Bio Prospecting and Indigenous Knowledge

Bio Prospecting - Introduction

Bio prospecting or biodiversity prospecting is the exploration of microorganisms, wild plants and animals for commercially valuable genetic and bio-chemical resources.

In many cases, bio prospecting is a search for useful organic compounds in microorganisms, plants and fungi that grow in extreme environments such as rainforests, deserts and hot springs.

Humankind has been studying, manipulating and exploiting natural diversity ever since the emergence of Homo sapiens over 150,000 years ago. Our early ancestors explored biodiversity and learned how to derive benefits from nature.

Early bio prospecting led to the improvement of methods for growing food, building shelters and maintaining health. Modern-day bio prospecting is simply an extension of our long history of exploring nature to improve the quality of our life.

Most of the raw materials for biotechnology oriented industry comes from the wild rainforests of the southern hemisphere. For example, many scientists believe that the cures for AIDS, cancer and other diseases lie hidden in these green vegetation.

The main objectives of bio prospecting are to fulfill economic and conservation goals and to enhance medical and agricultural advances needed to combat disease and sustain a growing human population.  Agencies- scientific and corporates, use the folk wisdom of indigenous people to locate and understand the use of medicinal plants. Then this knowledge is commercially exploited. There needs a partnership between business men, academicians and with indigenous people to effectively utilise the ‘green gold’ (the plants and animals with properties which businesses could use for new products and services) of bio prospecting.

Academic Work and Bio Prospecting- Case of Taq Polymerase:

Dr. Thomas Brock (1966) studied microorganisms living in yellow stone’s hot springs. He named one of the curious microorganism he isolated Thermus aquaticus. This microorganism lives and thrives in water so hot that it would kill an ordinary animal. Dr. Brock grew Thermus aquaticus in the laboratory and gave a living sample to the American Type Culture Collection for safe keeping. Dr. Brock’s work was an academic one.

In 1985, a biotechnology company named Cetus Corporation was developing a new way to duplicate genetic material. Dr. Kary Mullis, working at Cetus corporation invented a way to duplicate DNA, called Polymerase Chain Reaction (PCR).

But the high temperatures required by PCR destroyed the polymerase enzymes and fresh enzymes had to be added throughout the PCR process. Scientists at Cetus isolated an enzyme, named Taq polymerase, from Thermus aquaticus, which can withstand high temperatures of PCR process. PCR using Taq polymerase was so effective that a whole new scientific field has flourished as scientists finally had a convenient way to study DNA.

Thus, Dr. Brock’s academic work in Yellow stone had a practical application that he never imagined in his academic career.

 Bio Prospecting and Indigenous Knowledge

Indigenous Knowledge is the knowledge possessed by the local community- the unique knowledge confined to a particular culture or society. It is also known as local knowledge, folk knowledge, traditional wisdom or traditional science.

Indigenous traditional knowledge collected from places traditionally inhabited by indigenous people, help to easily identify valuable chemical compounds from plants, animals and microorganisms of that locality. A knowledge about the working of indigenous remedies and preparation offer clues to the nature of chemical compounds involved. This knowledge can be shared across different places, later

Usually, one in 10,000 chemicals derived from mass screening of plants, animals and microbes eventually results in a potentially profitable drug. Indigenous knowledge oriented approach is less expensive and more focused.

 In India, a well-known example of bio prospecting and benefit sharing is the Kani- TBGRI (Tropical Botanic Garden & Research Institute) – model in Kerala.

Kani is a tribal community inhabiting the southern Western Ghats in Kerala. In 1987, scientists from the TBGRI undertook an ethno botanical study in the tribal inhabited Western Ghat regions in Kerala.

They got an interesting ethno botanical information on a wild plant Trichophus zeylanicus, locally called “Arogyapacha” by the kani tribe. The Kani tribals accompanying the scientists, reported that eating fruits of this plant kept them energetic and agile. Pharmacological studies of the fruit confirmed its anti-fatigue properties. The leaves of the plant contained various glycolipids and other non-steroidal compounds with anti-stress and anti-hepatotoxic properties. 

The scientists developed a polyherbal formulation by Arogyapacha Ayurvedic pharmaceutical methods which was named ‘Jeevni’. After clinical evaluation this herbal drug was released for commercial production.

The manufacturing license of ‘Jeevni’ was transferred to the Aryavaidya Pharmacy, Coimbatore Ltd. for a license fee of Rs. 10 lakhs for a period of 7 years. TBGRI has agreed to share 50% of license fee and royalties with the tribal community. In November 1997, a number of Kanis with the assistance from TBGRI, registered a trust called Kerala Kani Samudaya Kshema Trust, comprising nine members from the tribals. 

Indigenous knowledge is thus a treasure house for bioprospecting

Pharmaceutical Industry in Bio Prospecting: 

Around 80% of people in developing countries rely on medicines largely based on plants and animals.

Today, there is greatly renewed interest in natural product screening for medicinal compounds. It is estimated that over 200 companies and research organizations worldwide are screening plant and animal compounds for medicinal properties.

Only less than 1% of the world’s 250,000 tropical plants have been screened for potential pharmaceutical applications.

A growing number of pharmaceutical corporations, biotechnological companies are stalking the forests, fields and waters of the developing world in search of biological riches and indigenous knowledge.

With the advances in molecular biology and the availability of more sophisticated tools for screening, it is increasingly cost effective for pharmaceutical industries to conduct natural product research. In a high-technology regime, extracts from biological specimens undergo rapid and precise screening procedures that allow for the isolation of active compounds displaying a specifically targeted activity.

However, two Fundamental Issues Concerning Bio Prospecting are:

(a) Sustainable use of biological resources and their conservation and

(b) The economic development of source countries and local communities.

Reference:

 https://www.biologydiscussion.com/biotechnology/fundamental-aspects-of-bio-prospecting/8594

Penicillin - Biosynthesis, Media preparation, fermentation

 Penicillin Biosynthesis

Penicillin is a modified tripeptide made from three amino acids aminoadipic acid, cysteine and valine. The principle enzyme involved in penicillin biosynthesis is ACV-synthetase. The β-lactam ring is formed  by isopenicillin-N synthetase; finally, side chains are added

 

Penicllin Fermentation- Medium preparation

Raw materials are primary requirement to design the fermentation broth. Fermentation broth contains all the necessary elements required for the proliferation of the microorganisms.  Generally, it contains a carbon source, nitrogen source, mineral source, precursors and antifoam agents if necessary.

Basic composition of inoculation/fermentation media

Corn steep liquor (3.5%)

Lactose (3.5%)

Glucose (1%)

CaCO₃ (1%)

KH₂PO₄ (0.4%)

Edible Oil (0.25%)

and Penicillin precursor in fermentation media (absent during inoculum preparation)

Inoculum preparation

Aim is to develop a pure inoculum in an adequate amounts. Inoculum is built up sequentially from small flasks to small fermenters. All the parameters like temperature, pH, aeration, agitation etc. should be properly maintained.  

pH: near 5.5 to 6.5

 Temperature: 26°C to 28°C

Aeration: a continuous stream of sterilized air is pumped into it.

Agitation: have baffles which allow constant agitation. 

Carbon Source

Lactose acts as a very satisfactory carbon compound for fermentation. Other carbohydrates like glucose is used during inoculum preparation. Lactose is preferred in fermentation media to provide slow feeding rate and starvation conditions to induce antibiotic formation. Sucrose may also be used. Complex as well as cheap sources like molasses, or soy meal can also be used.

Nitrogen Source

Another essential compound for metabolism of organisms is nitrogen. Ammonium salts such as ammonium sulfate, ammonium acetate, ammonium lactate or ammonia gas are used. Corn steep liquor also may also be used.

Mineral Source

Additionally, some minerals are necessary for the proper growth of these organisms like phosphorus, sulfur, magnesium, zinc, iron, and copper which are generally added in the form of water soluble salts. 

Precursors

Various types of precursors are added into production medium to produce specific type of penicillin. The most important naturally occurring penicillin is Penicillin-G. Depending upon the precursors added, the type of penicillin going to produced can be changed.

For example, if phenyl acetic acid is provided then  penicillin-G will be produced but if hydroxy phenyl acetic acid is provided then penicillin-X will be produced. Phenoxy acetic acid is provided as precursor for penicillin-V production. 

 When corn steep liquor is provided as nitrogen source, it also provides phenyl acetic acid derivatives; therefore, it is widely used in the production of penicillin-G.

Anti-foam agents

Anti-foaming agents such as lard oil, octadecanol and silicones are used to prevent foaming during fermentation. 

Following three points should be kept in mind before choosing raw materials for manufacture of penicillin,

1. An abundant growth of mycelium 

2. Maximum accumulation of penicillin 

3. Ease of extraction and purification of antibiotics.

 (contd..)

Protocols and selection of suitable methods according to work plan

The most difficult stage of conducting a research project is the preparation of a protocol  that clearly summarizes the project. If the protocol is poorly prepared and not followed, it is unlikely that the project will yield the desired results.

A research protocol should contain the following:

1. The protocol should adequately answer the research question. 
2. The research design must be sound enough to yield the expected knowledge. 
3. It should provide enough detail (methodology) that can allow another investigator to do the study and arrive at comparable conclusions. 
4. The proposed number of participants is reasonably justified and the scientific design is adequately described.

The key points

• What is the question? (Hypothesis)
• What is it to be investigated?
•  Why    is          the       study   important?       (Significance)
•  Where  and      when   it          will      take     place?
•   What   is the   methodology? (Procedures     and      methods          to         be used).
•   How    are       you      going   to         do        it?     (Research        design)            
•   Proposed time table and budget
•   Resources required  (technical, scientific and financial)

Methods and Materials 

 It should describe in detail the ‘Where’, ‘Who’, ‘How’ the research will be conducted. It explains the study design and procedures and techniques used to achieve the proposed objectives. It defines the variables and demonstrates in detail how the variables will be measured. It details the proposed methodology for data gathering and processing. 

Methodology composes an important part of the protocol. It assures that the hypothesis will be confirmed or rejected. It also refers to a thorough strategy to attain the objectives.

 The methods and materials are divided into various subheadings: 

a) study design (cross-sectional, case-control, intervention study, etc.): Proper explanation should be given as to why a particular design was chosen (on the basis of proposed objectives and availability of resources). A study design is in fact the researcher’s general plan to acquire the answer (s) to the hypothesis being tested. Here, strategies will be applied to develop balanced, correct, objective and meaningful information. It explains the methods that will be used to collect and analyze data.

Proper selection of the study design is important to attain reliable and valid scientific results. Ethics, logistic concerns, economic features and scientific thoroughness will determine the design of the study. The chief concern is given to the legality of the results including potential bias issues. Randomized controlled clinical trial is the best to document a causal relationship between an exposure and its outcome.

 b) study population (study subjects) Where are you going to do the research and who is the study population (why doing research in this place and why selecting this population?). It describes in detail about the study subjects, all aspects of the selection procedure and sample size calculation. Proper definition of eligibility, inclusion, exclusion and discontinuation criteria of the study subjects should be stated.

 c) sample size Sample size calculation is recommended for economical and ethical reasons. The calculation of the sample size must be explained. The sampling technique should be mentioned, 

e.g., randomization that will be used in order to obtain a representative sample for your target population. 

Each step involved in the recruitment of the study subjects should be described according to the selection criteria (inclusion and exclusion criteria).

 “Informed consent” should be mentioned (Permission granted in full knowledge of the possible consequences).

 d) Proposed intervention Full description of proposed intervention should be given. 

Here, all the activities and actions should be recorded and thoroughly explained in their order of occurrence.  

•When using    drugs,  both scientific and brandname should be mentioned followed by the name of the manufacturing company, city, and country. Drug route, dosage, frequency of administration, and total duration of treatment with the drug should be mentioned.

•When using apparatus its name should be given followed by the name of the manufacturer, city and country.

 e) Data collection methods, instruments used

Data collection tools are:

•Retrospective data     (medical          records)          

•Questionnaires                      

•Interviews (Structured,     Semi-Structured)

•Laboratory  test     

•Clinical  examinations  

•Description    of   instruments,     tools    used     for       data     collection, as well as the methods used to test the validity and reliability of the instrument should be provided 

Benefits 

A suitably drafted protocol has the following advantages

• ·         Allows the researcher to plan and review the project's steps.
• ·         Serves as a guide throughout the research.
• ·         Idea of time and budget estimates.

R    References

Protocol Writing in Clinical Research Journal of Clinical and Diagnostic Research. 2016 Nov, Vol-10 (11)

Penicillin Fermentation- Microorganisms

Penicillin was active against Gram positive bacteria, Nocardia, and Actinomycetes, but not against most Gram negative bacteria. It interferes with cell wall synthesis of actively growing sensitive organisms. It mainly inhibits the cross linking steps of peptidoglycan synthesis in the cell wall.

Structure of Penicillin

Penicillin is a group of compounds having common basic nucleus, 6-amino penicillanic acid (6-APA).  6-APA contains two rings- β-lactam &semithiazolidine ring.

                               

Penicillin F (2-pentenyl penicillin) was the original Penicillin obtained in more amount with P. notatum. Later, when P. chrysogenum was isolated and employed in fermentation, Penicillin K was obtained majorly along with dihydropenicillin F in the fermentation broth. With the addition of Corn steep liquor (CSL) as precursor, Penicillin G or benzyl penicillin was the major product in the mixture. It had better pharmacological properties and better stability.  

Thus penicillin is a group of compounds and the major compound depends on the microorganism and the media employed for fermentation.

Penicillins are of two different types,

i Natural Penicillin

ii Synthetic Penicillin

Natural penicillin is directly harvested from the Penicillium mold (P. notatum or P. Chrysogenum). 

Synthetic penicillin consists of the basic Penicillin nucleus (6-APA), but with new side chains that change properties of natural penicillin. Eg., Ampicillin, Methicillin, Penicillin V, etc.  These Penicillins could be produced by fermentation (biosynthetic) or chemical treatment (semi-synthetic).

Penicillin Fermentation -Microorganisms

Penicillium species are used in the Penicillin fermentation- P. notatum & P. chrysogenum.

P. notatum was used initially in stationary mat culture, however the strain was highly unstable and the yield variable. Stationary fermentation was replaced by submerged fermentation methods and P. notatum was found unsuitable.

    

                        P. chrysogenum                                     P. notatum

In 1943, P. chrysogenum was isolated from moldy fruit and was found as a high yielding strain and better suited for submerged fermentation. Descendants of P. chrysogenum is now widely used as production strain.

Strain development methods such as mutagenesis (x-ray/uv/alkylating agents, nitrosoguanidine), genetic recombination, protoplast fusion technique were employed to obtain high yielding variety- - P. chrysogenum NRRl 1951 Wis Q176 strain.

Fleming’s isolate, P. notatum gave a yield of around 2 IU/ml whereas P. chrysogenum NRRl 1951 WIS Q176 strain and its descendants yielded around > 85000 IU/ml after strain improvement. Such is the power of strain improvement techniques.

After strain improvement the production strains are carefully maintained by different preservation techniques like,

1. A spore suspension may be mixed with a sterile, finely separated inert support like soil or sand and then desiccated.

2. The spore suspension can be stored under liquid nitrogen (-196°C) i.e. in a frozen state.

3. The spore suspension can be lyophilized in appropriate media.

Along with improved yield, other properties were also modified, such as pigment production. P. chrysogenum produced yellow water soluble pigment which gave a yellow tint to the final preparations. With mutation and selection, strains that do not produce the pigment but produced penicillin in high yields could be selected.

Neisseria gonorrhoeae - pathogenicity, laboratory diagnosis and prevention of diseases

 The name Gonorrhea is derived from Greek words- Gonos (seed) rhoia (flow) - Describes a condition in which semen flows from the male organ without erection

 

Pathogenesis 

• Causes disease only in humans. Transmitted sexually both in males and females.

    Once inside the body the gonococci attach to the microvilli of mucosal cells by means of pili and protein II, which function as adhesins.  This attachment prevents the bacteria from being washed away by normal cervical and vaginal discharges or by the flow of urine  They are then phagocytosed by the mucosal cells and  transported through the intercellular spaces and subepithelial tissue. 

Incubation period of 2-8 days.

Phagocytes, such as neutrophils, also may contain gonococci inside vesicles. Because the gonococci are intracellular at this time, the host’s defenses have little effect on the bacteria. Following penetration of the bacteria, the host tissue responds locally by the accumulation of mast cells, more PMNs (polymorphonuceleocytes), and anitbody-secreting plasma cells. These cells are later replaced by fibrous tissue that may lead to urethral closing, or stricture, in males

Gonococci causes both localized infections, usually in the genital tract, and disseminated infections – Mainly 

1. Gonorrhea & Pelvic inflammatory disease (PID).

2. Neonatal conjunctivitis (ophthalmia neonatorum) 

Gonorrhea  

Gonorrhoea in men is characterized primarily by urethritis with mucopurulent discharge containing gonococci in large numbers, accompanied by dysuria (painful urination). Chronic urethritis lead to stricture formation. Other complications include epididymitis, prostatitis (painful condition that involves inflammation of the prostate), peri-urethral abscesses and “water can perineum” with multiple discharging sinuses.

:

(Epididymitis - inflammation of the epididymis, which is a tube located at the back of the testicles that stores and carries sperm

Stricture -narrowing of the urethra which restricts or slows the flow of urine in)

 

Gonorrhoea in women, infection is located primarily in the urethra and endocervix (cervicitis), causing a purulent vaginal discharge and inter-menstrual bleeding. Vaginal mucosa is not usually affected due to acidic pH. Infection may extend to Bartholin’s glands ( glands located near the opening of the vagina which secrete mucus to lubricate the vagina), endometrium and fallopean tubes.  The most frequent complication in women is an ascending infection of the uterine tubes (Salphingitis (inflammation of the fallopian tubes) and Pelvic Inflammatory Disease), which can result in sterility or ectopic pregnancy as a result of scarring of tissues.

Rarely, peritonitis and peri-hepatic inflammation is seen (Fitz-Hugh-Curtis syndrome). Also, Proctitis (inflammation of the anus and the lining of the rectum), conjunctivitis (due to autoinoculation) and rarely, blood invasion leading to arthritis, ulcerative endocarditis, or meningitis

 Clinical disease is less severe in women -Asymptomatic carriage common in women. Asymptomatic carriage rare in men.

 

Non-venereal infections/ Disseminated infections- Disseminated gonococcal infections occurs via the blood stream.  Septicemia, infections of skin and joints in 1-3 % of women (arthralgia/ arthritis) - more common in women due to untreated symptomatic infections.

 

In newborns, gonococcal ophthalmia in the newborn, due to direct infection during passage through birth canal (vertical transmission)

Ophthalmia neonatorum/ gonococcal ophthalmia - An eye infection which may develop within 2/3 days of vaginal delivery, affects cornea and can cause blindness, purulent conjunctivitis, acquired at delivery. This was once a leading cause of blindness in many parts of the world (now controlled by the practice of administering 1% silver nitrate solution into the eyes of all newborns)

 

 

Opthalmia neonatorum

 

  

Laboratory diagnosis of Gonorrhoea

 Sample: Urethral/Cervical/Vaginal discharge

§  To obtain a urethral specimen, swab is inserted approximately 2cm in urethra and rotated gently before withdrawing.

§  If there is profuse urethral discharge in male, it can be collected without inserting the swab.

§  A few drops of first voided urine can be used in males, but the sensitivity is low compared to discharge.

Transport: Swabs collected for isolation of gonococci may be transported to the laboratory in modified Staurt’s or Amie’s charcoal transport media and held at room temperature until inoculated to culture media. Good recovery of gonococci is possible if swabs are cultured within 12 hours of collection.

 

 

• Gram Staining

For men, a gram-stained smear of urethral discharge (exudate) showing intracellular Gram-negative diplococci is diagnostic.

Women may carry normal vaginal flora such as Veillonella or occasional gram-negative coccobacilli, may resemble gonococci. In case of women use of fluorescent antibody techniques for identification to increase sensitivity and microscopy for specifity.

• Culture

Specimens inoculated on a pre-warmed plate, immediately after collection, if not possible, collect on charcoal impregnated swabs and transport to laboratory on appropriate medium.

In acute gonorrhea, Chocolate agar/Mueller Hinton agar inoculated with the sample, incubation at 35-36^oC, under 5-10% CO₂

In chronic cases, where mixed infection can be seen, use selective media like,  Thayer Martin Medium {Chocolate agar containing antibiotics - vancomycin, colistin, trimethoprim, and nystatin or  Modified Newyork City Medium (MNC) .

 

 Biochemical tests for Neisseria gonorrhoeae identification •

• ·         Oxidase Test: Positive
• ·         Ferments glucose but not maltose, sucrose or lactose
• ·         DNase Test: Negative
• ·         Beta-galactosidase (ONPG) Test: Negative

Serological Tests

• Precipitation, complement fixation test, passive agglutination, immunofluorescence, radioimmunoassay etc-
• Not useful for diagnosis- it becomes positive only some weeks after infection is established, can remain positive for months/years afterwards. Can show positive with meningococcal infection.
• Enzyme-linked immunosorbent assay (ELISA) is also used as a rapid test and is sensitive to gonorrhea.

• Molecular Diagnosis:
• PCR method or Nucleic Acid Amplification Tests (NAATs) to detect the presence of gonococcal nucleic acids in patient specimens- highly sensitive and specific.
• Gonorrhea nucleic acid amplification (NAAT) testing-detects DNA of the gonococci and is considered the optimal test for gonorrhea infection- on a urine sample or a swab taken from a site of potential infection

•  Treatment
• Initially sulphonamides and penicillin but there was rapid development and spreading of drug resistance especially by Penicillinase producing Neisseria gonorrhoeae.
• Now, Cefixime/Ceftriaxone or Ciprofloxacin with  Doxycycline treatment for 7 days/Erythromycn single oral dose

• Prophylaxis

The prevention of gonorrhea involves the use of safety measures and the immediate treatment of symptomatic patients and their contacts.

• ·         Early detection of cases
• ·         Contact tracing
• ·         Health education
• ·         No vaccination (no immunity even by clinical infection)