Microbiology World Issue 3

Microbiology World Jan – Feb 2014 ISSN 2350 - 8774

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President Mobeen Syed, M.D.

King Endward Medical University Lahore

MSc. from ASD, BSc. from Punjab University

D-Lab from MIT MA USA

Vice-President Sudheer Kumar Aluru, Ph.D

Human Genetics, Sri Venkateswara University, India

HOD of Biology Department (Narayana Institutions)

Managing Director Dr. D K Acharya, Ph.D

Asst Prof., Biotech Dept.

A. M. Collage of Science, Management and Computer Technology, India

Chief Editor Mr. Sagar Aryal

Medical Microbiology (M.Sc), Nepal

Reviewers Mr. Samir Aga

Department of Immunological Diseases

Medical Technologist, Iraq

Mr. Saumyadip Sarkar

ELSEVIER Student Ambassador South Asia, Reed Elsevier (UK)

Ph.D Scholar (Human Genetics), India

Editors Dr. Sao Bang

Hanoi Medical University

Dean of Microbiology Department (Provincial Hospital)

Microbiology Specialist, Vietnam

Mr. Tankeshwar Acharya

Lecturer: Patan Academy of Health Sciences (PAHS)

Medical Microbiologist, Nepal

Mr. Avishekh Gautam Ph.D Scholar, Hallym University, South Korea

Medical Microbiologist, Nepal

Mr. Manish Thapaliya Lecturer: St. Xavier’s College Food Microbiologist, Nepal





Table of Content

Page No.

Mother Medicine of Nature, Tulsi 4-5

Cockroach brain can fight infections

against infectious diseases 6-9

The rise of antimicrobial resistant microorganisms 10-16

Adulteration in Food 17-18

The methods to detect point mutations

using real-time PCR 19-20

Catch a microbe in one hour 21

Book feature:

'Sterility, Sterilisation and Sterility Assurance for

Pharmaceuticals: Technology, Validation

And Current Regulations'. 22-24

Ferritin Nanocages to Encapsulate a Deliver

Photosensitizers for Efficient Photodynamic

Therapy against cancer 25-26





Mother Medicine of Nature, Tulsi

Tulsi is also known as “queen of plants”. In regards to its properties, it is regarded

as the mother of medicine of nature.

Medical plants are considered to be very rich sources of secondary metabolites and

oils which are of therapeutic importance. Ocimum sanctum, Tulsi is a plant with

enormous properties of curing and preventing diseases. It is regarded as deity in

Indian Subcontinents.

Different parts of Ocimum are used for medicinal purposes which includes flower,

fruits, stem and root. Various studies has been performed with Ocimum sanctum

for its antibacterial, antidiabetic, anti-inflammatory, anticipidemic, anticancer and

immunomodulatory, antipyretic, hypotensive and analgesic act.

Leaves are diaphoretic antiperiodic, they are also used in bronchitis, gastric and

hepatic disorders, and is also recommended for cough, malaise and in colds. Paste

of its leaves is applied on face to clear masks. The ursolic acid present in leaves

return elasticity and remove wrinkles. Leaf juice of Tulsi along with triphala is

used as in eye tonic and is recommended for glaucoma, cataract, chronic

conjunctivitis and other diseases associated with eyes.

Oil extracted from flowers is used in skin diseases and ring-worm infections. Oil of

Tulsi is used in skin diseases and ring worm infections. Oil of Tulsi is anticidal and

larvacidal.

Main constituents of Tulsi Oil

β-bisabolene = 13% - 15%

methyl chavicol = 3% - 19%

eugenol = 4% - 9%

(E) - α – bisabolene = 4% - 7%





Invitro-antifungal activities was observed against Candida species when oil from

Ocimum gratissimum was used. Ocimum also shows antibacterial functions

against bacteria like Klebsiella, E.coli, Proteus, Staph. and V. cholera.

Antiviral properties against DNA virus ( Herpes Viruses (HSV)), Adenovirus

(ADV) and Hepatitis B Virus.

Ocimum spp along with pepper, turmeric and onion is prophylactic against malaria.

Eugenol is the main constituent and is responsible for its repellent property. It

lowers the uric acid level and hence considered as potential anti-inflammatory

agent. It also helps to mobilize mucus in bronchitis and asthma. Tulsi, itself is a

good source of antioxidant therefore shows the properties of protection against free

radical induced damage. It also shows the stimulatory effect on physiological

pathways of insulin production. Ethanolic extract of Ocimum sanctum mediated a

significant reduction in tumor cell size.

Method of extraction of antimicrobial property of Tulsi.

1. 1gm of extract is dissolved in 10ml of dimethylformamide to obtain a 10%

concentration of extract.

2. 1ml of extract is then transferred to a sterilized test tube and labelled as 10%.

3. The remaining 9ml of the extract is then diluted further with

dimehylformamide to obtain seven different concentrations.

Cup and plate method are used to determine the zone of inhibition.

3 circular wells that could incorporate 3 different volumes (20ul, 30ul and 50ul) of

test agent (Tulsi extract) are cut in the agar plates using a template.

- Neelam Sharma

M.Sc. Microbiology, St. Xavier’s College

Kathmandu, Nepal





Cockroach brain can fight infections against infectious

diseases

The nuisance of antimicrobial resistance creates great havoc in the therapeutic

management globally. Millions of peoples dying from untreatable infections due to

the worsening trends of antimicrobial resistance that reflects that we are again

leading towards a pre antibiotics era. Thus, there is a dire need of newer, effective

and safer antibiotics. New antimicrobial agents are urgently needed to meet the

challenges posed by the re-emergence of infectious diseases. Pakistan even among

the third world countries is still under the grip of infectious diseases load and yet

no fruitful therapeutic management. The search for new antibacterial compounds

from novel natural sources is a vital research area and an area of interest all over

the world for leading scientific community. Animals living in a germ-infested

environment could serve as a potent source of antimicrobial activity.

Among the different animals, like insects represent 80% of all

fauna and the widest spread group with in animal’s kingdom. As we all know that

insects often live in unsanitary conditions, so it is not surprising that they produce

their own antimicrobial compounds to make them protected against the attack of

bugs. Our curiosity has now led the scientists to ponder over the role of cockroach

and locust and many other bugs. Some have a notion that probably these insect

creatures possess antimicrobial peptides and other substances in their brain tissues

that could have a killing effect against nasty pathogens.





“COCKROACHES ARE NASTY BUT THEY COULD HELP IN

FIGHTING OTHER BUGS”

Cockroach lives in unsanitary and unhygienic environments, so they kill different

bacteria even superbugs. It is therefore logical that they have developed ways of

protecting themselves was they must have a potent defense against that superbugs.

Logically speaking, if they did not

have antimicrobial activity so they

could not have ability to survive

dirty, dank, rotting environments in

which they often live. Cockroaches

can also thrive in spotlessly clean

homes and hospitals rooms, carrying

infection with them when they

forage in food storage areas and

rubbish bins. Even worse, they

secret chemicals that can provoke

allergies and asthma, tying up the immune system so, that it cannot respond to

disease and this smart way of defense mechanism allows the bugs to survive in the

most dirty places.

“THOUSANDS OF INFECTIOUS DISEASES; SOLUTION ARE

COCKROACHES”

Few brief researches have been conducted in the world that supports the idea that

probably brain has the ability to combat against infectious agents. According to

one of the research studies, cockroaches evolve to protect themselves against

micro-organisms by their vital part i.e. brain which controls all the activities of the

body so the crude extracts of cockroach’s muscles, ganglia and fat body and also

haemolymph produce to bactericidal effects. In contrast, lysates of locust ganglia

(head and thoraciac) and cockroach exhibited powerful antibiotics properties 90%

bactericidal as compared to other bugs. Present researchers found that the





rudimentary brain of cockroach produce variety of potent antimicrobial substances

that are effective against some bacterial pathogens E.coli and MRSA (Methicillin

resistant staphylococcus aureus). They could potentially lead to new antibiotics

without the unwanted side effects of drugs in human.

E. coli is a digestive tract bacterium of human and animals. Many strains of E. coli

are harmless but some can cause severe diseases like bloody diarrohea, anemia or

kidney failure and other strains E. coli can cause urinary tract infections or other

which can lead to death. Theses organisms can also spread from person’s hands to

other people or via objects or articles. Many antibiotics are recommended to treat

E.coli infections but mostly E .coli shows resistance against conventionally

employed antibiotics. Similarly, Staph.aureus is a bacterium commonly called

(Methicillin resistant Staph. aureus) MRSA. It

causes mild infections on the skin like sores or

boils. But it can also cause more serious skin,

lungs and urinary tract infections, soft tissue

infections, bacteremia, endocarditis, pneumonia,

bone and joints and central nervous system

(CNS) infections. Some of them are really

serious and also sometimes if unmanaged so

leads towards life threatening diseases. The emergence of antibiotics resistant

forms of pathogenic S.aureus (MRSA) is a worldwide problem in clinical

microbiology.

“COCKROACH’S BRAIN COULD ONE DAY SAVE YOUR LIFE”.

One of the research piece of work

highlighted that atleast nine molecules

present in the brain of cockroach which show

90% toxic for mostly bacteria specially

MRSA and E. coli which are more powerful

than others and also harmful for drug





resistant bacteria but without the unwanted side effects of drugs on human body.

So extensive research is in the next few years are needed to approach or realize

these expectations for the welfare of mankind. We hope that the discovery of

antimicrobial activity in the cockroach brain will stimulate research in finding

antimicrobials from unusual sources and has potential for the development of novel

antibiotics.

“ BRAINS BEHIND THE BRAIN”

This question about this new development has also been raised in many brains and

working on this topic has also started in many areas where different microbiologist

and researchers are trying to enhance the human efforts against different

pathogens. In Pakistan AKU, has done few aspects of such work. Moreover, our

research laboratory at Federal Urdu University, Department of Microbiology has

also initiated the work to trace out the antimicrobial substances from brain and

apply them against infectious agents.

Realizing the emergence of antibiotic

resistance among pathogens, the entire

is seriously thinking about exploring

natural substances and other products

that could play their role in treating

infections. Our endeavor is in the same

way to find out certain peptides or

molecules if isolated they could be

good candidates for pharmaceutical

industry to design future antibiotics.

- Miss Aleena Shahid, BS research student at Department of

Microbiology, FUUAST, Pakistan

- Sikandar K. Sherwani, Faculty member and BS supervisor of

projects at FUUAST, Pakistan





The rise of antimicrobial resistant microorganisms

Introduction

This article considers the risk posed by the emergence of antimicrobial resistant

microorganisms and the challenges this poses for the world's medical services. The

article also makes reference to some of the current initiatives which form part of

the quest for antimicrobial alternatives.

The current status

An antibacterial is an agent that inhibits bacterial growth or kills bacteria. The term

is often used synonymously with the term antibiotic(s) (1). Antibiotics have played

a key role, in improving life-expectancy around the world since the 1940s. The

first wave on antibiotics were beta-lactam antibacterials, which include the

penicillins (produced by fungi in the genus Penicillium), the cephalosporins, and

the carbapenems.

Humans face the very real risk of a future without antibiotics, a world of

plummeting life expectancy where people die from diseases easily treatable today.

One concern for the future is the re-emergence of classical diseases that we had

thought banished to history, for example tuberculosis. The other worry is that

without effective antibiotics, we will not any longer be able to conduct the many

types of modern medicine that lead to immunosuppression. These include therapies

for autoimmune disorders and cancer treatments.

Furthermore, many routine surgeries may also become too dangerous to perform

owing to the risk of untreatable infection.

The concept of a ‘post-antibiotic era’, where common infections can no longer be

successfully treated, has been worrying microbiologists since the early 1990s. At

that time, resistance amongst Gram-positive bacteria was rising rapidly. Penicillin-

resistant pneumococci were widespread internationally and vancomycin-resistant

enterococci were also circulating in hospital specialist units. Methicillin-resistant

Staphylococcus aureus (MRSA) were relatively uncommon in serious infections at





the start of the 1990s; however, these have since proliferated since the early 2000s.

To add to this catalogue, in 2013 it was reported that gonorrhoea is becoming

resistant to most antimicrobials.

The situation not only poses increased risks to patients due to the lack of

availability of effective antibiotics, there are also risks from the antibiotics

themselves. When first-line and then second-line antibiotic treatment options are

limited by resistance or are unavailable, healthcare providers have no choice but to

use antibiotics that may be more toxic to the patient and frequently more expensive

and less effective.

The phenomenon of resistance

Antimicrobial resistance describes the ability of a micro-organism to resist the

action of antimicrobial drugs. In a few instances some microorganisms are

naturally resistant to particular antimicrobial agents; however, a more common

problem is when microorganisms that are normally susceptible to the action of

particular antimicrobial agents become resistant. The resistance often arises as a

result of changes in the microorganism's genes (a spontaneous or induced genetic

mutation). In some cases, the genes causing resistance can be transferred between

different strains of microorganism (horizontal gene transfer via conjugation,

transduction, or transformation). When the latter happens the recipient organisms

will also become resistant.

Many antibiotic resistance genes reside on transmissible plasmids (a small DNA

molecule that is physically separate from, and can replicate independently of,

chromosomal DNA within a cell), facilitating their transfer (2). Exposure to an

antibiotic naturally selects for the survival of the organisms with the genes for

resistance. In this way, a gene for antibiotic resistance may readily spread through

an ecosystem of bacteria.

The causes of growing resistance

The causes of antimicrobial resistance include the over-prescribing by doctors,

often for conditions which do not require antibiotics, which causes the





effectiveness of these drugs to diminish. Antibiotics are the most commonly

prescribed drugs in medicine, and 50 percent of prescribed antibiotics are either not

needed or not effective as prescribed (3). In relation to this, a study published in

the journal JAMA Internal Medicine, in 2014, has found no letup in the over-use

(and some would say 'misuse') of antibiotics. The research found that the U.S.

prescribing rate for adults with sore throat held steady at around 60 percent from

1997 to 2010. But only around 10 percent of adults with sore throat are infected

with group A Streptococcus—the only common cause of the symptom requiring

antibiotics (4).

Another reason is environmental. Antibiotics have been polluting the environment

since their introduction through human waste (medication, farming), animals, and

the pharmaceutical industry. Along with antibiotic waste, resistant bacteria follow,

thus introducing antibiotic-resistant bacteria into the environment (5).

A further reason relates to the alarming decrease in antibiotic research and

development, with only four pharmaceutical companies working on antibiotics

today, compared with eighteen companies twenty years ago.

Actions being taken

In Europe and the U.S.A., one action to slowdown the growth of antibiotic

resistance has been to place restrictions on the use of antibiotics in farming and

agriculture. For example, in 2013 the U.S. Food and Drug Administration (FDA)

laid out a plan so that farmers will no longer use antibiotics to fatten up animals

(6). The Agency argues instead that good farm management, bio-security, and

animal husbandry systems underpin the health and welfare of food-producing

animals. When applied appropriately they minimize disease, reduce susceptibility

to bacterial disease and minimize the need for antibiotic use in animals.

Many countries have initiated antimicrobial stewardship programmes, designed to

reduce and focus the use of antibiotics. This is because indiscriminate or

inappropriate use of antibiotics is a key driver in the spread of antibiotic resistance.

Many countries have put in place an extensive range of guidance, education, tools

and initiatives to promote the responsible use of antibiotics in patients.





One reason for the misuse of antibiotics is that clinicians often do not know the

particular cause of an illness when a patient is first admitted. Understanding the

disease quickly will ensure that the correct antibiotic is used. This will soon be

possible through genomic technologies. Such technologies have the potential to

provide a valuable means to improve appropriate, prompt, patient treatment. In the

next few years, whole genome sequencing and other diagnostic technologies will

move from research laboratories into widespread use, enabling rapid identification

of bacteria pathogens and their genetic potential for drug resistance. This will help

the early tailoring of treatment, benefitting both the patient and helping the

conservation of antibiotics. Technologies like whole genome sequencing will also

increase the ability to investigate the epidemiology of bacterial outbreaks.

Ultimately, new drug discovery is the only way to overcome the rise of

antimicrobial resistance microorganisms. This is a slow process. The discovery and

development of new drugs takes time (about 10 to 15 years) and a barrier to

developing new antibiotics is their relatively low commercial return on investment,

relative to investments in other therapeutic areas. Barriers to new drug discovery

include:

The scientific difficulty of finding new agents,

The risk of inadequate return on investment given that duration of drug use

is limited compared to drugs for chronic conditions,

Concerns over the cost and complexity of the regulatory approval process,

Uncertainty about the regulatory environment for new antimicrobials.

There are some signals that new antimicrobial drugs could be emerging. For

example, in a paper by Geller et al (2013), a new bacterial-killing chemical is

described (7). The new antibacterial agent is called a PPMO and it appears to

function as well or better than many existing antibacterial chemicals. PPMO is an

acronym for a peptide-conjugated phosphorodiamidate morpholino oligomer. The

chemical is a synthetic analogue of DNA or RNA that has the ability to silence the

expression of specific genes within bacterial cells. PPMOs are completely

synthesized in the laboratory.





In animal studies, one form of PPMO showed significant control of two strains of

the bacteria Acinetobacter. This is a group of bacteria of global concern.

Acinetobacter are widely distributed in nature, and commonly occur in soil. They

can survive on moist and dry surfaces, including in a hospital environment. Some

strains have been isolated from foodstuffs. In drinking water. In

immunocompromised individuals, several Acinetobacter can cause life-threatening

infections. Such species also exhibit a relatively broad degree of antibiotic

resistance.

Various studies are being undertaken by Kenneth Keiler, an associate professor of

biochemistry and molecular biology at Penn State (USA). Here, the research team

are examining forty-six previously untested molecules as potential antibiotics.

Each of these chemicals targets and disrupts an important step in the process of

protein synthesis in bacteria, thereby making the bacteria incapable of replicating.

Essentially this stops growth and therefore an infection from spreading.

Although there are forty-six potential chemicals, the researchers began by testing

around 663,000 different molecules against a strain of Escherichia coli bacteria

and monitored how the chemicals affecting the growth and survival of the

bacterium (8).

Meanwhile, different parts of the globe are being studied for new sources of

antibiotics. This includes the depths of the oceans. Here, geophysicists, digging

127 meters below the Pacific Ocean floor into 100 million year-old sediments,

discovered several types fungi. Some of the fungi are closely related to the fungus

Penicillium. The fungi were isolated from sediments collected on a South Pacific

drilling expedition.

Taking another approach, some scientists argue that adding silver to antibiotics can

increase their effectiveness. Writing in Science Translational Medicine in 2013,

researchers have explained the cellular processes by which the precious metal

weakens bacteria and makes them more susceptible to antibiotics (9).





The ultimate goal

In order to address the problem of antimicrobial resistance, the world society needs

to reach the point where:

Good infection prevention and control measures to help prevent infections

occurring become the norm in all sectors of human and animal health.

Infections can be diagnosed quickly and the right treatment used.

Patients and animal keepers fully understand the importance of antibiotic

treatment

regimens and adhere to them. This requires improving professional

education, training and public engagement to improve clinical practice and

promote wider understanding of the need for more sustainable use of

antibiotics.

Surveillance is in place which quickly identifies new threats or changing

patterns in

resistance.

There is a sustainable supply of new, effective antimicrobials. This can only

be achieved through better collaboration between research councils,

academia, industry and others; and by encouraging greater public-private

investment in the discovery and development of a sustainable supply of

effective new antimicrobials, rapid diagnostics, and complementary tools for

use in health, social care, and veterinary systems.

Conclusion

There are few public health issues of greater importance than antimicrobial

resistance in terms of impact on society. This article has outlined how and why

antimicrobial resistance is a problem. The article has also considered some of the

root causes and has considered the steps necessary if human society is to overcome

this serious problem.

Microorganisms will inevitably find ways of resisting antibiotics; this is why

aggressive action is needed now to keep new resistance from developing and to

prevent the resistance that already exists from spreading.





References

1. Waksman, S.A. (1947). "What Is an Antibiotic or an Antibiotic Substance?".

Mycologia 39 (5): 565–569.

2. Lipps G (Ed.). (2008). Plasmids: Current Research and Future Trends.

Caister Academic Press

3. CDC (2013) Threat Report 2013, U.S. Centers for Disease Control and

Prevention, CDC, Washington, USA at:

http://www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats-2013-

508.pdf

4. Barnett ML, Linder JA. Antibiotic Prescribing to Adults With Sore Throat in

the United States, 1997-2010. JAMA Intern Med. 2014;174(1):138-140

5. Martinez, J. L., & Olivares, J. (2012). Environmental Pollution By

Antibiotic Resistance Genes. In P. L. Keen, & M. H. Montforts,

Antimicrobial Resistance in the Environment (pp. 151- 171). Hoboken, N.J.:

John Wiley & Sons

6. Sandle, T. (2013) ' U.S. aims to limit antibiotics for farm animals', Digital

Journal at: http://www.digitaljournal.com/news/environment/usa-aims-to-

limit-antibiotics-for-farm-animals/article/364044#ixzz2rWki4TkV

7. Geller BL, Marshall-Batty K, Schnell FJ, McKnight MM, Iversen PL,

Greenberg DE. (2013) Gene-silencing antisense oligomers inhibit

acinetobacter growth in vitro and in vivo, J Infect Dis.208(10):1553-60

8. Ramadoss, N.S., Alumasa, J.N., Cheng, L. et al (2013) Small molecule

inhibitors of trans-translation have broad-spectrum antibiotic activity, PNAS;

doi:10.1073/pnas.1302816110

9. Morones-Ramirez, J.R., Winkler, J.A., Spina, C.S., Collins, J.J. (2013)

Silver Enhances Antibiotic Activity Against Gram-Negative Bacteria, Sci

Transl Med; 5:190ra81

- Tim Sandle, PhD

[email protected]





Adulteration in Food

‘Adult your age, not your food’… I remember my first expression when I saw one

of my seniors jotting it down on the paper for some sort of food adulteration

project. I was completely blown away by the theme the proverb carried. It then

forced me to think more about the food products and its adulteration leading to

search some facts about the food which I have tried to summarize here.

Food adulteration might not have resulted if the consumers hadn’t have desired

higher commodity at low price. It gave an opportunity to the sellers to add different

adulterants in the food. If I have to say then it is terribly risky to consume any sort

of market foods available as food-adulterants has been synonymous these days.

Milk, the vital part of our nutrition, is adulterated the most. Whether it is to

increase the shelf life of the milk or to increase the carbohydrate content, various

chemicals are used. Table sugar is added in the milk to increase the carbohydrate

content so that milk can then be adulterated with the water which will not be

detected during the lactometer test. Starch is generally added in milk to increase

the solid content. Benzoic acid and Salicylic acid are added to milk to increase the

shelf life of milk. To increase the foamy nature of milk, soap is added. Though

formalin is considered to cause liver and kidney damage, it is added in the milk to

preserve the milk for long time. All these adulterants are not beneficial for the

health but then also they are being used in our milk which is a matter of great

concern.

Vegetables and fruits which are the main part of everybody meals, it is the center

for adulteration in every phase, starting from the farms and till it reaches to the

market for selling. To make them look fresh and attractive, wax coating is

generally practiced. Not only that but also they are dipped in chemicals for its long

term so- called fresh appearance. Copper sulphate, Malachite green, Rodamine- B

are some chemicals generally used for the purpose. Fatal infections in liver, kidney





damage, cancer are some aftermaths of consuming such chemical treated foods but

then also it is still in practice.

Pulse, another major part of our everyday meal is also prone to adulteration. In

order to lure the consumers with the shiny coating, various chemicals are used.

People are not aware about it but it has also been a matter of concern now.

Chili powder is often seen adulterated with the brick dust and turmeric powder

with the lead chromate and metanil yellow. These adulterants are very harmful for

the health but its use in food materials is still not under control. Sugar, salt are seen

adulterated with calcium carbonate which can be detected on the homely basis also

but some people are seen little bit ignorant in accepting the fact. Similarly parched

rice is adulterated with urea. Not only that kesari powder, metanil yellow color in

gram powder, coal tar dye in tea leaves, Sodium bicarbonate in Jaggery, saw dust

in coriander powder, cumin powder etc. are some examples of adulterations.

Food is the vital element of living organism without which we cannot think of the

survival. It therefore depicts its importance in our life. Adulterating the food even

after knowing its harmful outcome is a crime. Stoppage of adulteration practices

may not be an easy task but creating awareness among the people about these

practices is not that hard.

With the same motto, I along with four of my other friends took an opportunity in

using the platform of an exhibition for creating awareness among the people for

which we managed to gather some adulterated food samples, some of our findings

and lab results. The response was great. It had at least ignited a spark among the

visiting group of people in taking thee matter seriously. In the similar manner each

and every individuals need to be aware about food adulteration practices as ‘Health

is wealth’ and therefore we cannot compromise it with anything else.

Stay healthy, stay safe!!!

- Sachin Aryal

St. Xavier’s College, Kathmandu, Nepal





The methods to detect point mutations

using real-time PCR

1- Allele Specific real - time PCR:

Allele specific real - time PCR is one of the real-time PCR that primers are

optimized as well as the reaction conditions for the purpose of detection of mutant

that can detect alleles characteristic (allel - the different states of the same gene).

The only difference with one nucleotide is sufficient to alter the state of the

corresponding allele in the genome. To detect point mutations need to find

temperatures degree so extreme difference of one nucleotide at the 3 ' end of the

DNA template primer than enough to begin the process of primer pairs and

amplification cannot happen. Based on the signal fluorescence separates the

mutants and the

wild-type.

When designing

primers to detect

point mutations,

need to regard of

the final nucleotide

at the 3'OH end.

The difference of

this compared to the DNA nucleotide pattern often leads to primer cannot perform

amplification process. The reason is in the Taq polymerase enzyme activity. This

enzyme active towards additional nucleotides from 5 ' to 3' so it will be inserted

into the first nucleotide 3' end until the completion of the process of amplification.

A difference in the last three nucleotides at the 3 'end of DNA primer than molding

will inhibit this process. Take advantage of this feature, you're going to design

primer and optimization of real-time PCR reactions that distinguish point

mutations.

Primers catch pairs with mutations (ASP - of allele specific primers;

LST - TaqMan probe specific for locus; LSP - specific primers for locus)





In designing a complete novice pairs with mutations sequence, there is an error in

the first nucleotide from the 3' OH for mating with wild-type sequence is

sometimes capable of lasting no mutations in strains with lower rates. To remedy

this situation, the temperature can increase the temperature starts rising pairs of

primers for

specific products

or use more

probes coupled to

point mutations .

2 - Method TaqMan MGB real-time PCR ( real-time PCR with TaqMan is

attached red MGB - dihydrocyclopyrroloindole tripeptide minor groove

binder ) :

Real-time PCR mix in addition to the basic elements the two components are not

important to red fluoresce when in the presence of specific products amplified

from DNA translated as :

TaqMan probes, are oligonucleotides with complementary sequence but with an

additional specific sequences on the target DNA sequences and it is approx 24 -

30nu with the 5' starter attached with fluorescence reporter and the 3' end has

absorber quencher to adsorb the fluorescent light is emitted from the reporter.

Taq polymerase enzyme has activity 5' - 3 ' exonuclease will hydrolysis to

potentially cut off probe when it pairing on the mold DNA and inhibit the 3' end of

primer when enzyme lasting the primer for synthesis of complementary strand.

- Dr. Sao Bang

Ha Noi Medical University, Viet Nam

Probe pairs with mutations





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Book Feature

'Sterility, Sterilisation and Sterility Assurance for

Pharmaceuticals: Technology, Validation And

Current Regulations'.

A new book covering an entire range of different sterilization

methods, as well as exploring the related areas of sterility

assurance and the concept of sterility, has been published. The

book has been written by Dr. Tim Sandle, an experienced

pharmaceutical microbiologist.

The key features of the book are:

The main sterilization methods of physical removal, physical alteration and

inactivation

Discussions of medical devices, aseptically filled products and terminally

sterilized products

An examination of the bacterial, pyrogenic, and endotoxin risks to devices

and products.

Injections, infusions and pharmaceutical forms for application on eyes and on

mucous membranes must meet the requirement to be sterile. The most effective

means of reducing the risk of an infection is the provision of a sterile product

together with the complete prevention of microbial ingress up to and including the

time of administration to the patient. This includes using sterile items to

administer the drug (such as a sterile syringe and needle) and to administer the

drug under clean conditions, using trained medical or nursing practitioners. The

book explores the different ways by which sterile products are made, as well as

examining sterilisation methods.

In terms of the book's importance for pharmaceuticals, medical devices and

healthcare:





Failure to adequately control any microbial challenge associated within process or

product by robust sterilisation will result in a contaminated marketed product, with

potential harm to the patient. Sterilisation is therefore of great importance to

healthcare and the manufacturers of medical devices and pharmaceuticals.

Sterilisation can be taken to mean the use of a physical or chemical procedure to

destroy all microbial life, including highly resistant bacterial endospores. This

destruction of bacterial spores means that sterilisation is a complete process for the

destruction of life, unlike disinfection which refers to the reduction of a microbial

population by destruction or inactivation. Sterilisation can be divided into:

Physical removal: the complete removal of all microorganisms to achieve a

physical absence of microorganisms (such as filtration).

Physical alteration: including physical destruction, disintegration of

microorganisms. Altering, changing or deforming the physical cellular or

biochemical architecture to destroy all physiological functionality.

Inactivation: the permanent disruption of critical biochemical and

physiological properties, potential and the microorganisms propensity

(whether active or latent) to realize a clinical condition. Thus ensuring

impotency for generating an infection. For complete assurance of

inactivation the microorganisms must therefore be essentially ‘killed’ with

no residual metabolic activity.

From these important concepts, primary methods of sterilisation consist of the

following four main categories:

High temperature/pressure sterilisation (by dry heat or moist heat),

Chemical sterilisation (such as gassing using ethylene oxide),

Filtration,

Radiation sterilisation (such as gamma).

The book 'Sterility, sterilisation and sterility assurance for pharmaceuticals'

examines different means of rendering a product sterile by providing an overview

of sterilisation methods including heat, radiation and filtration. The book outlines

and discusses sterilisation technology and the biopharmaceutical manufacturing

process, including aseptic filling, as well as aspects of the design of containers and





packaging, as well as addressing the cleanroom environments in which products

are prepared. Consisting of 18 chapters, the book comprehensively covers sterility,

sterilisation and microorganisms; pyrogenicity and bacterial endotoxins; regulatory

requirements and good manufacturing practices; and gamma radiation. Later

chapters discuss e-beam; dry heat sterilisation; steam sterilisation; sterilisation by

gas; vapour sterilisation; and sterile filtration, before final chapters analyse

depyrogenation; cleanrooms; aseptic processing; media simulation; biological

indicators; sterility testing; auditing; and new sterilisation techniques.

The chapter list is:

Sterility, sterilization and microorganisms

Pyrogenicity and bacterial endotoxin

Regulatory requirements and Good Manufacturing Practices (GMP)

Gamma radiation

Electron beam processing

Dry heat sterilization

Steam sterilization

Gaseous sterilization

Hydrogen peroxide vapor sterilization

Sterilization by filtration

Other methods of sterilization

Depyrogenation and endotoxin

Cleanrooms, isolators and cleanroom technology

Aseptic processing and filling

Media simulation trials

Cleaning and disinfection of sterile processing facilities

Biological indicators

The Sterility Test

Investigating sterility test failures

Auditing sterilization processes and facilities.

The book has been published by Woodhead Publishing / Elsevier and is available

as a hardback and as an e-book.





Ferritin Nanocages to Encapsulate a Deliver

Photosensitizers for Efficient Photodynamic Therapy

against cancer

Photodynamic therapy is an emerging treatment modality that is under intensive

preclinical and clinical investigations for many diseases including cancer. Despite

the promise, there is a lack of a reliable drug delivery vehicle that can transport

photosensitizers (PSs) to tumors in a site specific manner. Previous efforts have

been focused on polymer or liposome based nanocarriers, which are usually

associated with a suboptimal PS loading rate and a large particle size.

Photodynamic Therapy (PDT) consists of three components, a photosensitizer,

light and oxygen. Photosensitizers are usually pharmacologically inactive in the

dark. When light at specific wavelength is applied PSs are activated, producing

reactive oxygen species (ROS) such as ‘O2, which are cytotoxic and capable of

killing nearby cells. Due to limited light penetration

PDT was first used in the clinic to treat superficial conditions such as vulgaris a

skin cancer. Limitation has changed due to methods that can deliver light to certain

internal organs. Optic fiber Emmitt’s laser that will be applied to illuminate the

tissue and to elicit PDT. PDT is also found to be effective in treating recurrence

prostate tumors after irradiation.

Here in report a surface-modified ferritin (FRT), a protein based nanoparticle can

serves as an efficient PS delivery vehicle. In particular research found that Cys-

Asp-Cys-Gly-Asp-Cys-PheCys (RGDC) - modified FRTs (RFRTS) can

encapsulate a large amount of zinc hexadecanfluorophthaolcyanine (ZnF16PC),a

potent hydrophobic photosensitizers and selectively deliver it to tumor to induce

efficient PDT against cancer. FRT is a major iron storage protein found in mast

living organisms including humans. Each FRT Nanocages is composed of 24

subunits self-assemble to form a cage-like nanostructure with external internal

diameter of 12 and 8 nm.





In vivo study with ZnF16 loaded RFRTs found a high tumor accumulation rate

(tumor to normal tissue ratio of 26.82± 4.07 at 24 hrs.), a good tumor inhibition

rate (83.64% on day 12) as well as minimal toxicity to the skin & other normal

tissues. All these features make FRT and its derivatives attractive new types of PS

carriers with great promise for selective against cancer.

The drug loading was achieved by ZnF16PC in DMSO into a RFRT solution in

0.01M PBS (pH 7.4) and after that, incubating at room temperature for 45 minutes.

The raw products were subjected to purification through a NAP-5 column to

remove unloaded ZnF16PC. Scientists found that 1mg of RFRTs can load upto

1.5mg of ZnF16PC , yielding a loading rate as high as 60%. For stability they used

formulation with a loading rate of 41.2% for the current investigation. The sizes of

the nanoparticles were studied by Atomic Force Microscopy (FAM) analysis.

PDT can target either tumor cells or tumor vasculature to cause damage. In first

mechanism PDT induces ‘O2 acts on tumor cell membrane or mitochondria to

cause necrosis or apoptosis. In next mechanism PDT cause vascular collapse and

embolization, terminating the supply of oxygen and nutrients to the tumor cells. In

the current study, ZnF16FC was delivered by RFRTsto both tumor vasculature &

U87MG tumor cells through RGD-integrin interactions. Hence both mechanisms

may have played a role in tumor destruction.

Over all, in this work RFRT nanoparticles are safe & efficient carriers for

ZnF16PC. The resulting conjugates can home to tumors through RGD-integrin

interactions and will light irradiations, induces photo toxicity to tumors while

leaving normal tissue unaffected. Boasting an extremely high as PS loading rate

and ultra-small particle size, this technology is expected to find widespread use in

PDT and holds great potential in clinical translation.

Key words:

RGD4C: Tumor Necrosis factor Fusion protein.

Ferritin: ubiquitous intracellular protein.

ZnF16PC: Zinc hexadecanfluoro-phthalocyanine.





You can also send your articles to

[email protected]

or

[email protected]

Selected ones will be published in

our next issue of Mar-Apr 2014.

Thanks,

Microbiology World Team



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Microbiology World Issue 3