. It has been reported that use of Polymyxin B can remove endotoxin from solutions to a certain extent (Van Miert and Van Duin, 1978). Cooperstock and Riegle, 1981). Morrison et al. (1976) have shown that the lipid A-associated protein (LAP) that is present in endotoxin inhibits the binding of Polymyxin B to the endotoxin (Morrison and Curry, 1979). As a result, one might surmise that the effectiveness of using Polymyxin B to remove endotoxins from solution would be of limited value. Despite the above data, within the past 2 or 3 years many investigators have suggested that Polymyxin B, either free or bound to a gel support, can be used to 'ensure' that a solution is free of endotoxins (for example, see Duff and Atkins, 1982; Dinarello, 1983; Issekutz, 1983; Dinarello et al., 1984). Amplification of Polymyxin B vacuole-targeting fungicidal activity has also been carried out in combination with allicin (an allylsulphur compound from garlic) against various yeasts and fungi. Work has also been carried out on Polymyxin B by combining it with certain fungal antibiotics. It has been observed that Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect, work carried out by - Bing Zhai, Henry Zhou, Liangpeng Yang, Jun Zhang, Kathy Jung, Chou-Zen Giam, Xin Xiang and Xiaorong Lin,

Cerexin A, comparatively a very new antibiotic isolated from Bacillus cereus has not been used till date for clinical trials because of its high toxicity. The question now arises that why is Cerexin A so toxic and how its toxicity level can be reduced? There can be a lot of scope for researchers on this but nobody have ever thought about the same. May be Cerexin A can prove to be a better alternative for the multi-drug resistant microbes and it may solve the issues that have cropped up regarding public health due to these microbes.

Objective:
1. 
To isolate Bacillus polymyxa and Bacillus cereus from the rhizosphere of grass.
2.
To produce the antibiotics – Polymyxin B and Cerexin A from Bacillus polymyxa and Bacillus cereus respectively.
3.
To optimize the production medium of Polymyxin B and Cerexin A.
4.
To purify the crude antibiotics obtained by various purification techniques (Adsorption through activated charcoal, Acetone Precipitation, Dialysis, Ion Exchange Chromatography and Sephadex Column Chromatography) and comparing the results to find the best way out to purify them.
5.
To check and compare the antibiotic activity of the crude and purified antibiotics.
6.
To determine the amount of Polymyxin B present in the purified sample by performing an assay.

Materials & Methods:
Source: Soil sample from rhizosphere of grass.
Location: (Neredmet ‘X’ Road Secunderabad), Andhra Pradesh.

1. Serial Dilution:
Materials Used
- Sample, NaCl, Distilled Water, Test Tubes, Micropipette, Micro tips, Autoclave, Laminar Air Flow.
Procedure
- 8 test tubes first one with 10 ml & the rest with 9 ml each of normal saline solution (0.89 % of NaCl in distilled water) were taken. The test tubes along with the saline solutions were autoclaved at 121oC for 45 min. Then 1gm of the soil sample was mixed to the first test tube containing 10 ml of the saline solution, mixed thoroughly and was marked as blank. 1 ml of the solution from the blank was taken by the help of a micropipette and poured into the second test tube, mixed thoroughly and was marked as 10-1. Similarly 1ml from 10-1 was taken and poured into the next test tube and was marked as 10-2 and so on till 10-7. This continuous dilution give well separated surface colonies. For isolation of bacteria generally the dilutions of 10-6 and 10-7 were considered.

2. Spread Plating:
Materials Used
- 2 Petri plates, Nutrient Agar Media (Peptone = 5 %, NaCl = 5 %, Beef Extract = 3 %, Agar Agar = 18 %), Ethyl Alcohol, Spreader, Micropipette, Micro tips.
Procedure
- Take 1ml of solution each from the test tube marked 10-6 and 10-7 and spread them on two separate plates containing solidified nutrient agar by the help of a spreader until the surface of the agar becomes dry. Incubate the plates for 24 hours in an incubator.

NOW YOU CAN ALSO PUBLISH YOUR ARTICLE ONLINE.

SUBMIT YOUR ARTICLE/PROJECT AT articles@pharmatutor.org

Subscribe to Pharmatutor Alerts by Email

FIND OUT MORE ARTICLES AT OUR DATABASE


Pages

FIND MORE ARTICLES