Acute Otitis Media

This infection is usually caused by S. Pyogenes, Pneumococcus, H. infuenzae, Branhlamella catarrhalis or, in many cases, One of the respiratory tract viruses . The organism spreads to the middle ear via the Eustachian tube from the nasopharynx which is the primary site of infection As the eardrum remains intact, none of the infected exudates can be collected on an ear swab, through culture of a throat swab may give a provisional indication of the causal organism. Antibiotic therapy is urgently required to prevent a possible bacterial infection damaging the hearing mechanism and amoxycillin erythromycin or cotrimoxazole may be used when the causal organism is unknown. Amoxycillin is the drug of choice unless a β-lactamase-producing variety of H. influenzae is the cause, When the absence of a rapid response will indicate the need for a change of drug.
If the eardrum has ruptured spontaneously, or a myringotomy has been performed to relieve pressure exudates may be collected on a thin swab introduced carefully into the external meatus. It should be examined in a Gram film and by aerobic and anaembic Culture plates of heated blood agar and blood agar.

Automated Method for Antibiotics Susceptibility

Automated method In the last several years, a variety of instrument-assisted identification and susceptibility test methods have been developed that permit generation of test results in a period of 6—9 hours. as opposed to the 15—24 hour time frame required with traditional overnight method. These newer ‘rapid” methods have, in general, been shown to provide test results nearly as accurate as those derived from traditional overnight tests, d0t the newer tests are more expensive. The clinical impact of this newer technol­ogy and whether it truly facilitates faster and more cost-effective patient care is undergoing clinical study. One study suggests the rapid tests have a positive impact on patient care. The exact role of rapid tests versus traditions’ awaits further clinical experience and comparative studies 4-Antimicrobial concentration gradient methods -The spiral gradient endpoint method employs an agar plate containing a continuous gradient of antibiotic concentration from the center of the plate to the edge; the test organism is applied to the plate in a radial streak, and the MIC is determined by measuring the distance of growth from the edge of the plate -E test E test (AB Biodisk, Solnia. Sweden) is a method based on the diffusion of a continuous concentration gradient of an antimicrobial agent from a plastic strip into an agar medium . This newly developed in Vitro technique was created to overcome several of the disadvantages of the disc diffusion and dilution techniques and also to retain the principle of the agar dilution method by producing on accurate, reproducible, quantitative MIC result

Antimicrobial Agents Used in Treatment of Infectious Disease

Most microbiologist distinguish two groups of antibiotics agents used in the treatment of infectious disease Antibiotics, which are natural substances produced by certain groups of microorganisms Chemotherapeutic agents, which are chemically synthesized. A hybrid substance is a semisynthetic antibiotic, where in a molecular version produced by the microbe is subsequently modified by the chemist to achieve desired properties Antibiotics may have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that is affected by a certain antibiotic is expressed as its spectrum of action. Antibiotics effective against procaryotes which kill or inhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum If effective mainly against Gram-positive or Gram-negative bacteria, they are narrow spectrum. If effective against a single organism or disease, they are referred to as limited spectrum A clinically-useful antibiotic should have as many of these characteristics as possible -It should have a wide spectrum of activity with the ability to destroy or inhibit many different species of pathogenic organisms. -It should be nontoxic to the host and without undesirable side effects. -It should be no allergenic to the host. -It should not eliminate the normal flora of the host. -It should be able to reach the part of the human body where the infection is occurring. -It should be inexpensive and easy to produce. -It should be chemically-stable (have a long shelf-life). -Microbial resistance is uncommon and unlikely to develop. Microbial Resistance to Antibiotics 1-The Genetic Basis of Bacterial Resistance to Antibiotics -Natural Resistance. Bacteria may be inherently resistant to an antibiotic. Gene that is responsible for resistance to its own antibiotic; or a Gram-negative bacterium has an outer membrane that establishes a permeability barrier against the antibiotic; Organism lacks a transport system for the antibiotic; or it lacks the target or reaction that is hit by the antibiotic. -Acquired Resistance. Bacteria can develop resistance to antibiotics, e.g. Acquired Resistance. Bacteria can develop resistance to antibiotics, e.g. bacterial populations previously-sensitive to antibiotics become resistant. This type of resistance results from changes in the bacterial genome. Acquired resistance is driven by two genetic processes in bacteria: - Mutation and selection (sometimes referred to as vertical evolution); - Exchange of genes between strains and species (sometimes called horizontal evolution). Some bacterial specieses are able to spread drug resistance to other strains and species during genetic exchange processes Acquired Resistance. Bacteria can develop resistance to antibiotics, e.g. bacterial populations previously-sensitive to antibiotics become resistant. This type of resistance results from changes in the bacterial genome. Acquired resistance is driven by two genetic processes in bacteria: - Mutation and selection (sometimes referred to as vertical evolution); - Exchange of genes between strains and species (sometimes called horizontal evolution). Some bacterial specieses are able to spread drug resistance to other strains and species during genetic exchange processes Antifungal Drugs Antifungal drugs are classified according to mode of actions into three groups: -Drugs acting on ergosterol on cell wall of fungus. Azole Terbinafine Amphotricine Polyene-Nystatin -Drugs blocks protein and DNA synthesis -Flucytosine -Drug acting on microtubules and spindle synthesis of DNA Grisoflavin Chemotherapeutic agents for viral infections Agents that inactivate intact viruses (virucidal) Agents that inhibit viral replication at cellular level (antivirals) Agents that augment the host response to infection (immunomodulators) Virucidal agents May cause direct inactivation in a single step Can damage host cells as well as virus so use is limited Can be used in preventing transmission of viral infections Examples include detergents organic solvents ultraviolet light Antiviral agents Viral replication depends on host cell metabolic functions Useful antiviral agents: must inhibit virus-specific events must not interfere with host metabolism which would result in toxicity to the host/person Typically have a restricted spectrum of activity Site of action ِِAattachment to the host cell Uncoating of the viral genome Nucleic acid synthesis Assembly of progeny virions Drugs inhibit ongoing replication at host cell level and ٌReplication will resume on removal of drug Agents are not effective in elimination of non-replicating or latent virus Site of action ِِAattachment to the host cell Uncoating of the viral genome Nucleic acid synthesis Assembly of progeny virions Drugs inhibit ongoing replication at host cell level and ٌReplication will resume on removal of drug Agents are not effective in elimination of non-replicating or latent virus Host Immune Responses and immunomodulators Intact host immunologic response is essential for recovery from viral infections. I t can be used in the following situations; Immunosuppression due to cancer chemotherapy, transplantation or HIV infection are associated with higher rates of chronic viral infection (HBV)or reactivation (HSV) Response to antiviral therapy may be delayed Drug resistance viruses may be higher Replace deficient host immune responses using exogenous antibodies interferon augment cell-mediated immunity (CMI) Antiviral drug resistance Resistance results from mutations within the viral genome and the presence of selective drug pressure Factors favouring emergence of resistance high replicative load high intrinsic mutation rate: RNA>DNA viruses degree of selective drug pressure (higher in prolonged or repeated courses of drug therapy Antiviral drug resistance Resistance results from mutations within the viral genome and the presence of selective drug pressure Factors favouring emergence of resistance high replicative load high intrinsic mutation rate: RNA>DNA viruses degree of selective drug pressure (higher in prolonged or repeated courses of drug therapy Laboratory Methods of Antimicrobial Susceptibility Tests I-Phenotypic Method II-Genotypic Method I-Phenotypic Method: 1-Disc diffusion tests Kirby – Bauer method Tests with diffusion gradients of concentration . By this method the organism is seeded uniformly on the agar surface and exposed to a continuous concentration gradient of antibiotic diffusing from a paper disk (disk diffusion test) Medium used :- Must support good growth of the isolated bacteria e.g Muller – Hunton agar . Blood used in agar may interference with antibiotic activity which are highly bounded to protein . Inoculum's preparation :- Diluted 5-10 colonies in sterile saline or nutrient broth . Colonies used depend on the number of organisms needed to produce semi-confluent growth e.g. – few numbers in rapidly growing bacteria e.g Klebsiella species than slowly growing species e.g enterococci . Inoculation :- The inoculums can be distributed evenly over the test plate by flooding the plate with the bacterial suspension and drying the surface while the plate in horizontal position . By sterile swab , squeezed the tube and run over the plate . By 100p from the suspension of the organism Number of disk 6 in 8.5 cm diameter plate Inoculum's preparation :- Diluted 5-10 colonies in sterile saline or nutrient broth . Colonies used depend on the number of organisms needed to produce semi-confluent growth e.g. – few numbers in rapidly growing bacteria e.g Klebsiella species than slowly growing species e.g enterococci . Inoculation :- The inoculums can be distributed evenly over the test plate by flooding the plate with the bacterial suspension and drying the surface while the plate in horizontal position . By sterile swab , squeezed the tube and run over the plate . By 100p from the suspension of the organism Number of disk 6 in 8.5 cm diameter plate Choose of antibiotic disks :- Members of antibiotics used for particular species by the preferred method . Infections at a particular body site . First line antibiotics that are commonly used are first tested . Isolated of species should not be tested with drugs that are valueless in therapy e.g. Gram positive not to be tested to polymyxin or aztroneam . One antibiotic representive of each group . Interpretation :- Measure the radial diameter around the disc not including the disk itself . Sensitive : Zone equal or larger to the sensitive zone of the control organism Intermediated : the Zone is equal to reported intermediate which equal 3mm less than the control organism . Resistant the zone size of the test strain is smaller than 3mm of the test strain . Limitation of disc diffusion tests:- Not applied to slowly –growing, Fastidious organisms or anaerobes . -Mycobacterial and fungus susceptibility testing requires specific techniques - The reported sensitivity tests results not applied to clinical sites infections, e.g. –Salmonella Typhi to aminoglycosids. -Not related to the achieved serum levels or body fluid levels of antibiotics. Intermediated : the Zone is equal to reported intermediate which equal 3mm less than the control organism . Resistant the zone size of the test strain is smaller than 3mm of the test strain . Limitation of disc diffusion tests:- Not applied to slowly –growing, Fastidious organisms or anaerobes . -Mycobacterial and fungus susceptibility testing requires specific techniques - The reported sensitivity tests results not applied to clinical sites infections, e.g. –Salmonella Typhi to aminoglycosids. -Not related to the achieved serum levels or body fluid levels of antibiotics. Sensitivity of isolated bacteria in vitro may not coordinate with activity in vivo:- Drug is not adequately absorbed . The drug unable to penetrate in effective concentration into the least accessible site of multiplication of pathogen . Inactivation of drug by a concomitant drugresistant bacterium . Resistant to drug may prove effective if :- Administered in high dose . Elimination of bacteria is helped by the immune system of the body. Primary sensitivity tests :- In these tests the specimen serves as the inoculum. When mixed well a portion of it is spread uniformly over part or whole of one or more plates and antibiotics discs are applied before the plates are incubated. Advantages: -Rapid results of susceptibility in the second day e.g day earlier than test on pure subculture. - In mixed culture, help to separate bacteria of different species with different susceptibility patterns. - Primary sensitivity tests :- In these tests the specimen serves as the inoculum. When mixed well a portion of it is spread uniformly over part or whole of one or more plates and antibiotics discs are applied before the plates are incubated. Advantages: -Rapid results of susceptibility in the second day e.g day earlier than test on pure subculture. - In mixed culture, help to separate bacteria of different species with different susceptibility patterns. - Help in rapid identification of bacteria with diagnostic susceptibility patterns e.g. MRSA. - Can be used to isolate pure organisms growth around certain antibiotics as the disc of antibiotic can act as selective media inhibiting the growth of certain bacteria e.g. yeasts around antibiotics discs. Disadvantages: -The primary inoculum's can not be measured. -The choice of antibiotics is difficult as the identity of the organisms is not known, so the choice will be suggestive on the organisms possible to be found. Uses: -Urine -Swabs from wounds or pus in emergency clinics. 2-Dilution susceptibility tests:- Micro-minimal inhibitory and minimal bactericidal activity methods. How to choose MIC ? It is equal to or less than quarter or half the concentration of the antibiotic found in the infected tissues of the patient give the usual schedule of doses . Laboratory it the concentration of MIC measurement of antibiotics that inhibit the growth of isolated organism . MBC measure the concentration of antibiotic that prevent absolutely the bacterial culture . 2-Dilution susceptibility tests:- Micro-minimal inhibitory and minimal bactericidal activity methods. How to choose MIC ? It is equal to or less than quarter or half the concentration of the antibiotic found in the infected tissues of the patient give the usual schedule of doses . Laboratory it the concentration of MIC measurement of antibiotics that inhibit the growth of isolated organism . MBC measure the concentration of antibiotic that prevent absolutely the bacterial culture . Methods 1. Broth dilution tests. Serial, twofold dilutions of an antimicrobial ore incorpo­rated into broth-containing tubes, which are then inoculated with standard number of organisms, usually l05—l06 colony-forming units (CFU) per milliliter. After the culture has been incubated at 35oC for 16—20 hours with traditional technology, the tubes are inspected for visible growth. (Rapid techniques are also available. See sec. E.) The MIC of the drug is the lowest concentration that prevents visible growth. If the tubes with no visible growth are subcultured quantitatively to a drug-free medium, the MBC of the antimicrobial can be determined. also available. See sec. E.) The MIC of the drug is the lowest concentration that prevents visible growth. If the tubes with no visible growth are subcultured quantitatively to a drug-free medium, the MBC of the antimicrobial can be determined. Microdilution susceptibility testing em­ploys the same principles but uses wells on a microtiter tray rather than diludon tubes, permitting miniaturization and automation of the MIC determination Agar dilution test. The agar dilution test is very similar to the broth technique except that the antibiotic dilutions are incorporated into a solid medium and the inoculum, usually 104 CFU/ml, is applied as a spot to a small portion of the agar plate. The MIC again is recorded as the lowest antibiotic concentration that prevents visible growth. In contrast to the broth dilution technique, an MBC cannot be determined with agar dilution Application:- Serious infection where endpoint concentration is ended Disc diffusion yield inter mediate susceptibility Life threatening infection due to organisms with unpredictable susceptibility pattern. Fastidious or slowly growing organisms. Failure of antibiotic therapy Serious infections caused by organisms susceptible only to toxic agents Limitation Limitation -Difficult It needs the knowledge about the achievable level in serum or body fluid Automated method In the last several years, a variety of instrument-assisted identification and susceptibility test methods have been developed that permit generation of test results in a period of 6—9 hours. as opposed to the 15—24 hour time frame required with traditional overnight method. These newer ‘rapid” methods have, in general, been shown to provide test results nearly as accurate as those derived from traditional overnight tests, d0t the newer tests are more expensive. The clinical impact of this newer technol­ogy and whether it truly facilitates faster and more cost-effective patient care is undergoing clinical study. One study suggests the rapid tests have a positive impact on patient care. The exact role of rapid tests versus traditions’ awaits further clinical experience and comparative studies

Streptomyces and antibiotics

Streptomyces are Gram-positive, aerobic, high-GC, filamentous soil bacteria known for the production of secondary metabolites and biologically active materials (Stackebrandt et al., 1991).
Streptomycetes are members of the order Actinomycetales within the classes Actinobacteria (Stackebrandt et al., 1997) and have a DNA G+C content of 69±78 mol% (Korn-Wendisch and Kutzner, 1992).
Streptomycetes produce an extensive branching substrate and aerial mycelium. The substrate hyphae are approximately 0.5–2.0 μm in diameter and often lack cross-walls during the vegetative phase. Growth occurs at the hyphal apices and is accompanied by branching, thus producing a complex tightly woven matrix of hyphae during the vegetative growth phase. As the colony ages, aerial mycelia (sporophores) are produced which develop into chains of spores (conidia) by the formation of crosswalls in the multinucleate aerial filaments. This is followed by separation of individual cells directly into spores (Wildermuth and Hopwood, 1970).
The surface of the conidial wall often has convoluted projections which, together with the shape and the arrangement of the spore-bearing structures, are characteristic of each species and were often used for the separation of Streptomyces species (Pridham et al., 1958; Korn-Wendisch and Kutzner, 1992). They can be distinguished from other actinomycetes by their cell wall type which is characterized as Type I sensu (Lechevalier and Lechevalier, 1970b).
Besides antibiotics, which present the largest group of bioactive secondary metabolites, the streptomycete compounds show several other biological activities . The secondary metabolites from streptomycetes can be broadly separated into four classes according to their biological activity: (1) antagonistic agents, including antibacterials, antifungals, antiprotozoans as well as antivirals, (2) pharmacological agents, including antitumorals, immunomodulators, neurological agents and enzyme inhibitors, (3) agrobiologicals, including insecticides, pesticides and herbicides, and (4) compounds with regulatory activities, such as growth factors, siderophores or morphogenic agents. To detect simultaneous bioactivities for a given compound, pharmacological and agricultural screens are increasingly being used in combination with antimicrobial tests. This has revealed several novel therapeutic and agrobiological agents and previously unknown biological activities for antibiotics (Sanglier et al., 1996; Bérdy, 2005). Many reports have shown that since streptomycetes are frequently screened for antimicrobial activity, the existence of secondary metabolites with other activities may have been missed (Garcia et al., 2000; Nunes et al., 2005).

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