Tag: #microbiology

MICROBIOLOGY AND ITS BRANCHES

MICROBIOLOGY:
microbiology, study of microorganisms, or microbes, a diverse group of generally minute simple life-forms that include bacteria, archaea, algae, fungi, protozoa, and viruses. The field is concerned with the structure, function, and classification of such organisms and with ways of both exploiting and controlling their activities.

HISTORY OF MICROBIOLOGY:

The 17th-century discovery of living forms existing invisible to the naked eye was a significant milestone in the history of science, for from the 13th century onward it had been postulated that “invisible” entities were responsible for decay and disease. The word microbe was coined in the last quarter of the 19th century to describe these organisms, all of which were thought to be related. As microbiology eventually developed into a specialized science, it was found that microbes are a very large group of extremely diverse organisms.
Microorganisms have played a key role in the evolution of the planet earth.
Microorganisms affect animals, the environment, the food supply and also the healthcare industry. There are many different areas of microbiology including environmental, veterinary, food, pharmaceutical and medical microbiology, which is the most prominent.
Microorganisms are very important to the environment, human health and the economy. Few have immense beneficial effects without which we could not exist. Others are really harmful, and our effort to overcome their effects tests our understanding and skills. Certain microorganisms can be beneficial or harmful depending on what we require from them.
There are both useful and harmful microorganisms in the environment.

Microbiology Careers :
Most jobs in microbiology require at least a bachelor’s degree. An individual who is interested in microbiology may obtain a bachelor’s degree in biology or microbiology. The courseload is very similar for each of these majors; while a microbiology major may be more specific to the interests of someone who wants to study microbiology, it is also possible to achieve a similar level of specificity in the biology major by taking upper-level microbiology courses. The biology major may be preferred if one has interests in other subfields of biology, or if he or she is double majoring in biology and in another field. In both the microbiology and biology majors, students must take numerous biology courses and laboratories, and usually they must also take courses in chemistry (including organic), physics, mathematics, and statistics.
With a bachelor’s degree, one can become employed as a research technician in an academic or industry laboratory and provide technical support. One could also become a quality assurance technician in the food, environmental, pharmaceutical, or biotechnology industries, or with some additional training, become a medical technologist. However, many individuals with bachelor’s degrees in microbiology or biology go on to do further schooling. With a master’s degree in microbiology, an individual may go on to become a laboratory manager/coordinator or a biosafety officer. Further schooling leading to a PhD opens up opportunities in teaching and doing research at a university. Being a professor requires a PhD. Most heads of research laboratories in industry have PhDs as well. Other high-level careers involving microbiology include becoming a consultant/adviser, administrator, or lab director.

BRANCHES OF MICROBIOLOGY:
There are various different branches of microbiology and these include the following:
1. Bacteriology- The study of bacteria
2. Mycology –The study of fungi
3. Phycology- The study of photosynthetic eukaryotes. (Algae- Seaweed)
4. Protozoology – The study of protozoa (Single-celled eukaryotes)
5. Virology- The study of viruses, non-cellular particles which parasitize cells.
6. Parasitology- The study of parasites which include pathogenic protozoa certain insects and helminth worms.
7. Nematology- The study of nematodes.

VACCINE TECHNOLOGY

BY DAKSHITA NAITHANI

ABSTRACT

The immune system is a system that operates 24 hours a day, seven days a week to keep assaults at bay and diseases at bay. The whole system is made up of organs, tissues, and a variety of cell types that work together to defend the body. Immune cells must be able to tell the difference between native and non-native cells and proteins. Microbial cells have antigens that serve as identifiers. Antigens can induce an immune response in the human body. Each species has its own set of characteristics. Vaccines function by inducing an antibody memory response in the body without producing illness. As a result, you build immunity without becoming sick. It must include at least one antigen from the target species to trigger a response.

INTRODUCTION TO VACCINE TECHNOLOGY

A vaccination, often known as an immunisation, is a biological substance that protects people from disease-causing microorganisms. They make advantage of our immune system’s built-in ability to fight infection.

They’re produced from the same pathogens that cause the disease. They have, however, been destroyed or reduced to the point that they are no longer a source of it. Certain medicines just contain a part of the microorganism.

This is why they work so well as medications. They don’t treat or cure diseases like conventional medications; instead, they prevent them. They deceive the immune system that it has been invaded by a real intruder. When real germs enter our bodies, the same thing happens, but you don’t become ill. If you ever come into touch with a pathogen, your immune system will remember it and eradicate it before it can damage you.

TYPES

Vaccines are made using a number of techniques. Various vaccine types need different techniques to development. Antigens can be used in a variety of ways, including:

These can be delivered by a needle injected into the human skin, or ingested orally or through the nasal route.

LIVE (CHICKEN POX AND MMR)

Attenuated vaccines can be made in a variety of ways. All methods involving the transmission of a virus to a non-human host result in a virus that can be recognised by the immune system but cannot replicate in humans. When given to a human, the resulting will not be able to proliferate sufficiently to cause disease, but it will protect the individual from infection in the future. Its protection outlasts that of a dead or inactivated vaccination in most cases.

INACTIVATED (POLIO VIRUS)

A pathogen is inactivated using heat or chemicals to create this sort of vaccination. Because destroyed viruses are unable to replicate, they cannot revert to a more virulent form capable of causing disease. They are, however, less effective than live vaccines and are more likely to require renewals in order to acquire long-term protection.

RECOMBINANT (HPV)

They have been genetically modified in a lab. This method may be used to duplicate a certain gene. The HPV vaccine may be tailored to protect against strains that cause cervical cancer.

SUBUNIT (INFLUENZA AND ACELLULAR PERTUSSIS) AND CONJUGATE VACCINES (HAVING ONLY PIECES OF THE PATHOGEN)

Subunit vaccines use only a fraction of a target pathogen to elicit a response. This can be accomplished by isolating and administering a specific pathogen protein as a stand-alone antigen.

Conjugate vaccines, like recombinant vaccines, are made up of two different components. The “piece” of microbe being supplied would not typically elicit a substantial reaction on its own, but the carrier protein would. The bacterium is not the sole cause of the disease, but when combined with a carrier protein, it can render a person resistant to subsequent infections.

TOXOIDS (DIPHTHERIA AND TETANUS)

Some diseases are caused by a toxin produced by bacterium rather than by the bacterium themselves. Toxoids are inactivated toxoids that are used in vaccinations. Toxoids are classed as killed vaccines, although they are sometimes given their own category to emphasise the fact that they include an inactivated toxin.

DEVELOPMENT AND PRODUCTION

Vaccine development is a lengthy process that involves both public and private parties and takes almost a decade. Millions of individuals receive them each year, and the most of them have been in use for decades. Before being included in a country’s vaccination programme, they must undergo extensive testing to ensure their safety. Each vaccine in development must first go through screenings and evaluations to determine which antigen should be utilised to elicit a reaction. This step is completed without the use of humans. Animals are used to assess the safety and disease-prevention potential of experimental vaccinations.

STAGE 1

It takes around 2-4 years to produce and necessitates some fundamental research. Antigens, whether natural or synthetic, are identified by scientists and may help in disease prevention or therapy. Antigens might be virus-like particles, attenuated viruses or bacteria, weakened bacterial toxins, or other pathogen-derived substances.

STAGE 2

Using tissue or cell-culture techniques and animal testing, studies assess the candidate vaccine’s safety or ability to elicit an immune response. Animal topics include fish, monkeys, and mice. These studies give an idea of what to expect in terms of cellular responses in people. This period often lasts 1-2 years.

PHASE I TRIALS

The vaccine is administered to a small number of volunteers to determine its safety, confirm that it induces a reaction, and determine the optimum dosage. This round of testing is carried out on young, healthy adult participants. The goals are to determine the type and number of reactions generated by the candidate vaccine, as well as to assess the candidate vaccine’s safety.

PHASE II TRIALS

The vaccine is then given to several hundred participants to assess its safety and ability to elicit a response. Participants in this phase share the same traits as the vaccine’s intended recipients. Several studies are often undertaken during this phase to test various age groups and vaccination formulations. In most studies, a non-vaccinated group is included as a comparison group to check if the changes in the vaccinated group were due to chance or medicine.

PHASE III TRIALS

The goal is to assess vaccine safety in a large group of patients. Certain rare side effects may not have showed themselves in the low numbers of people tested in the first phase. Thousands of volunteers are given the vaccination compared to a similar number of individuals who did not receive the injection but received a comparator product to assess the vaccine’s efficacy against the illness. It is meant to protect against and to examine its safety in a much bigger group of people. To guarantee that the performance findings are applicable to a wide variety of persons, the bulk of phase three trials are conducted across various countries and different sites within a country.

PHASE IV TRIALS

Firms may conduct optional studies following the launch of a vaccine. The producer may do additional testing to determine the vaccine’s safety, efficacy, and other potential applications.

REVERSE VACCINOLOGY

Reverse vaccinology is the use of genetic information combined with technology to make vaccines without the use of microorganisms. It assists in the study of an organism’s genome for the purpose of identifying novel antigens and epitopes that may be utilised as prospective candidates. This method has been around for at least a decade. By unravelling the entire genomic sequence, it is possible to determine what molecules make up the genomic sequence. Without needing to grow the pathogen for a longer amount of time, candidate antigens can be discovered.

Reverse vaccinology has been used to create vaccines for meningococcal and staphylococcal diseases all over the world. Infections are caused by Staphylococcus bacteria, which can be found on the skin or in the nose of even healthy persons. The bacteria Neisseria meningitidis causes a serious infection of the thin covering of the brain and spinal cord.

PRODUCTION QUALITY CONTROL AND COMMERCIALIZATION

Vaccines are biological compounds that are frequently hybridised and complex to understand. They are made through a succession of manufacturing and formulation steps, with the finished product often containing a large number of component items. As a result, unlike a tiny molecule medicine, the finished product is impossible to classify. This needs a highly controlled production system as well as a personnel capable of performing such processes on a continual basis. Control testing takes over two years and occupies more than half of the time in the subsequent manufacturing process.

 STEP 1- PRODUCTION

Following clinical trials, when a vaccine reaches the pre-approval stage, it is evaluated by the applicable regulatory authority for quality, safety requirements.

STEP -2 MAKING

Businesses will create development plans for a vaccine on their own. Once a vaccine is approved, production begins to pace up. The antigen has been rendered inactive. All of the components are mixed to make the final product. The entire process, from testing to manufacturing, can take a lengthy time to complete.

STEP- 3 PACKAGING

It is then bottled in glass vials and packed for safe cold storage and transportation once it is produced in bulk. It must be able to resist severe temperatures as well as the dangers associated with international shipping. As a result, glass is the most often used material for vials since it is robust and can keep its integrity under severe extrinsic factors.

 STEP- 4 STORAGE

When it is excessively hot or cold, it loses its effectiveness and may even become inert. Vaccinations can be destroyed or rendered dangerous to use if kept at the improper temperature. Most vaccinations must be kept chilled between 2 and 8 degrees Celsius, necessitating the use of specialist medical freezers.

STEP-5 SHIPPING

They are transported out using particular equipment so as to maintain its integrity. Lorries deliver them from the airport to the warehouse cool room after supplies arrive in the market. New innovations have resulted in the development of portable devices that can keep vaccines cold for several days without the need of power.

QUALITY CONTROL

Once they are given out, authorities continuously check for – and assess the severity of – any potential side effects and responses from the recipients. Safety is a top priority, with frequent reviews and post-approval clinical trials reporting on its effectiveness and safety.

CAREER SCOPE

There are several prospects in vaccine research and development, clinical trials, vaccine manufacturing, and public distribution. These jobs are available at universities, companies, government laboratories and agencies, hospitals, and on the front lines of vaccine distribution all around the world. When different components of a project are handled by different groups at the same time in industry, greater teamwork is usually required, whereas a scientist in an academic lab may be a lone worker overseeing all parts of a project.

The balance between creative science and all of the business administration that comes with securing money, maintaining a budget, and overseeing other scientists or assistants is the most challenging aspect.

 Research allows scientists to work on a project that has the potential to have a direct influence on public health, whether it’s on a lab bench, a production line, or to support a clinical trial.

Cholera the infection and related pandemics.

Cholera is considered as a gastro-intestinal disease. An acute, secretory diarrhea caused by infection with Vibrio cholerae of the O1 and O139 serogroups. This bacterium is transmitted via contaminated food or water that has come in contact with fecal matter of the infected person. In some severe form, cholera can be a very terrifying illness in which profuse painless watery diarrhea and copious effortless vomiting may lead to hypovolemic shock and death in less than 24 hours, if untreated. Management of patient with cholera include aggressive fluid replacement, antibiotics. Prevention include safe water and good sanitary conditions. Two oral vaccines are available. Researchers have estimated that each year there are approximately 1.3 million to 4.0 million cases of cholera, and 21 000 to 143 000 deaths occurring in world due to cholera. Total of seven cholera pandemics have occurred in the past 200 years. The first pandemic originated in India.

Morphology and Identification

A. Typical Organisms V.cholerae is a gram negative, comma-shaped, curved rod 2–4 μm long. It is actively motile shows presence of polar flagellum.

(Vibrio cholerae, the bacterium that causes cholera)

B. Cultural characteristics and Plating media.

V.cholera are strongly aerobic. They grow well at 37°C on many kinds of media, including defined media containing mineral salts and asparagine as sources of carbon and nitrogen. On Mac Conkeys agar the colonies are colorless at first then become pink on prolonged incubation due to slow fermentation of lactose. V.cholerae grows on thiosulfate-citrate-bile-sucrose (TCBS) agar, a media selective for vibrio’s, on which it gives yellow-colored colonies that are readily visible against the dark-green background of the agar. Monsur’s gelatin taurocholate trypticase tellurite agar (GTTA) medium is also used. They produce small, translucent colonies with a greyish black Centre and a turbid halo. Most Vibrio species are halotolerant, and NaCl often enhances their growth. Some vibrios are halophilic, requiring the presence of high concentration of NaCl to grow. Vibrio species are susceptible to the compound O/129 (2,4-diamino-6,7di-isopropylpteridine phosphate)

C. Holding or Transport Media. Cary-Blair medium is used as a transport medium, it is a buffered solution of sodium chloride, calcium chloride, sodium thioglycolate, disodium phosphate at pH 8.4. Venkatraman-Ramakrishnan (VR) medium, in this the organisms do not multiply but remain viable for few weeks. It is dispended in screw capped bottles in 10-15 ml amounts. About 1-3 ml of stool is added to each bottle. Autoclaved sea water can also be used as a holding medium.

D. Biochemical Reactions. V.cholerae shows following features: It is catalase positive and oxidase positive. V.cholerae ferments sugars with production of acid only no gas formation. It ferments glucose, sucrose, maltose, mannitol, and mannose. It is a late lactose fermenter ferments lactose on incubation for several days. It does not ferment arabinose, inositol, and dulcitol. It forms indole and reduces nitrates to nitrites. It gives methyl red positive and urease test negative. It liquefies gelatin and decarboxylates lysine and ornithine, but not arginine. A positive oxidase test is a basic step in the identification of V.cholerae and other vibrios.

E. Antigenic Structure and Biologic Classification. Many vibrio’s possess a single heat-labile flagellar H antigen. They are classified as Group A vibrio’s, and the rest as Group B. Based on major somatic O antigen, Group A vibrio were further classified into subgroups or serovars also called as serogroups. Antibodies to the H antigen are not involved in the protection of susceptible hosts. V.cholerae contain an O lipopolysaccharide that confer serologic specificity. There is a minimum of 206 O antigen groups. V.cholerae strains of O group 1 and O group 139 that cause classic cholera; non-O1/non-O139 V.cholerae causes cholera-like disease. The V.cholerae serogroup O1 antigen has determinants that make further typing possible; the serotypes are Ogawa, Inaba, and Hikojima. V. cholerae O139 is similar to V.cholerae O1 El Tor biotype. V.cholerae O139 does not produce the O1 lipopolysaccharide and is incapable of making this antigen. V.cholerae O139 produce a polysaccharide capsule, but V.cholerae O1 does not produce a capsule.

Virulence factor and Resistance. Virulence factor of V.cholerae include cholera toxin, adhesin factor, toxin regulated pilus, siderophores, hemagglutination-protease, neurotransmidase and some others also. They produce a heat labile enterotoxin. Which consists of subunits A and B. Ganglioside GM1 act as the mucosal receptor for subunit B, which promotes entry of subunit A inside the cell. Activation of subunit A1 yields increased levels of intracellular cyclic adenosine monophosphate (cAMP) and results in hypersecretion of water and electrolytes. Electrolyte-rich diarrhea occurs—as much as 20–30 L/day—which results in dehydration, shock, acidosis, and death. The genes for V.cholerae enterotoxin are present on the bacterial chromosome. Cholera enterotoxin can stimulate the production of neutralizing antibodies. Toxin regulated pilus, helps in adherence to mucosal cells of intestine. Hemagglutination- protease, splits mucus and fibronectin and cholera toxin. Thereby inducing intestinal inflammation and helps in releasing free vibrios from bound mucosa to the intestinal lumen. Neuraminidase, destroys muramic acid and increases toxin receptors for V. cholerae. Siderophores is responsible for sequestration of iron. These organisms are susceptible to heat, drying and acids, but resist high alkalinity. Survival in water is influenced by pH, temperature, salinity and organic pollutants.

Immunity and Pathogenesis. After ingestion of V.cholerae, the majority are killed by gastric acid. Specific IgA antibodies are found in the lumen of the intestine. Similar antibodies in serum develop after infection but last only for few months. Vibriocidal antibodies in serum are associated with protection against colonization.

The pathogenesis of cholera and of diarrhea caused by enterotoxigenic bacteria other than V.cholerae 01 comprises three main stages: (1) bacterial colonization; (2) production and delivery of enterotoxins; and (3) toxin action and intestinal fluid secretion. (Ananthanarayan and Paniker, 1948;)

The structure and function of cholera toxin (CT) and its effects on fluid transport processes have been particularly well elucidated. It is believed that colonization may involve, sequentially: (1) chemotactic attraction of the bacteria to the surface of the mucus gel; (2) penetration of the mucus gel;'(3) adhesion to the epithelial cell surface; and (4) multiplication of mucus gel- and mucosa-associated bacteria. The bacterial cell surface receptor for CTXφ is the toxin-co-regulated pilus, which is itself encoded within a genomic island, vibrio pathogenicity island (VPI-1). Evolution of virulence in V.cholerae involves sequential acquisition of VPI-1 followed by CTXφ. Under normal conditions, V.cholerae is pathogenic only for humans. A person with normal gastric acid secretion may have to ingest as many as 1010 or more V.cholerae to become infected. When the medium is food, as few as 102–104 organisms are necessary because of the buffering capacity of food. Any medication that decreases stomach acidity makes a person more susceptible to infection with V cholerae. The organisms do not invade the bloodstream but remain within the intestinal tract. Pathogenic V cholerae organisms attach to the microvilli of epithelial cells. They multiply and secrete cholera toxin and also mucinases and endotoxin.

Laboratory Diagnosis.

  1. Specimens

Fresh stool specimen collected before administration of antibiotics is the specimen of choice. 

  1. Microscopy 

Dark field microscopy and phase contrast microscopy is preferred to check out motility and inhibition by antisera. Direct immunofluorescence is another rapid method used for detection of vibrios in the stool sample. 

  1. Culture 

The specimen collected in holding media is inoculated in enrichment media for 6-8 hrs., before inoculating on selective and general-purpose media. The specimen collected in transport media are incubated for 6-8 hrs. The inoculated plates are incubated at 37oC for a period of 24 hrs.

4. Specific Tests

V.cholerae organisms are also identified by slide agglutination tests using anti-O group 1 or group 139 antisera and also by biochemical reaction patterns. The diagnosis of cholera under field conditions has been reported to be facilitated by a sensitive and specific immunochromatographic dipstick test.

(Antisera to the O1 serogroup of V. cholerae will agglutinate homologous organisms (left). A normal serum or saline control (right) does not show agglutination)

Transmission.

Both contaminated water and contaminated food can serve as medium for the transmission of cholera. In Bangladesh and India, water appears to play a major role. In other areas, such as the South Pacific islands, foodborne outbreaks have occurred. In situations where water is the medium, it need not only be drinking-water that is responsible, since contaminated water may be consumed in other forms. In addition, contaminated water may inoculate food, leading to foodborne cholera. The role of fomites, fingers, bed linen, or other soiled objects in the transmission of cholera remains unclear. Type of transmission more often when there is overcrowding and hygiene is very poor. Children who acquire nosocomial cholera may be more susceptible than normal children because of their underlying illness.

Diagnosis and Treatment.

Physicians in endemic areas diagnose cholera based on its manifestations, particularly so-called “rice-water stool,” which is watery, colorless, odorless, and flecked with mucus, which looks like bits of rice. The necessary and immediate part of therapy consists of water and electrolyte replacement to correct the severe dehydration and salt depletion. Oral tetracycline and doxycycline tend to decrease stool output in cholera and shorten the period of excretion of vibrios. In some areas, tetracycline resistance of V.cholerae has emerged; the genes are carried by plasmids. In children and pregnant women, alternatives to the tetracyclines are erythromycin and furazolidone.

Epidemiology, Prevention and control. 

In endemic regions, the major cases occur among children below 5 years of age and in reproductive-age women. In some countries like Bangladesh and India, cholera infections occur every year. It is found that environmental factors such as climate, temperature, and salinity play a major role in cholera transmission. Reoccurrence of epidemic cholera has also been related to population density, urbanization, and overcrowding. For the prevention and control of cholera, it is necessary to understand the factors that are responsible for initiation and transmission of cholera in a community. Measures for the preventing cholera include provision of clean water, hygienic food and proper sanitary conditions to the cholera-endemic communities. Health education regarding personal hygiene and food safety should be provided. Media, community leaders, and religious leaders should participate in health education and social mobilization campaigns. Today, there are two oral cholera vaccines, namely Dukoral and Shanchol. Dukoral is made up of killed whole cell vaccine including V. cholerae O1 serogroup and recombinant B subunit of cholera toxin. This vaccine can be given to children above 2 years and to adults. Shanchol is a killed bivalent whole‐cell vaccine suspension. It can be dosed to 1 year of age and above. he primary methodologies for cholera control are suitable administration of cholera cases; fortifying research centers; preparing and limit working of medical care laborers; and accessibility of sufficient clinical supplies for the executives. Likewise, admittance to safe water, legitimate disinfection, suitable waste administration; individual cleanliness and food cleanliness rehearses; improved correspondence and public data are required for the control of cholera episodes.

Pandemics. 

Despite the fact that cholera has been around for a long time, the illness came to conspicuousness in the nineteenth century, when a deadly flare-up happened in India. There have since been various flare-ups and seven worldwide pandemics of cholera. Every year, cholera taints 1.3 to 4 million individuals around the globe, slaughtering 21,000 to 143,000 individuals. The primary cholera pandemic rose out of the Ganges Delta with a flare-up in Jessore, India, in 1817, coming from polluted rice. The infection immediately spread all through the majority of India. The pandemic ceased to exist 6 years after it started. The second cholera pandemic started around 1829. The pandemic would vanish and reappear all through various nations for almost twenty years until it died down around 1851. Six resulting pandemics executed huge number of individuals over all mainland. The seventh pandemic began in South Asia in 1961, and arrived at Africa in 1971 and the Americas in 1991. Cholera is presently endemic in numerous nations.

Prophylactic use of antimicrobials – a debatable issue

An ancient and quiet honourable practise has been the use of preventive medicine. For example, the ancient Chinese use to pay their doctors while they remained healthy, however as soon as they felt sick this payment would not be given. The effectiveness of antibiotics as a prophylactic means for protecting healthy individuals exposed to pathogenic bacteria, preventing the development of an infection in chronically ill patients and preventing an infection in patients who undergo surgery is a debatable issue. Many surgeons reported significant reductions in post-operative infection following antibiotic use, and a few did not even reported infections for a period of twenty years. Despite this success, prophylaxis presents certain hazards, including the evolution of antibiotic resistance, superinfections and drug side effects, for the individual patient and for the general public. Therefore, physicians have broad views on the responsible preventive use of antibiotics. However, antimicrobial prophylaxis (AP) should be confined to specific well-accepted evidence for the prevention of excess costs, toxicity and antimicrobial resistance in order to effectively prevent infections. Initial or secondary (recurring prevention or reactivating infections) prophylaxis may also be regarded as primary (prevention of initial infections) or may be administered to prevent infection by killing a colonising organism. Patients should know in detail the potential risks and benefits of AP. The potential risks are allergic reactions with the use of antibacterial agents that can be serious or life-threatening, and clostridium difficile colitis. The risk of tendinitis, including the rupture of the tendon of Achilles should be alerted to patients taking fluoroquinolones.

The pros and cons of using antimicrobials as a prophylactic.

  1. THE PROS: In the diagnosis of life-threatening acute bacterial infections, surgical infectious diseases and if there is an effective use of antimicrobials as prophylaxis, antibiotics can have many benefits.

In bacterial infections: In acute bacterial infections, which were highly mortal before introduction of antibiotics, the benefits of antibiotics as prophylactics is most clearly indicated. Mortality in endocarditis was about 100% prior to 1990 and about 20% total in 2010 although the death rate is usually caused not by unsuccessful antibiotic therapy but by cardiac failure or embolic complications. In bacterial meningitis in 1990, the mortality rate was reduced to 8% to 20% in 2010 and acute osteomyelitis mortality decreased from 50% to less than 1%. Many other infections, in both individual patients and others within the community, have significantly reduced morbidity and serious effects of spraying. In high-risk patients, this included the use of antibiotic prophylaxis for bacterial meningitis. During the systemic inflammatory response (SIR) stage of the infection, the early initiation of broad- spectrum antibiotics was proved critical for preventing the development of sepsis. When appropriate antibiotics are prescribed early in the surgical sepsis, mortality is significantly reduced.

In surgical site infections: Although the technique is still less than good surgical and aseptic technique, the risk of surgical site infection is considerably decreased by antibiotic prophylaxis in high-risk surgical patients such as operations over 2 hours, abdominal procedures, endogenous or exogenous contamination and co-morbidity. The choice of antibiotics depends on the organisms that are most likely to be affected; the kind of operation; the probability of resistance development and the financial cost involved. In felines, the rate of postoperative infections was reduced in the course of the optional orthopaedic surgery by preoperative antibiotic prophylaxis. Therefore, it is usually advisable to treat routine perioperative prophylactic antibiotics, even if numerous orthopaedic operations are categorised as clean. Orthopaedic procedures normally last longer than 90 minutes and the potential infection may be influenced by local wound factors like implants and tissue trauma. In the presence of implants, bone and joint infections are very difficult to treat, increase morbidity and may adversely affect the result. Cefazolin is currently seen as a choice antibiotic because of its outstanding effectiveness, low toxicity and reasonable costs against most surgical wound pathogens. The first dose should be given at a concentration of 22 mg/kg 30–60 minutes before surgery. The dose is usually recommended to be repeated every 90–120 minutes, but there is evidence that the frequency is enough every three hours.

The selection criteria of the antibiotic are:

  • The most prone bacteria that could cause infection should be identified. A prophylactic against frequently found skin bacteria (skin flora) is used when only an incision in the skin is made. An antibiotic is chosen to treat both the skin and the mucosal flora if the mucosal incision is involved.
  • Chemical and drug toxicity characteristics.
  • The least likely antibiotic that is required for serious infections is chosen if different antibiotics are equally helpful for prophylaxis. This helps prevent antibiotic resistance from developing.
  • Sensitivities specific to the environment of the specific hospital. Some hospitals may be very frequent with methicillin-resistant infections, while vancomycin or clindamycin-resistant infections in other hospitals may be more frequent.
  • CONS: The drawbacks of prophylactic antimicrobials are shown by side effects, resistance development and opportunistic pathogens.

Side effects: Their ability to cause serious or fatal adverse reactions sometimes provides a reason to limit the use of antibiotic agents for true therapeutic indications. For example, the most commonly used antibiotics for UTI prevention are nitrofurantoin, trimethoprim/sulfamethoxazole (TMP / SMX), but these drugs have negative reactions in children. Gastrointestinal disturbance, skin reactions such as urticaria, maculopapular rash are the common adverse reactions to nitrofurantoin. Almost exclusively because of sulfamethoxazole, most commonly dermal, adverse events related to trimethoprim/sulfamethoxazole. Serious side effects are extremely rare and mostly reversible when treatment is discontinued but they do exist. The long-term use of low-dose urinary prophylaxis antibiotics is therefore not completely safe. While adverse reactions existed in children to these medications, the lower dose of prevention and the lack of significant co-morbidities and medicinal interactions in children are much less common in children than in adults. In 1% of patients, penicillin causes death from type I anaphylactic shock in sensitive allergic patients and have other harmful consequences. High dose of penicillin may be associated with serum sickness (type III reaction), penicillin, thrombocytopenia, and haemolytic anaemia from cytotoxic antibodies. There is 10 percent cross-sensitivity between the derivatives of penicillin, cephalosporin and carbapenems because they share a similarity between the side chain rather than the beta-lactam structure. Therefore, the same or closely related drug must be avoided to which the patient has shown sensitivity in the past. In certain circumstances, certain drugs are more likely to be toxic. Ampicillin and amoxicillin rash are more common when lymphoid tissue is ebullient, in the case of lymphomas or glandular fever. The following are commonly used antimicrobials for prophylaxis along with their side effects:

  • Penicillin: side effects are reactions of hypersensitivity, renal damage, low potassium (hypokalemia)
  • Cephalosporin: side effects are reactions of hypersensitivity, reduction of blood cell levels such as: neutrophils, leucocytes (leucopoenia) and thrombocytopenia, nausea and vomiting, gastrointestinal problems diarrhoea, anorexia.
  • Metronidazole: side effects are toxicity of the CNS, problems in gastrointestinal tract, neutropenia, blood clotting problems, and alcohol reactions
  • Antibiotic resistant: The bacterial resistance mechanisms are known to include genetic changes, antibiotic metabolism by bacteria, like beta lactamase (beta lactamases), altered receptor site affinity, cell wall permeability alterations (antibiotic efflux pump) and the environmental influence at infection sites. In pus, most bacteria are relatively resistant in the dormant phase. The slow cellular immune mechanism does not affect the intracellular microbes such as tubercle bacillus, Brucella abortus, and Salmonella typhi. This partly explains the slowness of antibiotics in these infections. Infections on heart valves and the meninges, for example, are more resistant to antibiotics than infections elsewhere because the concentrations of polymorphs and macrophages are low. Inappropriate antibiotic treatment facilitates the spread of resistance. In many countries, UTI-associated antibiotic resistance has become widespread. Previous studies showed an increased rate of antibiotic resistance. Antimicrobial resistance in enteric and oropharyngeal flora may be developed through the use of antibiotics for prophylaxis. A recent study has reported a high rate of resistance against third generation cephalosporins in children who received prophylactic antibiotics. Clinicians are advised to carefully use prophylactic antibiotics. Additional hazards are present in antibiotics that inhibit the growth of a range of different types of bacteria. These medications also eliminate benign bacteria that help protect us from diseases by competing with pathogenic bacteria and limiting their propagation. Wide ranging antibiotics may produce deep changes in bacterial population composition and lead to the outgrowth and invasion of so-called superinfections of antibiotic-resistant strains.

Since the V. Cholerae infection dose is high, proper hygiene generally makes immunisation and prophylaxis unnecessary, hence antimicrobial prophylaxis in endemic areas has not proved effective. In the prevention of sexually transmitted diseases, chemical prophylaxis is ineffective. The use of antimicrobials to prevent genital diseases may, indeed, deteriorate the situation by selecting harder resistant strains. Antibiotic products, especially those of a wide activity range, alter the normal flora of the body, allowing resistant and opportunistic pathogens to colonise and multiply. These could cause secondary infections in a healthy female, such as candida vaginitis, or fungal and systemic infection in a highly susceptible patient, such as an immunosuppressive treatment. Clostridium difficile, anaerobic bacterium which can multiply after normal flora is suppressed and is relatively resistant to many commonly employed antibiotics but metronidazole or vanzomycin, causes the severe complication of pseudomembraneous colitis. Between 2000 and 2007, 400 percent of the increased mortality from Clostridium difficile infection was partially due to the emergence of the insusceptible C. fluoroquinolone strain. When pseudomembraneous colitis is developed in transplant patient, it is impaired to absorb the immunosuppressive medications which increase allograft-rejection susceptibility.

Other disadvantages of prophylactic use of antimicrobials are:

  • More expensive treatment: Antibiotics are costly and shouldn’t be used inappropriately. However, the cost of the antibiotics is negligible compared to the cost of hospitalisation for a long stay due to a wound infection in patients at clearly reduced risk of wound infections which were found by prophylaxis. The least cost-effective agent for a short period of time is selected if antibiotic prophylaxis is used.
  • Allergies and toxic reactions: When antibiotics are used, toxic or allergic reactions may occur. These can be reduced for brief periods by using safe agents.
  • The use of antibiotics can lead to a false sense of safety. Careful surgery and precautionary and postoperative care are essential if wound infections are to be minimised.
  • Infection due to side effects by drugs, especially clindamycin, with bacteria such as the Clostridium difficile.

Conclusion:

The use of antimicrobial prophylaxis has led to a large number of infections being prevented and significant declines in surgical infections at the site. Specific, accepted indications should be limited to antimicrobial prophylaxis to avoid excess cost, toxicity and resistance to antimicrobials. The potential risks and benefits of any antimicrobial prophylaxis system should be understood by patients. Although there is evidence based antimicrobial prophylaxis practises, many are based on low evidence or expert advice. Additional antimicrobial prophylaxis studies are necessary. There remain significant controversies in antimicrobial prophylaxis, with many opportunities to practise improvement through rigorous studies. More antibiotics do not always reduce surgical site infection more effectively. There are significant gaps between directives and practises, mainly over the current guidelines on antibiotic prophylaxis.

ANTI-CELL WALL ANTIBACTERIAL DRUGS

Selective toxicity is the important characteristic of antimicrobial drugs which means that any drug is selective against a particular microorganism and also selectively act on a particular site. Not all drugs can act on every site. There are many sites at which any drug acts such as cell wall, cell membrane of the bacterial cell. Basically selective toxicity explains that any drug will only act on the pathogen and not on the host.
ANTI-CELL WALL DRUGS
Anti-cell drugs are those drugs which act on the cell wall of the bacterial pathogen and not the host. There are variety of drugs which fall under this category. The major class is of beta-lactam antibiotics among which penicillin is the drug which is studied the most. The drugs can be administered into the patient’s body by different ways like intramuscular, intravenous, or can be applied as topical preparations. But mostly, these drugs are intramuscular or intravenous drugs. The following points explain the further different mechanisms of anti-cell wall drugs.
There are 3 different mechanisms by which anti-cell wall drugs work and thus they are also classified as following:

  1. First classification involves the drugs that directly interact with Penicillin-Binding-Proteins (PBPs) and inhibit the transpeptidase activity which in turn inhibits the attachment of newly formed peptidoglycan subunit to the pre-existing one.
    This is the main mechanism of β-lactam antibiotics. These antibiotics include Penicillin (penams), cephalosporins, Penems, Carbapenems, and monobactams.
    These antibiotics bind to the penicillin-binding proteins which are enzymes present in the bacterial cell wall. Different β-lactam antibiotics bind in a different way. After the antibiotics bind to the enzyme, it changes the morphological response of the bacteria to the antibiotic.
  2. Second classification involves the drugs that bind to the peptidoglycan subunit, blocking different processes.
    The important class of compounds called as glycopeptides are mainly involved in this mechanism of anti-cell wall antibiotics.
    Vancomycin and Teicoplanin are the major examples of glycopeptide antibiotics.
    Vancomycin kills only gram-poitive bacteria whereas Teicoplanin is active against both. The overall mode of action of glycopeptides antibiotics is blocking transpeptidation i.e. similar to β-lactam antibiotics, they also inhibit the transpeptidase activity, and transglycosylation i.e. they being large in size attach to the peptidoglycan subunits thus creating a blockage which does not allow the cell wall subunits to attach to the growing peptidoglycan backbone.
  3. Third classification involves the drugs that block the transport of peptidoglycan subunits across cytoplasmic membrane.
    The main example of such type of drugs is bacitracin, which is a simple peptide antibiotic originally isolated from Bacillus subtilis.
    The mode of action of these class of drugs is blocking the activity of specific cell membrane lipid carriers which act as the attachment surface for peptidoglycan precursors and help in their movement from cell cytoplasm to exterior of the cell. This activity of lipid carriers is inhibited by bacitracin like drugs and they finally prevent the incoroporation of those precursors into cell wall thus inhibiting its biosynthesis.

Although, its route of administration is mostly oral or intramuscular, bacitracin is also known to show its effects when used as topical ointments like Neosporin.

GRAM STAINING

Bacteria can be classified into two different categories i.e. gram-positive bacteria and gram-negative bacteria. To differentiate the type of bacteria present in any collected sample there is a technique known as GRAM STAINING OR GRAM STAIN. It is sometimes also known as gram’s method. This technique is a different step process which can easily distinguish and classify between different types of bacteria. This was named after the great scientist Hans Christian Gram.
Gram Staining method differentiates bacteria on the basis of their physical and chemical structure of cell wall. They are stained with different reagents and are observed in different colors when seen under the microscope. It is due to the fact that gram positive bacteria have a thick layer of peptidoglycan which allows it to retain the primary stain which is crystal violet and thus they appear purple whereas on the other hand, gram-positive bacteria have thin peptidoglycan cell wall and thus only retain the secondary or counter stain which is Safranin and thus they appear slightly pinkish when observed under microscope. Gram staining is the basic technique which is widely used in the microbiology labs to distinguish between both the types of bacteria. It provides a great help to microbiologists to perform their clinical tasks. If any infection is suspected in the patient then after the collection of sample, the gram staining is done and the type of bacteria is observed.
Gram staining is completed in the various steps –

  1. Fixation of clinical materials i.e. the sample collected are being fixed on the glass slide by first making a smear using water and then that smear is either heat fixed or methanol fixed. It is a first and a very important step. Methanol fixation was later discovered keeping in mind its advantage of not destroying the morphology of host cell, as well as bacteria present in that. It is majorly used for the testing of blood samples collected from patients.
  2. Application of primary stain i.e. crystal violet. Primary stain means the first stain which is applied onto the fixed smear which stains all the calls purple or blue.
  3. To distinguish the slide is then washed off in a gentle and indirect stream of water for 2 seconds which removes the extra stain that is not absorbed by the cells.
  4. The next step involves the application of a mordant i.e. Iodine solution. This forms a complex with crystal violet due to which all the cells start appearing blue.
  5. Again the slide is washed for 2 seconds to remove extra stain.
  6. Addition of a decolorizing agent is the next step which will ultimately remove the excess stain which bacteria has not absorbed. The decolorizer contains the combination of acetone and alcohol. In this step, the gram positive bacteria continue appearing violet or blue in color whereas gram negative bacteria start appearing colorless.
  7. Application of counter stain or secondary stain i.e. safranin is the next step. It should let remain on the slide for 30 second to 1 minute. This will stain all the colorless gram negative bacteria pink and gram positive bacteria remains blue in color.
  8. Again the slide is washed off in a gentle stream of water.
  9. The prepared stained slide is then observed under a microscope using immersion oil (for observing under 100x).

VIRUS AND ITS TRANSMISSION

WHAT IS A VIRUS?????
A virus is referred as an infectious agent that can only replicate inside the living cells of an organism i.e. a virus is something which can not at all grow or replicate by its own. It always needs a living cell for its replication process. It is a microorganism which cannot be seen by naked eyes and can infect any life form. It can be infectious for humans, plants and even for other microorganisms like bacteria and archea. Viruses infecting bacteria are known as bacteriophage. Viruses are not restricted to a place and they can be found everywhere at every place of ecosystem whether land, or water or in air. They can cause various infections including air-borne, water-borne or even food-borne. The science dealing with the study of viruses is known as Virology and it is a branch of microbiology. A complete virus particle ranges in size from about 10-400nm in its diameter.
Viruses are near to dead when outside the living cell but once entered any living cell of an organism, they are forced to replicate using the life machinery of that particular organism and thus they produce thousands of their multiple copies and in this way infect the organism. Outside the living cells they are present in the free, independent form which may also be known as a virion.
There are 3 main parts in the structure of a virus i.e. –

  1. Genetic core which is also known as nucleic acid core containing all the genetic material whether DNA or RNA, but not both. It is known as genome.
  2. A protein coat, which is also known as capsid which surrounds the genome of a virus particle.
  3. An envelope which is made of lipid. It is an external coat surrounding the genome as well as capsid.

VIRUS TRANSMISSION
Transmission of virus particles is important for them to survive because as discussed above they can only replicate themselves inside a host living organism. The virus transmits from one organism to another in order to survive, reproduce and continue their species. The effectiveness of the transmission of viral particle depends on 2 main factors i.e. the concentration of virus and its route of transmission. More concentration of virus leads to more transmission.
There are several ways by which a virus particle may get transmitted from one organism to another.

  1. Blood – Virus particles can get transmitted through the blood. The one way is direct viral infection in blood and the other way is by arthropods like dengue or malaria is transmitted. Arthropods bite one organism and collect viral particles from them and then when they bite other organism, the same viral particles are being transmitted to the next organism and this way transmission and infection occurs. Another way is direct viral infection in blood which can be via direct infected blood exposure to a healthy individual. It may be transmitted via sexual contacts with infected person like HIV is transmitted.
  2. Saliva – It is the most commonly seen in kissing the infected individual. The saliva contains the viral particles and thus they are transmitted to healthy individual.
  3. Respiratory secretions – If any infected individual sneezes, or coughs or in any other way its respiratory secretions come in contact with the healthy individual, he may get infected by the same. It may also occur by singing or even breathing.
  4. Feces – This is not a very common method in developed countries but can infect those who do not take sanitary actions after using toilets. The virus particles secreted in feces can infect other healthy individuals if they come in contact with them.

GOOD LABORATORY PRACTICES AND BIO-SAFETY METHODS

  1. When you arrive the laboratory, the first thing is that you must wash your hands with a disinfectant soap for your immediate sanitization.
  2. Eating anything in the laboratory area and smoking is strictly prohibited. Do not put anything in your mouth such as pencils, labels, or fingers. Do not store food in areas where microorganisms are stored.
  3. Purchase a lab coat and safety glasses and use them. Leave protective clothing in lab and do not wear it in non-lab areas.
Photo by Chokniti Khongchum on Pexels.com
  1. Avoid loose fitting items of clothing. Wear appropriate shoes (sandals not allowed) in the laboratory.
  2. Backpacks, purses and quotes should be placed in the cubbyhole by the front door of the lab. Place needed items on the floor near your feet, but not in the aisle.
  3. Disinfect work areas before and after use with 70% ethanol and fresh 10% bleach. The regular disinfection of the laboratory surfaces must be done using appropriate disinfectants like hypochlorite solution which kills almost pathogenic microorganisms.
  4. Label everything clearly.
  5. Caps and lids of reagents, solution bottles, and bacterial must be replaced properly in order to prevent contamination and petri dishes must not be opened directly in the lab unless absolutely necessary.
  6. Inoculating loops and needles should be flame sterilize in a bunsen burner before you lay them down.
  7. Turn of bunsen burner when not in use. Long hair must be restrained if bunsen burner are in use.
  8. Flame sterilization using alcohol must be done so carefully and it must be kept in mind that no papers or similar materials that can catch fire easily are nearby.
  9. Treat all microorganisms as potential pathogens and culturing of microorganisms must be done inside a special sterilized laminar flow hood and not outside it because many air-borne microorganisms can be spread.
  10. Wear disposable gloves when working with potentially infectious microbes and samples. If you are surely working with a pathogenic sample, you must handle it with extra care so that it doesn’t spill out on you or on any surface of the laboratory.
  11. Sterilize equipment and materials.
  12. Never pipette by mouth.
  13. Consider everything a bio hazard. Do not pour anything down the sink. Autoclave liquids and brought cultures to sterilize them before discarding.
  14. Dispose off all solid waste material in a biohazard bag and autoclave it before discarding in the regular trash.
  15. There are a special column of safety equipment in the laboratory which you must be aware of so that in case of any emergency you can make use of those safety equipments.
  16. Dispose of broken glass in the broken glass container.
  17. Dispose of razor blades, syringes, and sharp metal object in the “sharps” container.
  18. If by any chance, there is any type of spill of sample or culture or any media, you must immediately contact your instructor or mentor so that he/she can help you and find a solution to remove it from the surface. If you’re able to clean the spill by yourself do it immediately.
  19. In the same way, in case of any mishappening or sudden accident, you must immediately report to your instructor for the immediate help.

MECHANISM OF DIFFERENT TYPES OF ANTIBIOTICS

Antibacterial Drugs are classified according to their site of action which are as follows :

CELL WALL SYNTHESIS INHIBITORS
There are 3 different mechanisms by which anti-cell wall drugs work and thus they are also classified as following:

  1. First classification involves the drugs that directly interact with Penicillin-Binding-Proteins (PBPs) and inhibit the transpeptidase activity which in turn inhibits the attachment of newly formed peptidoglycan subunit to the pre-existing one.
    This is the main mechanism of β-lactam antibiotics. These antibiotics include Penicillin (penams), cephalosporins, Penems, Carbapenems, and monobactams.
    These antibiotics bind to the penicillin-binding proteins which are enzymes present in the bacterial cell wall. Different β-lactam antibiotics bind in a different way. After the antibiotics bind to the enzyme, it changes the morphological response of the bacteria to the antibiotic.
  2. Second classification involves the drugs that bind to the peptidoglycan subunit, blocking different processes.
    The important class of compounds called as glycopeptides are mainly involved in this mechanism of anti-cell wall antibiotics.
    Vancomycin and Teicoplanin are the major examples of glycopeptide antibiotics.
    Vancomycin kills only gram-poitive bacteria whereas Teicoplanin is active against both. The overall mode of action of glycopeptides antibiotics is blocking transpeptidation i.e. similar to β-lactam antibiotics, they also inhibit the transpeptidase activity, and transglycosylation i.e. they being large in size attach to the peptidoglycan subunits thus creating a blockage which does not allow the cell wall subunits to attach to the growing peptidoglycan backbone.
  3. Third classification involves the drugs that block the transport of peptidoglycan subunits across cytoplasmic membrane.
    The main example of such type of drugs is bacitracin, which is a simple peptide antibiotic originally isolated from Bacillus subtilis.
    The mode of action of these class of drugs is blocking the activity of specific cell membrane lipid carriers which act as the attachment surface for peptidoglycan precursors and help in their movement from cell cytoplasm to exterior of the cell. This activity of lipid carriers is inhibited by bacitracin like drugs and they finally prevent the incoroporation of those precursors into cell wall thus inhibiting its biosynthesis.

Although, its route of administration is mostly oral or intramuscular, bacitracin is also known to show its effects when used as topical ointments like Neosporin.

INHIBITORS OF PROTEIN SYNTHESIS
Protein Inhibitors can be divided into 2 parts:

  1. Inhibitors binding to 30S subunits
    • Aminoglycosides bind to the bacterial ribosome, after which they cause tRNA mismatching and thus protein mistranslation.
    This occurs by mismatching between codons and anticodons, which synthesize proteins with incorrect amino acid. This mistranslated protein, along with correctly translated proteins move into move into the periplasm where most of the mistranslated proteins are degraded and some of them are inserted into cytoplasmic membrane. This causes disruption of the membrane, ultimately killing the bacterial cells.
    • Tetracyclines are bacteriostatic and block the binding of tRNAs with the ribosome during translation thus inhibiting protein synthesis. Most of the tetracycline class of drugs are broad spectrum and are active against wide range of bacteria.
  2. Inhibitors binding to the 50S subunit
    • Macrolides are the large class of naturally produced secondary antibiotics. They are basically broad spectrum, bacteriostatic antibiotics. Their main mode of action is blocking peptide chain elongation and they inhibit the formation of peptide bond.
    Patients allergic to penicillins are recommended erythromycin which is a macrolide.
    • Lincosamides include lincomycin and clindamycin. Though they are structurally different but functionally similar to macrolides. They are specifically known to inhibit streptococcal and staphylococcal infections.
    • Chloramphenicol also inhibits peptidyl transferase reaction inhibiting peptide bond formation. It was the first broad spectrum antibiotic and is very much active against a broad range of bacterial pathogens but is very toxic and can cause side.

INHIBITORS OF MEMBRANE FUNCTION
Biological cytoplasmic membranes are basically composed of lipids, proteins and lipoproteins. The cytoplasmic membrane acts as a selective barrier which allows the transport of materials between inside the cell and the environment.
A number of antibacterial agents work by targeting the bacterial cell membrane. They basically are involved in the disorganization of the membrane. Polymyxins and Lipopeptides are the main anti- cell membrane agents.

NUCLEIC ACID SYNTHESIS INHIBITORS
These drugs inhibit nucleic acid synthesis function by either of the following:

  1. Interfere with RNA of bacterial cell
    Antibacterial drugs of this mechanism are selective against bacterial pathogenic cells.
    For example: The drug rifampin, belonging to the drug class rifamycin blocks the bacterial RNA polymerase activity. It is also active against Mycobacterium tuberculosis and thus id used in the treatment of tuberculosis infection. It also shows side effects.
  2. Interfere with DNA of bacterial cell
    There are some antibacterial agents that interfere with the activity of DNA gyrase.
    The drug class fluoroquinolones show this mechanism. They are borad spectrum antibacterial agents. Some examples of drugs in fluoroquinolone family are Ciprofloxacin, Ofloxacin, Moxifloxacin, etc

INHIBITORS OF METABOLIC PATHWAYS
There are some antibacterial drugs which act as ANTIMETABOLITES and inhibits the metabolic pathways of bacteria.
• The sulfonamides block the production of dihydrofolic acid.
This blocks the production of purines and pyrimidines required for nucleic acid synthesis by blocking the biosynthesis of folic acid. Their mechanism of action is bacteriostatic and they are broad spectrum antibacterial agents. Though humans also obtain folic acid but these drugs are selective against bacteria.
Sulfones are also structurally and functionally similar to sulfonamides.
• Trimethoprim is used in the same folic acid synthesis pathway but at a different phase, in the production of tetrahydrofolic acid.
• There is another drug, Isoniazid which is an antimetabolite only selective against mycobacteria. It can also be used to treat tuberculosis when used in combination with rifampin and streptomycin.

INHIBITORS OF ATP SYNTHASE
There is a class of drug compounds called as Diarylquinolones that are specifically active against mycobacterial growth. They block the oxidative phosphorylation process and finally leading to reduced ATP production which either kill or inhibit the growth of mycobacterial species.

TYPHOID FEVER AND IT’S SYMPTOMS

Typhoid Fever or Typhoid is a systemic enteric infection caused by bacteria usually through ingestion of contaminated food or water. The disease is also referred by several other names such as Gastric Fever, Enteric Fever, Abdominal Typhus, Infantile Remittent Fever, Slow Fever, Nervous Fever, and Pythogenic Fever. The disease causes several symptoms causing mild to severe problems for the patients. The symptoms are generally seen from six to thirty days after exposure. Generally there is the gradual onset of high fever for several days, weakness, abdominal pain, constipation, headaches, and vomiting. Some people also develop a skin rash with red colored spots.
The cause of the disease is the bacterium Salmonella typhi which is also known as Salmonella enterica serotype typhi , mainly growing in the intestines and blood. Risk factors include poor sanitation and poor hygiene. Diagnosis of the disease is done by either culturing the bacteria or detecting the bacterium’s DNA in the blood, stool, or bone marrow. Culturing the bacterium is little bit difficult so Bone marrow testing is the mostly used method. It has been observed that a typhoid vaccine can prevent about 40-90% of the infection during the first 2-7 years. But this vaccine is always recommended for the people at high risk or travelling to the places where this disease is so common. Other efforts which can be done to prevent the disease are clean drinking water, good sanitation, and hand washing. The disease is usually treated with several antibiotics such as Azithromycin, Fluoroquinolones or third generation cephalosporins.

SIGNS AND SYMPTOMS
Usually, the complete course of untreated typhoid fever is divided into 4 different stages, where the each stage lasts for a week which makes the patient completely exhausted.
• In the first week, the body temperature of the patient rises slowly, and the fever fluctuations are generally seen with relative bradycardia, malaise, headache, and cough.
A bloody nose is also seen in this quarter of the disease and abdominal pain is also possible. There is also a decrease in the number of WBCs. Widal test is negative in the first week of the disease.

• In the second week, the person is often too tired to get up, with a very high fever and bradycardia is continued in this stage also with dicrotic pulse wave. In this stage, Delirium is frequent which gives the typhoid another name i.e. NERVOUS FEVER.
Rose spots also appear on the lower chest and abdomen. Rhonchi are heard in lung bases.
Diarrhea can occur in this stage : six to eight stools in a day, green, comparable to pea soup, with a characteristic foul smell. However, constipation is also frequent. The spleen and liver is enlarged and liver transaminases are elevated. Patients can still test positive.

The major symptoms of this fever is that the fever usually rises in the afternoon up to the first and second week.

• In the third week of the fever, a number of complications can occur like :
 Intestinal hemorrhage due to bleeding in congested Peyer’s patches: it can be very serious but is not usually fatal.
 Intestinal perforation in the distal ileum: this is a very serious complication and is usually fatal.
 Encephalitis.
 Respiratory diseases such as pneumonia and acute bronchitis.
 Neuropsychiatric symptoms with picking at bedclothes or imaginary objects.
 Metastatic abscesses, cholestasis, endocarditis, and osteitis.
 The fever is usually very high and oscillates very little over 24 hours. Dehydration is also caused.
 Platelet count goes down and risk of bleeding rises.

BASICS OF A MICROBIOLOGY LAB

Microbiology is the study of microbes i.e. the organisms which we can’t see with the naked eyes. Although many microorganisms are beneficial for the human use, some are pathogenic also which causes diseases. Clinical Microbiological Laboratory is concerned with finding of those infectious, pathogenic microbes.

MATERIALS USED IN MICROBIOLOGY LAB
Laminar flow hood, Incubator, Autoclave, Refrigerator, Bunsen Burner, Wire loop, Petri plates, Glass slides, Weighing balance, Media plates, Sensitivity disks, Staining rack, Microscope, Bio safety Cabinet, Centrifuge etc.

INTRODUCTION TO DIFFERENT MEDIA
Some of the media used in the microbiology lab are :

  1. MacCONKEY AGAR : It is the selective and differential media used for the isolation of Gram-negative Bacteria. This media can be used for differentiating Lactose fermenting and Non-lactose fermenting bacteria.
  2. BLOOD AGAR : It is the enriched media for the growth of bacteria such as streptococci.
  3. CHOCOLATE AGAR : It is the lysed Blood Agar. The only difference in blood agar and chocolate agar is that in blood agar RBCs are lysed. This enriched media is suitable for the growth of bacteria that are unable to grow on Blood Agar.
  4. THIOSULFATE CITRATE BILE SALT AGAR (TCBS) : It is the selective as well as differential media for the growth of vibrio cholerae , a causative organism for cholera.

GRAM STAINING
Gram staining is the process for differentiating Gram positive and Gram negative bacteria. When the whole procedure of gram stain is followed and the slide is observed under the microscope, Gram positive bacteria appear Violet in color and Gram negative bacteria appear Pink in color.
For the gram staining we need Glass slide, Normal Saline, Inoculating loop, Bunsen burner, Crystal Violet, Gram’s Iodine, Acetone, Safranine.

PROCESS :

  1. The isolated colony of the microorganism is taken and in the drop of normal saline on the glass slide the colony is mixed with the help of inoculating loop to make a smear. The prepared smear is heat fixed.
  2. A staining rack is taken and on the smear, Crystal Violet is added. After 1 minute, the stain was removed by washing the slide in running water.
  3. After that, Gram’s Iodine is added on the smear as a decolorizing agent which is again washed after 1 minute under the running tap water.
  4. The next step is to add Acetone on the smear which is added in the hand to hand process.
  5. After the decolorisation is done, Safranine is added on the smear which is also washed after 1 minute.
  6. The glass slide is then air dried and observed under the microscope.

RESULTS :
It was observed under the microscope that the Gram positive bacteria appear Violet in color due to Crystal Violet stain whereas Gram negative bacteria appear Pink in color due to safranine.

TESTS ANALYZED
The tests analyzed in the microbiology section of the laboratory are basically the culture and sensitivities tests of urine, stool, sputum, pus swab etc.
The basic procedure of performing all the tests are :

  1. First of all, all the tests are performed inside the laminar flow hood.
  2. The samples collected from the patients and the media plates are kept inside the laminar flow.
  3. The inoculating wire loop is heat sterilized and with the help of it, the samples are cultured or streaked on the media plates.
  4. After inoculation, the cultured media plates are incubated for 24 hours (48 hours if necessary) for allowing the growth of bacteria.
  5. After the growth, staining is done or sensitivities are checked according to the requirement by the doctor.
  6. The report is prepared for the patient.