2. Introduction – Aseptic techniques.
3. History of aseptic techniques.
4. Common aseptic techniques.
5. Antimicrobial agents.
6. Types of antimicrobial agents.
7. Use of antimicrobial agents in treatment of infectious diseases.
8. Effectiveness of antimicrobial agents.
9. Antimicrobial agents and their actions.
10. Antimicrobial therapy.
11. Challenges for the development of antimicrobial agents. `
INTRODUCTION –Aseptic techniques:
Aseptic technique is a set of specific practices and procedures performed
Under carefully controlled conditions with the goal of minimizing
Contamination by pathogens.
Purpose and Description:
Aseptic technique is such a technique which is employed to maximize and maintain asepsis, the absence of pathogenic organisms, in the clinical setting. The goals of aseptic technique is to protect patient from infection and to prevent the spread of pathogens. Often, practices that clean (remove dirt or other impurities), sanitize or disinfect are not sufficient to prevent infection.
The centers for disease control and prevention (CDC) estimates that over 27 million surgical procedures are performed in United States each year. Surgical site infections are the most common infections and can lead to longer hospital stays and increased costs to the patient and hospital.Aseptic technique is vital in reducing the morbidity and mortality associated with surgical infections.
Aseptic techniques are also widely used in laboratories in order to conduct all the experiments with proper results and prevent contamination to get into the culture.
In pharmacy aseptic techniques are practiced by technicians to prevent bacterial contamination and other impurities from getting into certain pharmaceutical products.
HISTORY OF ASEPTIC TECHNIQUE:
Ancient records shows that antiseptics date far back into his troy; the ancient Chinese ,Persians, Egyptians had methods for water sanitation and antisepsis for wounds.The ancient Greeks and Romans used silver vessels to store fresh liquids and wine. Settlers in the Australian outback put silverware and Pioneers of the American West put silver and copper coins in drinking water to keep it fresh and prevent algae; settlers in the Australian outback put silverware in drinking water for the same purpose. Mercuric chloride was used to prevent sepsis in wounds by Arabian physicians in the middle Ages. Hypocrite and iodine were introduced as a treatment for open wounds in 1825 and 1839, respectively.
In 1861 Louis Pasteur proved that microorganisms caused spoilage and could be transported via the air. He placed broth in flasks with long S-shaped necks, then boiled the broth and observed that no microorganism grew in the flasks. These experiments were the basis for the development of aseptic techniques. Pasteur showed that heat could kill microorganisms; this process was later named pasteurization.
Using the knowledge gained from Louis Pasteur, a scientist named Dr. Ignaz Semmelweis reduced the number of postpartum infections (puerperal sepsis) in the wards of Vienna’s lying-in hospitals by urging doctors to wash their hands between patients.
Bythe mid-nineteenth century, post-operative sepsis infection accounted for the death of almost half the patients who underwent major surgery. Later in 1860’s, an English surgeon named Joseph Lister heard about Pasteur’s work. He began soaking his surgical dressings in carbolic acid (phenol) because he had heard the previous year that carbolic acid had been used to treat sewage in Carlise and the fields that had been treated were now free of parasite-causing disease. This led to a dramatic decrease in the number of post-operational infections. Before the discovery of antisepsis by Lister, about 80% of surgical patients contracted gangrene. In 1870, Lister’s antiseptic methods were used by Germany during the Franco-Prussian war, where they saved the lives of many Prussian soldiers. Although Germany and several other countries followed Lister’s procedure of sterilization, England and America were still in opposition to his “germ theory.” The turning point for Lister came on October 26, 1877, when he had the opportunity to perform a simple knee operation (wiring a fractured kneecap, which entailed deliberate conversion of a simple fracture into a compound fracture), which often resulted in generalized infection and death. News of this operation was widely publicized; its success forced people to accept that his methods greatly added to the safety of operative surgery. The culmination of his emphasis on the principle of preventative medicine was the opening of the Institute of Preventative Medicine in 1891.These is a few of the reasons why Joseph Lister is often referred to as the “father of antiseptic surgery.
Paul Ehrlich, a German scientist, later advanced the idea of using chemicals to kill microorganisms by testing many more compounds. He eventually found a chemical that was successful against syphilis. Another scientist that had a significant impact on the field of sterilization was Ernst von Bergmann. He is credited with introducing steam sterilization unde pressure for treating instruments and all other medical equipment used for a surgical patient.
A famous surgeon from John Hopkins, William Stewart Halsted introduced sterile rubber gloves to the field of medicine when his hands became irritated from constant washing and antiseptics.
COMMON ASEPTIC TECHNIQUES:
1. ASEPTIC TECHNIQUES IN LABORATORIES.
2. ASEPTIC TECHNIQUES IN CLINICAL UNITS.
3. ASEPTIC TECHNIQUES IN PHARMACEUTICALS.
ASEPTIC TECHNIQUES IN LABORATORIES:
Aseptic technique requires the use of certain procedures in order to maintain pure cultures for growth within a laboratory setting. Following are some common aseptic practices:
1.The first rule of any lab experiment is no food or drink this is the best way to contaminate yourself as well as an experiment.So any lab equipment that could possibly be used for food should not be used for food preparation. This includes Bunsen Burners, refrigerators, or microwaves.
2. Before beginning any experiments all participants should wash their hands to reduce the number of bacteria that they bring into the lab. Experimenters should wash their hands before leaving the lab after an experiment as well.
3. Another preventative technique to limit contamination is to wipe down the lab bench before and after with a disinfectant, like 70% ethanol.
4.Proper lab coat along with gloves and mask should be worn in order to reduce the risk of leaving with microbes on your person as well as reducing the risk of bacteria into the lab with you.For these reasons, gloves and lab coats should be worn all the times.As our skin has it’s own bacteria everyone working with cultures should also wear clothes that completely cover their bodies these are either long pants and closed shoes.Long hair should be pulled back to reduce the risk catching it on fire.
5. Cross contamination can occur if experimenter is not careful to flame test tube cultures before taking samples.This is because the air in the lab also contains bacteria that contaminate results.By keeping the test tube at an angle you also reduce the risk of contamination by air.
6. Experimenters should also flame loops and stabs before culture sampling, between sampling of cultures, and after a sample is taken to reduce any contamination to the culture or the surrounding work area.
Sterile equipment’s: Sterile equipment’s are used in laboratories for performing experiments for this purpose a number of devices are utilized.
2. Oven (microwave).
4. Ethylene Oxide sterilizer.
5. Laminar flow biological safety cabinets.
A device like pressure-cooker which is used to sterilize media and equipment’s like testtube etc. Steam sterilization is carried out by the autoclave. It is operated at 121 degree Celsius and 15 pounds of pressure for 15 minutes.
2. Oven (microwave):
It is possible to sterilize items in microwave. Most of the laboratories sterilize glassware and pipettes with dry heat. It is usually operated at 160 -170 degree celcius for 2-3 hours.
3. UV- Radiations:
Around 260 nm is quite suitable but it does not penetrate glass, dirt films, water and other substances effectively.UV lamps are placed on the ceiling of rooms or in biological safety cabinets to sterilize the air and any exposed surface.
4. Ethylene Oxide Sterilizer:
Many heat sensitive items such as disposable plastics petri dishes and syringes are sterilized with ethylene oxide gas.EtO is both microbicidal and sporicidal. This process is carried out in special sterilizer somewhat like autoclave. A clean object can be sterilized for 5-8 hours at 38 degree celsius or 3-4 hours at 54 degree celsius when relative humidity is maintained at 40- 50% and EtO concentration at 70mg/lit.
5. Laminar flow biological safety cabinets:
These employ high efficiency particulate air filters (HEPA) to remove 99.97% of 0.3 micro- meter particles.This protect a worker from microorganisms being handled with in the cabinet and prevent contamination of room. They are also employed in pharmaceutical industries.
ASEPTIC TECHNIQUES IN CLINICAL UNITS:
Aseptic technique can be applied in any clinical setting.Pathogens may introduce infection to the patient through contact with the environment, personnel, or equipment.All patients are potentially vulnerable to infection, that can disturb body’s natural defences.Typical situations that call for aseptic measures includes surgery and the insertion of intravenous lines, urinary catheters, and drains.
‘Operating rooms’ strictly follows aseptic techniques because of direct and often extensive disruption of skin and underlying tissue.Aseptic technique helps to prevent or minimize post-operative infection.
A surgical scrub is also performed by members of the surgical team who will come into contact with the sterile field or sterile instruments and equipment.This procedure requires use of long- acting, powerful, antimicrobial soap on the hands and the forearms for a longer period of time than used for typical hand washing.CDC recommends at least 2- 5 minutes of scrubbing.
‘Isolation units’ are also a clinical setting that requires a high level of attention to aseptic technique.Isolation is use of physical separation and aseptic technique for a patient who has a contagious disease.For the patient with contagious disease, the goal of isolation is to prevent the spread of infection to others.
Other clinical settings outside the operating rooms generally don’t follow the strict rules, however avoiding the potential infection remains the goal in every clinical setting.
ASEPTIC TECHNIQUES IN PHARMACEUTICALS:
Pharmaceutical companies are involved in the production of drugs and medicines.In such kind of working areas aseptic techniques are practiced to maintain the quality and purity of drugs or medicines
Sterility is achieved with a flash-heating process (temperature between 91-146 degree celsius which retains morenutrients and uses less energy than conventional techniques.Pharmaceutical sterile processing includes use of clean rooms, bacteria retaining filters, dry or steam heat.
In pharmaceutical production; aseptic techniques will include mechanical aspects like making sure that the utilities, equipment and clean rooms are maintained and operated in such a way that aseptic surroundings in production areas is achieved.
Antimicrobial agents are defined as any chemical or biological agents that either destroy or inhibit the growth of microorganisms.
Antimicrobial products kill or slow the spread of microorganisms that include bacteria, viruses, protozoans, and fungi such as mold and mildew.
The U.S Environmental Protection Agency (EPA) regulates antimicrobial agents as pesticides, and the U.S. Food and Drug Administration (FDA) regulates antimicrobial drugs as drugs/ antiseptics. As pesticides, antimicrobial products are used on objects such as counter tops, toys, grocery, carts and hospital equipments.As antiseptics, antimicrobial products are used to treat or prevent diseases on people, pets, and other living things.
TYPES OF ANTIMICROBIAL AGENTS:
Antiseptics are microbicidal agents harmless enough that they can be applied to the skin and mucous membrane; should not be taken internaslly. Examples: mercurials, silver nitrate, iodine solution, alcohols and detergents.
Agents that kill microorganisms, but not their spores, it is not a safe aplication for living tissues; they are applied on inanimate objects such as tables, floors, utensils.
Examples: chlorine, hypochlorites, chlorine compounds and quaternary ammonium compounds.
Static agents used to inhibit the growth of microorganisms, most often in foods. If eaten should be nontoxic. Examples: calcium propionate, sodium benzoate, formaldehyde, nitrate, sulphur dioxide.
4. Chemotherapeutic agents:
Antimicrobial agents of synthetic origin useful in the treatment of microbial or viral disease.
Examples: sulphonilamides, isoniazid, ethambutol, AZT, chloramphenicol.
Antimicrobial agents produced by microorganisms that kill or inhibit other microorganisms.They can also be defined as any chemical of natural origin from any type of cell that has the ability to kill or inhibit the growth of other typr of cells. Examples: pencillin, streptomycin, erythromycin, tetracyclines, polymyxin and bacitracin.
ANTIMICROBIAL AGENTS USED IN THE TREATMENT OF INFECTOUS DISEASES:
The modern era of antimicrobial chemotherapy began in 1929 with Fleming’s discovery of the powerful bactericidal substance penicillin, and Domagk’s discoery in 1935 of synthetic chemicals (sulfonamides) with broad antimicrobial activity. In early 1940’s penicillin was isolated, purified and injected into experimental animals, where it was found to not only cure infections but also possess low toxicity for animals. This fact ushered into being the age of antibiotic chemotherapy and an intense search for antimicrobial agents of low toxicity that might be used to treat infectious diseases. The rapid isolation of streptomycin, chloramphenicol and tetracycline soon followed , and by the 1950’s, these and several other antibiotics were in clinical usage.Antibiotics may have a cidal or static effect on the range of microbes.The range of bacteria or other microorganisms that are affected by a certain antibiotic are expressed as its spectrum of action.Antibiotics effective against prokaryotes that kill or inhibit a wide range of Gram- postive and Gram- nagative bacteria are said to be broad spectrum.
EFFECTIVENESS OF ANTIMICROBIAL AGENTS:
Chemical agents used to treat diseases caused by microbes. There are three groups of antimicrobial agents: 1.Synthetic agents – produced in laboratories.
2. Natural agents – metabolic products produced by certain group of fungi and fungal- like bacteria that are antibacterial in action; commonly called antibiotics.
3. Semi-synthetic agents – derivatives of natural agents altered in laboratory by adding chemical groups to improve effectiveness. Criteria for the effectiveness of antimicrobial agents:
Criteria that determines the effectiveness of antimicrobial agents is as follows:
1. Selective toxicity – destroys or inhibits microbes without affecting host cells.
2. Broad spectrum – effective against a wide variety of organisms.
3. Non-mutagenic – does not induce development of resistant strains.
4. Soluble in body fluids – not easily broken down or excreted, to maintain constant and effective levels.
5. Stable in body fluids – not easily broken down or excreted, to maintain constant and effective levels.
6. Absorbed by tissues – to reach the site of infection.
7. Non-alergenic to host – should not cause adverse reactions in host.
8. Should not disturb host’s normal flora (organisms normally living in body) causing secondary infections produced by opportunists.Modes of action:
1.Interfere with microbe’s vital metabolic process that does not occur in host cells (incomprehensibility) Act by:
A. Competitive inhibition – competes with essential substrate to act with microbial enzyme.
B. Noncompetitive inhibition – reacts directly with enzyme.
2. Taregets structural/ metabolic diffrences between eukaryotic, prokaryotic cells.
3. Actionofantimicrobialagents – interferewith:
A. Metabolic pathways – production of an essential metabolite (by competitive inhibition)
B. The cell wall (murein) synthesis.
C. Cause damage to cell membrane.
D. Protein synthesis (enzymes).
E. Nucleic acid replication/transcription.
They produce metabolites, compete with the substrate of microbial enzyme acts with microbial enzyme by taking place of it’s substrate and thus affect the metabolic pathway.
Cell wall synthesis Inhibitors:
Cell wall synthesis inhibitors generally inhibit some step in the synthesis of bacterial peptidoglycan. Generally they exert selective toxicity against eubacteria because human cells lack cell wall.
Cell membrane Inhibitors:
They disorganize the structure or inhibit the function of bacterial membranes. The integrity of the cytoplasmic and outer membranes is vital to bacteria, and compounds that disorganize the membranes rapidly kill cells.However, due to similarities in phospholipids in eubacteria and eukaryotic membranes, this action is rarely specific enough permit these compounds to be used systemically.
Protein synthesis Inhibitors:
Many therapeutically useful antibiotics owe their action to the inhibition of some step in the complex process of translation. Their attack is always at one of the events occuring on the ribosome and rather than the stage of amino acid activation or attachment to a particular tRNA. Most have an affinity or specificity for 70S (as opposed to 80S) ribosomes, and they achieve their selective toxicity in this manner.
Some chemotherapeutic agents affect the synthesis of DNA or RNA, or can bind to DNA or RNA so that their messages cannot be read. Either case, of course, can block the growth of cells. The majority of these drugs are unselective, however, affect animal cells and bacterial cells alike and therefore have no therapeutic application.
ANTIMICROBIAL AGENTS AND THEIR ACTIONS:
a. Sulfonamides – Metabolic path way
1. Inhibits synthesis of folic acid by competitive inhibition.
2). Similar in structure to PABA – substrate for folic synthesis.
3). Sulfa drugs compete with PABA for active site on enzyme.
4). Sulfa drugs react with active site on enzyme.
5). Inhibits enzyme and folic acid synthesis.
b. Penicillin – Cell Wall Synthesis
1). Binds to & inhibits enzyme – penicillin-binding proteins.
2). Inhibits formation peptide (amino acid) cross bridges.
3). Inhibits formation bond between amino acid & muramic acid.
4). Weakens cell wall – lysis bacterial cell.
c. Erythomycin – Protein Synthesis
1). Binds to bacterial ribosome (protein)
2). Inhibits translocase – catalyzes movement codons (mRNA) on active site ribosome.
3).“Freezes” mRNA on ribosome.
4). Stops synthesis of protein (enzyme).
d. Tetracyclines– Protein Synthesis
1). Binds to bacterial ribosome.
2). Blocks attachment tRNA-amino acid to ribosome-mRNA.
3). Amino acid not added to a. a. chain (protein).
4). Stops protein synthesis.
e. Chloramphenicol – Protein Synthesis
1). Binds to bacterial ribosome – transferase.
2). Prevents transfer of a.a on tRNA to a.a. chain.
3). Stops protein synthesis.
f. Streptomycin – Protein Synthesis
1). Binds to active site on ribosome.
2). Distorts active site on ribosome _ misreading.
3). Wrong a.a. bonded together.
4). Nonsense protein (nonfunctional) protein produced.
g. Quinolones – DNA Synthesis
1). Inhibits DNA gyrase (produced by prokaryotic cells).
2).Prevents replications bacterial DNA.
h. Rifampin – RNA Synthesis
1). Inhibits RNA polymerase (produced by prokaryotic cells).
2). Prevents transcription (synthesis mRNA).
i. Polymixins – Damage Cytoplasmic Membrane
1). Structure – two solubilities
a). One end – water soluble (hydrophilic)
b). One end – lipid soluble
2). As absorbed across cell membrane:
a). Water soluble end – affinity for water soluble proteins, water in cytoplasm.
b). Lipid soluble end – affinity to phospholipid layers.
3).Causes cleavages in cell membrane.
4). Loss of cell contents.
All the examples given above are effective antimicrobial agents.
Antimicrobial therapy is the treatment of infectous disease using, typically, chemotherapeutic agents that either kill microbes or otherwise interfere with microbial growth
“Infectious disease claimed the lives of about one in every 100 U.S. residents per year as late as 1900 but only about one in every 300 in 1990. Although antimicrobial agents still don’t save all patients, they have drastically lowered the death rate from infectious disease. A period of increased infectious diseases could return, however, if patients and the medical community fail to protect the effectiveness of antimicrobial agents. As many pathogens develop resistance to available antimicrobial drugs, our ability to fight infectious diseases is dwindling.” In 1922, Alexander Fleming, a bacteriologist in London, had a cold. He was not one to waste a moment and consequently used his cold as an opportunity to do an experiment. He allowed a few drops of his nasal mucus to fall on a culture plate containing bacteria. He was excited to find some time later that the bacteria near the mucus had been dissolved away. Fleming showed that the antibacterial substance was an enzyme, which he named lysozyme lyso because of its capacity to lyse bacteria and zyme because it was an enzyme. Flemming found that tears are a rich source of lysozyme. Volunteers provided tears after they suffered a few squirts of lemonan ordeal by lemon. Fleming was disappointed to find that lysozyme was not effective against the most harmful bacteria. But seven years later, he did discover highly effective antibiotic penicillin. Complexity of antimicrobial therapy: Complex process of therapy can be explained by the following diagram:
In humans, there are three independent case reports on treatment of PCR-confirmed MAP infections in individual Crohn’s patients. In the first report an adolescent male was treated with clarithromycin (500 mg/day) and rifabutin (300 mg/day) for 32 months resulting in long term remission of his Crohn’s disease symptoms. In the second report a 21-year-old Canadian male was treated with clarithromycin and rifabutin (dosages not given) for 12 months with marked improvement both clinically and endoscopically. In the third report, a 63 year old male patient with long-standing Crohn’s disease experienced clinical remission of his symptoms after a six month course of treatment with clarithromycin (1,000 mg daily), rifabutin (300-450 mg daily), and levofloxacin (500 mg daily). The patient relapsed, however, after cessation of the antibiotics. Several therapeutic trials involving Crohn’s disease patients using antimycobacterial drugs yielded conflicting and controversial results.
CHALLENGES FOR THE DEVELOPMENT OF ANTIMICROBIAL AGENTS: Micro-organisms exist to survive. Even in the absence of antimicrobial agents, many have determinants of resistance that may be expressed phenotypically. With the advent of the antibiotic age, as more and more drugs were developed to treat serious infections, micro-organisms (particularly bacteria) rapidly developed resistance determinants to prevent their own demise.The most important determinants of resistance have been in the Gram-positive and Gram-negative bacteria. Among Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) have taxed researchers and pharmaceutical companies to develop new agents that are effective against these resistant strains. Among the Gram-negative bacteria, extended-spectrum beta-lactamase (ESBL) enzymes, carbapenemases (CREs) and the so-called amp-C enzymes that may be readily transferred between species of enterobacteriaceae and other facultative species have created multi-drug resistant organisms that are difficult to treat. Other resistance determinants have been seen in other clinically important bacterial species such as Neisseria gonorrhoeae, Clostridium difficile, Haemophilus influenzae and Mycobacterium tuberculosis. These issues have now spread to fungal agents of infection. A variety of modalities have been used to stem the tide of resistance. These include the development of niche compounds that target specific resistance determinants. Other approaches have been to find new targets for antimicrobial activity, use of combination agents that are effective against more than one target in the cell, or new delivery mechanism to maximize the concentration of antimicrobial agents at the site of infection without causing toxicity to the host. It is important that such new modalities have been proved effective for clinical therapy. Animal models and non-mammalian systems have been developed to determine if new agents will reach sufficient concentrations at infection sites to predict clinical efficacy without toxicity. It will also be key to consider antimicrobial stewardship as an important component of the continuing battle to prevent the development of antimicrobial resistance.
Microorganisms are present on all intimate surfaces creating possible contamination. In order to achieve the required goals aseptic techniques are utilized for the prevention of contamination. Aseptic techniques are actually precautionary measures taken to prevent contamination. Ancient records shows that aseptic techniques date far back in history Chinese, Persians, Egytptians, Greeks, Romans and Arabian physicians use antiseptics for different purposes. Louis Pasteur and several other scientists contributed to the basic development of aseptic techniques however, an English surgeon named Joseph Lister is often referred as ‘father of antiseptic surgery’. Aseptic techniques are widely used in laboratories, clinical units and pharmaceutical industries. In laboratories aseptic techniques are utilized to prevent contamination from an area of work, equipments, cultures and experimenter it’s self. Different devices like autoclave, oven, laminar biological safety cabinets etc are used to sterilize equipments, gloves and labcoats etc. Hospitals strictly follow aseptic techniques in clinical as well as surgical units to prevent infections caused by contamination (microorganisms). Pharmaceutical industries also apply certain aseptic methods to ensure the quality of medicines or drugs. Antimicrobial agents are those chemical agents that destroys or inhibit the growth of microorganisms. There are different types of antimicrobial agents which includes Antiseptics, Disinfectants, Preservatives, Chemotherapeutic agents and Antibiotics.Most of the antimicrobial agents are used to treat infectous diseases. Modern era of antimicrobial chemotherapy begins with the discovery of powerful bactericidal penicillin. After this wonderful discovery of Flemming a number of antibiotics were discovered for clinical usage. Antimicrobial agents are very effective because they are selectively toxic, non mutagenic, soluble in body fluids, easily absorbed by body tissues and non alergenic to host. They interfere with metabolic pathway, cell wall synthesis, cell membrane, protein synthesis, DNA and RNA processes in order to destroy microbe causing a disease or an infection. There are number of antimicrobial agents of common use in clinical units like Sulfonamides, Penicillin, Erythomycin, Tetracyclines, Chloramphenicol, Streptomycin, Quinolones, Rifampin and polymixins etc. All of these are very effective against microbes. Antimicrobial therapy is used to treat number of infectious diseases mostly done by chemotheraputic agents.It has evolved with penicillin effectiveness on staphlococcus aureus.Therapy is a complex phenomena. As there are number of bacterial strains that are resistant to antimicrobial agents which include gram positive, gram nagative bacteria and clinical bacterial species like Neisseria gonorrhoeae etc for them scientists are trying to develop efficient antimicrobial drugs despite of many challenges.