Microbiology World
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Microbiology World
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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
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Table of Content Page No. 4-5
Mother Medicine of Nature, Tulsi 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
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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% www.microbiologyworld.com
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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 www.microbiologyworld.com
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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. www.microbiologyworld.com
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“COCKROACHES ARE NASTY FIGHTING OTHER BUGS”
BUT
THEY
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HELP
IN
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 COCKROACHES”
INFECTIOUS
DISEASES;
SOLUTION
ARE
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 www.microbiologyworld.com
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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 www.microbiologyworld.com
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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 www.microbiologyworld.com
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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. Penicillinresistant 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 www.microbiologyworld.com
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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 www.microbiologyworld.com
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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. www.microbiologyworld.com
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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. www.microbiologyworld.com
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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).
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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. www.microbiologyworld.com
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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-2013508.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-tolimit-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 timsandle@btinternet.com
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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
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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 www.microbiologyworld.com
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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 Primers catch pairs with mutations (ASP - of allele specific primers; at the 3'OH end. LST - TaqMan probe specific for locus; LSP - specific primers for locus) 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. www.microbiologyworld.com
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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 . Probe pairs with 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 www.microbiologyworld.com
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Catch a microbe in one hour The Dutch company Innosieve Diagnostics has the best, most sensitive and fastest rapid method for your hygiene screening! Based on Solid Phase Cytometry (SPC). The Sieve-ID® Viable count PLUS assay allows a bio burden/ hygiene screening test within just one hour (60 minutes). After applying any given sample onto the microsieve surface and performing an easy handling staining procedure the robust MuScan device searches the microsieve surface for viable microbes. Catching the microbe is easy for the MuScan due to the generated fluorescence of the cell that indicates metabolism (viability) in cells. With a sensitivity of 0.3 cells and detection range between 1 and 20,000 cells per sample the ease of use has never been higher or more efficient. The dedicated sample volume depends on how you do your assay now. If you spread 0.5mL on a plate, apply this on the microsieve and see the microbes appear on your screen within 60 minutes. The microsieve can process volumes between 0.100mL and 100mL depending on the sample type. The Sieve-ID® Viable count PLUS test detects and enumerates all living microbes, including the viable but nonculturable (VBNC).
- Innosieve Diagnostics BV The Netherlands www.innosieve.com www.microbiologyworld.com
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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: www.microbiologyworld.com
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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 www.microbiologyworld.com
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Microbiology World
Jan – Feb 2014
ISSN 2350 - 8774
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. www.microbiologyworld.com
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Microbiology World
Jan – Feb 2014
ISSN 2350 - 8774
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 CysAsp-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. www.microbiologyworld.com
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Microbiology World
Jan – Feb 2014
ISSN 2350 - 8774
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. www.microbiologyworld.com
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Microbiology World
Jan – Feb 2014
ISSN 2350 - 8774
You can also send your articles to info@microbiologyworld.com or broneps1@gmail.com Selected ones will be published in our next issue of Mar-Apr 2014. Thanks, Microbiology World Team
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