Disease Control Measures and Microbial Diagnostics.pptx

Disease Control Measures and Microbial Diagnostics

1. Disease Control Measures and Microbial Diagnostics Unit 5

2. INTRODUCTION Infections and diseases may be caused by different types of organisms like bacteria, fungi, and viruses, etc., in humans and animals. The drug used to prevent the pathogenicity of microorganisms is called an  antimicrobial agent . Examples: Antibiotics, antiseptics, and disinfectants.

3. TYPES Antimicrobial agents are used to preventing infections and diseases caused by pathogens. Different types of antimicrobial drugs are commonly available. These are as follows: Antibacterial drug:   A drug that is used to inhibit the pathogenic activity of bacteria is called an antibacterial drug. Example: Zithromax. Antifungal drug:   A drug that is used to prevent fungal activity in the host is called an antifungal drug. Example: Miconazole Antiviral agent:   A drug which is used to stop the pathogenic action of a virus is called antiviral agents. Example: Tamiflu. Antiparasitic drug:   A drug that is used to prevent the growth of pathogenic parasites. Example: Anthelmintics

4. ANTIBIOTICS Antibiotics are the substances which are derived from one microorganism in order to kill another microorganism. Antibiotics are effective against bacterial, fungal and parasitic infections. But, antibiotics are not helpful against viral infections. The development of chemical synthesis has helped to produce the synthetic components which act as an antimicrobial agent against the pathogenic bacteria. These synthetic components are also called as antibiotics. Pathogenic bacteria can be killed by synthetic components at low concentrations. Examples: Ampicillin and amoxicillin. In 1908, a German bacteriologist, Paul Ehrlich developed a synthetic component from an arsenic-based structure for the treatment of syphilis, which is called as arsphenamine or salvarsan . Then, in 1929, Alexander Fleming discovered Penicillin from the fungus  Penicillium notatum . Penicillin is used to treat different types of bacterial infections.

5. TWO TYPES OF ANTIBIOTICS ARE COMMONLY AVAILABLE. These are as follows: Bactericidal antibiotics –   These antibiotics had killing effects on bacteria. Example: Penicillin, Aminoglycosides, Ofloxacin . Bacteriostatic antibiotics –  These antibiotics have an inhibitory effect on bacteria. Example: Erythromycin, Tetracycline, Chloramphenicol. Depending on the spectrum of action, antibiotics are further classified into three types. These are as follows: Broad-spectrum antibiotics:  These antibiotics are widely used to kill or inhibit the Gram-positive and Gram-negative bacteria. Example: Chloramphenicol Narrow spectrum antibiotics :  These antibiotics are widely effective against specific groups of bacteria. Example: Penicillin G Limited spectrum antibiotics:  These antibiotics are effective against a single organism or a single disease. Example: Dysidazirine .

6. Antiseptics and Disinfectants Antiseptics and disinfectants are the chemical components which are used as antimicrobial agents. Antiseptics are applied to the injured tissues, cuts, and infected skin surfaces. Antiseptics are not prescribed to be taken orally. A few examples are given below: Dettol –  It is a mixture of chloroxylenol and terpineol . It is used to apply in the wounds. Iodine tincture and iodoform –  It has very good antiseptic properties. Boric acid  –  It is used as an antiseptic agent for eyes. Disinfectants are used to destroy the pathogenic microorganisms in the non-living objects such as floors and drainage systems. Example: Chlorine and sulphur dioxide at low concentration.

7. CLASSIFICATION AND MECHANISM OF DRUG ACTION Inhibition of cell wall synthesis, Inhibition of Nucleic acid synthesis, Inhibition of Protein synthesis, Inhibition of Enzyme action and metabolism;

8. INHIBITION OF CELL WALL SYNTHESIS beta-lactam antibiotic : A broad class of antibiotics that inhibit cell wall synthesis, consisting of all antibiotic agents that contains a β- lactam nucleus in their molecular structures. This includes penicillin derivatives ( penams ), cephalosporins ( cephems ), monobactams , and carbapenems . Glycopeptide antibiotic : Glycopeptide antibiotics are composed of glycosylated cyclic or polycyclic nonribosomal peptides. Significant glycopeptide antibiotics include vancomycin , teicoplanin , telavancin , bleomycin , ramoplanin , and decaplanin . This class of drugs inhibit the synthesis of cell walls in susceptible microbes by inhibiting peptidoglycan synthesis. peptidoglycan : A polymer of glycan and peptides found in bacterial cell walls. Two types of antimicrobial drugs work by inhibiting or interfering with cell wall synthesis of the target bacteria. Antibiotics commonly target bacterial cell wall formation (of which peptidoglycan is an important component) because animal cells do not have cell walls. The peptidoglycan layer is important for cell wall structural integrity, being the outermost and primary component of the wall. The first class of antimicrobial drugs that interfere with cell wall synthesis are the β- Lactam antibiotics (beta-lactam antibiotics), consisting of all antibiotic agents that contains a β- lactam nucleus in their molecular structures. This includes penicillin derivatives ( penams ), cephalosporins ( cephems ), monobactams , and carbapenems . β- Lactam antibiotics are bacteriocidal and act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls. The final step in the synthesis of the peptidoglycan is facilitated by penicillin-binding proteins (PBPs). PBPs vary in their affinity for binding penicillin or other β- lactam antibiotics.

9. Bacteria often develop resistance to β- lactam antibiotics by synthesizing a β- lactamase, an enzyme that attacks the β- lactam ring. To overcome this resistance, β- lactam antibiotics are often given with β- lactamase inhibitors such as clavulanic acid . The second class of antimicrobial drugs that interfere with cell wall synthesis are the glycopeptide antibiotics, which are composed of glycosylated cyclic or polycyclic nonribosomal peptides. Significant glycopeptide antibiotics include vancomycin , teicoplanin , telavancin , bleomycin , ramoplanin , and decaplanin . This class of drugs inhibit the synthesis of cell walls in susceptible microbes by inhibiting peptidoglycan synthesis. They bind to the amino acids within the cell wall preventing the addition of new units to the peptidoglycan.

10. INHIBITION OF NUCLEIC ACID SYNTHESIS Some antimicrobial drugs interfere with the initiation, elongation or termination of RNA transcription. Some antimicrobial drugs interfere with various aspects of DNA replication. The antimicrobial actions of these drugs are a result of differences in the prokaryotic and eukaryotic enzymes involved in nucleic acid synthesis.

11. Contd …. Antimicrobial drugs can target nucleic acid (either RNA or DNA) synthesis. The antimicrobial actions of these agents are a result of differences in prokaryotic and eukaryotic enzymes involved in nucleic acid synthesis. Prokaryotic transcription is the process in which messenger RNA transcripts of genetic material are produced for later translation into proteins. The transcription process includes the following steps: initiation, elongation and termination. Antimicrobial drugs have been developed to target each of these steps. For example, the antimicrobial rifampin binds to DNA-dependent RNA polymerase , thereby inhibiting the initiation of RNA transcription. Other antimicrobial drugs interfere with DNA replication, the biological process that occurs in all living organisms and copies their DNA and is the basis for biological inheritance. The process starts when one double-stranded DNA molecule produces two identical copies of the molecule. In a cell, DNA replication begins at specific locations in the genome, called “origins. ” Uncoiling of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination. Any of the steps in the process of DNA replication can be targeted by antimicrobial drugs. For instance, quinolones inhibit DNA synthesis by interfering with the coiling of DNA strands.

12. INHIBITION OF PROTEIN SYNTHESIS Protein synthesis is a complex, multi-step process involving many enzymes as well as conformational alignment. However, the majority of antibiotics that block bacterial protein synthesis interfere with the processes at the 30S subunit or 50S subunit of the 70S bacterial ribosome. P rocess that are attacked the formation of the 30S initiation complex (made up of mRNA, the 30S ribosomal subunit, and formyl - methionyl -transfer RNA ) are affeced ( 2) the formation of the 70S ribosome and the 50S ribosome is inhered, ( 3) the elongation process of assembling amino acids into a polypeptide. Tetracyclines , including  doxycycline , prevent the binding of aminoacyl-tRNA by blocking the A ( aminoacyl ) site of the 30S ribosome. They are capable of inhibiting protein synthesis in both 70S and 80S (eukaryotic) ribosomes, but they preferentially bind to bacterial ribosomes due to structural differences in RNA subunits. Additionally, tetracyclines are effective against bacteria by exploiting the bacterial transport system and increasing the concentration of the antibiotic within the cell to be significantly higher than the environmental concentration .

13. Contd ….. Aminoglycoside antibiotics have an affinity for the 30S ribosome subunit.  Streptomycin , one of the most commonly used aminoglycosides, interferes with the creation of the 30S initiation complex.  Kanamycin  and  tobramycin  also bind to the 30S ribosome and block the formation of the larger 70S initiation complex. Erythromycin , a macrolide, binds to the 23S rRNA component of the 50S ribosome and interferes with the assembly of 50S subunits. Erythromycin, roxithromycin , and clarithromycin all prevent elongation at the transpeptidation step of synthesis by blocking the 50S polypeptide export tunnel. Elongation is prematurely terminated after a small peptide has been formed but cannot move past the macrolide roadblock. Peptidyl transferase is a key enzyme involved in translocation, the final step in the peptide elongation cycle.  Lincomycin  and clindamycin are specific inhibitors of peptidyl transferase , while macrolides do not directly inhibit the enzyme.  Puromycin  does not inhibit the enzymatic process, but instead competes by acting as an analog of the 3′-terminal end of aminoacyl-tRNA , disrupting synthesis and causing premature chain termination. Hygromycin B  is an aminoglycoside that specifically binds to a single site within the 30S subunit in a region that contains the A, P, and E sites of tRNA . It has been theorized that this binding distorts the ribosomal A site and may be the cause of the ability of hygromycin to induce misreading of aminoacyl-tRNAs as well as prevent the translocation of peptide elongation.

14. INHIBITION OF METABOLISM

15. Types of drugs – Antibacterial Antifungal A ntiprotozoans

16. Antifungal drugs Fungi can be found throughout the world in all kinds of environments. Most fungi don’t cause disease in people. However, some species can infect humans and cause illness. Antifungal drugs are medications that are used to treat  fungal infections . While most fungal infections affect areas such as the skin and nails, some can lead to more serious and potentially life threatening conditions like  meningitis  or  pneumonia . There are several types of antifungal drugs available to fight fungal infections.

17. Types of drugs – Antifungal A ntifungal drugs can work in two ways: by directly killing fungal cells or by preventing fungal cells from growing and thriving. Antifungal drugs target structures or functions that are necessary in fungal cells but not in human cells, so they can fight a fungal infection without damaging your body’s cells. Two structures that are commonly targeted are the fungal cell membrane and the fungal cell wall. Both of these structures surround and protect the fungal cell. When either one becomes compromised, the fungal cell can burst open and die.

18. Types Azoles: Azoles are some of the most commonly used antifungals. They interfere with an enzyme that’s important for creating the fungal cell membrane. Because of this, the cell membrane becomes unstable and can leak, eventually leading to cell death. Example 1: Clotrimazole and Miconazole :  skin and mucous membrane infections Example 2: Isavuconazole :   aspergillosis and  mucormycosis Polyenes : Polyenes kill fungal cells by making the fungal cell wall more porous, which makes the fungal cell prone to bursting . Example 1 : Amphotericin B:  various formulations are available to treat aspergillosis , blastomycosis , cryptococcosis , histoplasmosis (off-label), mucosal or invasive Candida infections, and coccidioidomycosis Example 2 : Nystatin :  Candida infections of the skin and mouth

19. Contd ……. Allylamines : Like the azole antifungals, allylamines interfere with an enzyme that’s involved in the creation of the fungal cell membrane. One example of an allylamine is terbinafine , which is often used to treat fungal infections of the skin. Echinocandins : Echinocandins are a newer type of antifungal drug. They inhibit an enzyme that’s involved in the making of the fungal cell wall . Example 1: Anidulafungin : mucosal and invasive Candida infections Example 2: Caspofungin : mucosal and invasive Candida infections, aspergillosis Example 3: Micafungin : mucosal and invasive Candida infections Miscellaneous There are also some other types of antifungal medications. These have mechanisms different from the types we’ve discussed above. Flucytosine  is an antifungal that prevents the fungal cell from making nucleic acids and proteins. Because of this, the cell can no longer grow and thrive. Flucytosine can be used to treat systemic infections with  Candida  or  Cryptococcus  species. Griseofulvin  works to prevent the fungal cell from dividing to produce more cells. It can be used to treat infections of the skin, hair, and nails.

20. ANTIPROTOZOAL DRUG Antiprotozoal drug , any agent that kills or inhibits the growth of organisms known as  protozoans . Protozoans cause a variety of diseases, including  malaria and   Chagas ’ disease . While protozoans typically are microscopic, they are similar to  plants  and  animals  in that they are  eukaryotes  and thus have a clearly defined cell  nucleus . This distinguishes them from  prokaryotes , such as  bacteria . As a result, many of the  antibiotics  that are effective in inhibiting bacteria are not active against protozoans. Metronidazole is usually given orally for the  treatment  of vaginal infections caused by  Trichomonas vaginalis , and it is effective in treating bacterial infections caused by anaerobes (organisms that can survive without oxygen). It affects these organisms by causing nicks in, or breakage of, strands of  DNA  or by preventing DNA replication. Metronidazole is also the drug of choice in the treatment of  giardiasis , an infection of the intestine caused by a flagellated  amoeba . Iodoquinol inhibits several  enzymes  of protozoans. It is given orally for treating asymptomatic  amoebiasis and is given either by itself or in combination with metronidazole for intestinal and hepatic amoebiasis . Trypanosomes  are flagellated protozoans that cause a number of diseases.  Trypanosoma cruzi , the causative agent of Chagas ’ disease, is treated with nifurtimox , a nitrofuran derivative. It is given orally and results in the production of activated forms of oxygen, which are lethal to the parasite. Other forms of  trypanosomiasis  (African trypanosomiasis , or  sleeping sickness ) are caused by  T. brucei gambiense  or  T. brucei rhodesiense . When these parasites invade the  blood  or  lymph , the drug of choice for either form is  suramin , a nonmetallic  dye  that affects  glucose  utilization and hence energy production. Because suramin is not absorbed from the  gastrointestinal tract , it is given by intravenous injection.

21. Contd.. Pneumocystis carinii  causes pulmonary disease in immunocompromised patients. These infections are treated with trimethoprim- sulfamethoxazole , which inhibits  folic acid  synthesis in protozoans. An alternative agent for treatment of these diseases is  pentamidine isethionate , which probably affects the parasite by binding to DNA. Chloroquine  phosphate , given orally, is a  drug  used for the prevention and treatment of uncomplicated cases of  malaria , which is caused by species of  Plasmodium . In regions where chloroquine -resistant  P. falciparum  is encountered, mefloquine or doxycycline may be used for prevention of the disease. Infection with chloroquine -resistant  P. falciparum  may be treated with  quinine  sulfate, often in combination with  pyrimethamine  and sulfadoxine , or with  artemisinin , in combination with agents such as mefloquine or amodiaquine . A high level of quinine in the plasma frequently is associated with  cinchonism , a mild adverse reaction associated with such symptoms as a ringing noise in the ears ( tinnitus ), headache, nausea, abdominal pain, and visual disturbance.  Primaquine  phosphate is given orally to prevent malaria after a person has left an area where  P. vivax  and  P. ovale  are endemic and to prevent relapses with the same organisms.

22. Antibacterial drugs Antibacterial drugs are derived from bacteria or molds or are synthesized de novo. Technically, “antibiotic” refers only to antimicrobials derived from bacteria or molds but is often (including in THE MANUAL) used synonymously with “antibacterial drug.” Antibiotics have many mechanisms of action, including the following: Inhibiting cell wall synthesis Increasing cell membrane permeability Interfering with protein synthesis, nucleic acid metabolism, and other metabolic processes ( eg , folic acid synthesis) Antibiotics sometimes interact with other drugs, raising or lowering serum levels of other drugs by increasing or decreasing their metabolism or by various other mechanisms. The most clinically important interactions involve drugs with a low therapeutic ratio ( ie , toxic levels are close to therapeutic levels). Also, other drugs can increase or decrease levels of antibiotics . Many antibiotics are chemically related and are thus grouped into classes. Although drugs within each class share structural and functional similarities, they often have different pharmacology and spectra of activity.

23. Contd ….   Antibiotic should be used only if clinical or laboratory evidence suggests bacterial infection. Use for viral illness or undifferentiated fever is inappropriate in most cases; it exposes patients to drug complications without any benefit and contributes to bacterial resistance. Certain bacterial infections ( eg , abscesses, infections with foreign bodies) require surgical intervention and do not respond to antibiotics alone. In general, clinicians should try to use antibiotics with the narrowest spectrum of activity and for the shortest duration. In vivo  antibiotic effectiveness is affected by many factors, including Pharmacokinetics : The time course of antibiotic levels, which are affected by factors such as  absorption ,  distribution  (concentration in fluids and tissues, protein binding),  rate of metabolism , and  excretion Pharmacodynamics : The antimicrobial activity of local antibiotic concentrations on the target pathogen and that pathogen's response including resistance Presence of foreign materials Control of source of infection Drug interactions  or inhibiting substances Host defense mechanisms

24. Resistance to an antibiotic Resistance to an antibiotic may be inherent in a particular bacterial species or may be acquired through mutations or acquisition of genes for antibiotic resistance that are obtained from another organism. Different mechanisms for resistance are encoded by these genes. Resistance genes can be transmitted between 2 bacterial cells by the following mechanisms: Transformation (uptake of naked DNA from another organism) Transduction (infection by a bacteriophage) Conjugation (exchange of genetic material in the form of either plasmids, which are pieces of independently replicating extrachromosomal DNA, or transposons, which are movable pieces of chromosomal DNA) Plasmids and transposons can rapidly disseminate resistance genes. Antibiotic use preferentially eliminates nonresistant bacteria, increasing the proportion of resistant bacteria that remain. Antibiotic use has this effect not only on pathogenic bacteria but also on normal microbiota ; resistant normal microbiota can become a reservoir for resistance genes that can spread to pathogens.

25. Contd …. Bactericidal  drugs kill bacteria.  Bacteriostatic  drugs slow or stop in vitro bacterial growth. These definitions are not absolute; bacteriostatic drugs may kill some susceptible bacterial species, and bactericidal drugs may only inhibit growth of some susceptible bacterial species. More precise quantitative methods identify the minimum in vitro concentration at which an antibiotic can inhibit growth (minimum inhibitory concentration [MIC]) or kill (minimum bactericidal concentration [MBC]). An antibiotic with bactericidal activity may improve bacterial killing when host defenses are impaired locally at the site of infection ( eg , in meningitis or endocarditis) or systemically ( eg , in patients who are neutropenic or immunocompromised in other ways). However , there are limited clinical data indicating that a bactericidal drug should be selected over a bacteriostatic drug simply on the basis of that classification. Drug selection for optimal efficacy should be based on how the drug concentration varies over time in relation to the MIC rather than whether the drug has bactericidal or bacteriostatic activity.

26. Time vs concentration of a single dose of a theoretical antibiotic Antibiotics can be grouped into 3 general categories ( 1 ) based on the pharmacokinetics that optimizes antimicrobial activity (pharmacodynamics): Concentration-dependent: The  magnitude  by which the peak concentration exceeds the MIC (typically expressed as the peak-to-MIC ratio) best correlates with antimicrobial activity Time-dependent: The  duration  of the dosing interval in which the antibiotic concentration exceeds the MIC (typically expressed as the percentage of time above MIC) best correlates with antimicrobial activity Exposure-dependent: The  amount of drug  given relative to the MIC (the amount of drug is the 24-hour area under the concentration-time curve [AUC24]; the AUC24-to-MIC ratio best correlates with antimicrobial activity)

27. Examples Aminoglycoside, fluoroquinolones , and  daptomycin  exhibit concentration-dependent bactericidal activity. Increasing their concentrations from levels slightly above the MIC to levels far above the MIC increases the rate and extent of their bactericidal activity. In addition, if concentrations exceed the MIC even briefly, aminoglycosides and fluoroquinolones have a post-antibiotic effect (PAE) on residual bacteria; duration of PAE is also concentration-dependent. If PAEs are long, drug levels can be below the MIC for extended periods without loss of efficacy, allowing less frequent dosing. Consequently, aminoglycosides and fluoroquinolones are usually most effective as intermittent boluses that reach peak free serum levels ( ie , the portion of the antibiotic not bound to serum protein) ≥ 10 times the MIC of the bacteria; usually, trough levels are not important. Beta-lactams , clarithromycin, and erythromycin  exhibit time-dependent bactericidal activity. Increasing their free serum concentration above the MIC does not increase their bactericidal activity, and their in vivo killing is generally slow. In addition, because there is no or very brief residual inhibition of bacterial growth after concentrations fall below the MIC ( ie , minimal post-antibiotic effect), beta-lactams are most often effective when serum levels of free drug (drug not bound to serum protein) exceed the MIC for ≥ 50% of the time. Because ceftriaxone has a long serum half-life (about 8 hours), free serum levels exceed the MIC of very susceptible pathogens for the entire 24-hour dosing interval. However, for beta-lactams that have serum half-lives of ≤ 2 hours, frequent dosing or continuous infusion is required to optimize the time above the MIC.

28. I n vitro and in vivo methods of testing drug sensitivity Kirby Bauer, Broth dilution and checker board method

29. Kirby Bauer Aim: Determine the susceptibility of various bacterial species to various antibiotics and synthetic agents. A true antibiotic is an antimicrobial chemical produced by microorganisms against other microorganisms. Mankind has made very good use of these antimicrobials in its fight against infectious disease. Many drugs are now completely synthetic or the natural drug is manipulated to change its structure somewhat, the latter called semisynthetics . Bacteria respond in different ways to antibiotics and chemosynthetic drugs, even within the same species. For example, Staphylococcus aureus is a common normal flora bacterium found in the body. If one isolated this bacterium from 5 different people, the 5 isolates would likely be different strains, that is, slight genetically different. It is also likely that if antibiotic sensitivity tests were run on these isolates, the results would vary against the different antibiotics used. The Kirby-Bauer test for antibiotic susceptibility (also called the  disc diffusion test ) is a standard that has been used for years. First developed in the 1950s, it was refined and by W. Kirby and A. Bauer, then standardized by the World Health Organization in 1961. It has been superseded in clinical labs by automated tests. However, the K-B is still used in some labs, or used with certain bacteria that automation does not work well with. This test is used to determine the resistance or sensitivity of aerobes or facultative anaerobes to specific chemicals, which can then be used by the clinician for treatment of patients with bacterial infections. The presence or absence of an inhibitory area around the disc identifies the bacterial sensitivity to the drug.

30. Contd …… The bacterium is swabbed on the agar and the antibiotic discs are placed on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc. If the organism is killed or inhibited by the concentration of the antibiotic, there will be  NO growth  in the immediate area around the disc: This is called the  zone of inhibition  (Figure 9.1). The zone sizes are looked up on a standardized chart to give a result of sensitive, resistant, or intermediate. Many charts have a corresponding column that also gives the  MIC  (minimal inhibitory concentration) for that drug. The  MIC  is currently the standard test run for antibiotic sensitivity testing because it produces more pertinent information on minimal dosages. The  Mueller-Hinton medium  being used for the Kirby-Bauer test is very high in protein(non differential media and non selective media).

31. Protocol Swab a Mueller-Hinton plate with  ONLY   2  of the bacteria (tables will run different combinations of the 4 bacteria). Dip a sterile swab into the broth and express any excess moisture by pressing the swab against the side of the tube. Swab the surface of the agar  completely  ( you do not want to   leave any unswabbed agar areas at all ). In the pictures below, you can see what happens when the plate is not swabbed correctly with even coverage of the bacterium over the entire agar. After completely swabbing the plate,  turn it 90 degrees and repeat the swabbing process . (It is not necessary to re-moisten the swab.) Run the swab around the circumference of the plate before discarding it in the discard bag. Allow the surface to dry for about 5 minutes before placing antibiotic disks on the agar. You are using individual antibiotic dispensers. will probably have to use a pair of forceps to remove an antibiotic disc from the dispenser: the forceps have to be sterile. Place the forceps in alcohol, flame the forceps until they catch on fire, let the flame go out---- sterile forceps . Lightly touch each disc with your sterile inoculating loop to make sure that it is in  good   contact with the agar surface . Incubate upside down and incubate at 37 o   C.

32. INTERPRETATION Place the metric ruler across the zone of inhibition, at the widest diameter, and measure from one edge of the zone to the other edge. HOLDING THE PLATE UP TO THE LIGHT MIGHT HELP. Use millimeter measurements. The disc diameter will actually be part of that number. If there is NO zone at all, report it as 0---even though the disc itself is around 7 mm. Zone diameter is reported in millimeters, looked up on the  chart , and result reported as sensitive, resistant, or intermediate.

33. Broth Dilution method The  lowest concentration at which the isolate  is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration (MIC). Minimum inhibitory concentration (MIC) is determined when a patient does not respond to treatment thought to be adequate, relapses while being treated or when there is immunosuppression. Dilution methods can be carried out in 2 ways;  broth dilution and agar dilution.

34. Contd.. Broth dilution testing allows providing both quantitative (MIC) and qualitative (category interpretation) results. MIC can help establish the level of resistance of a particular bacterial strain and can substantially affect the decision to use certain antimicrobial agents. Broth dilution can again be performed in 2 ways. Macro dilution:  Uses broth volume of  1 ml  in standard test tubes. Microdilution : Uses about  0.05 to 0.1 ml  total broth volume and can be performed in a microtiter plate or tray. The procedure for macro and microdilution is the same except for the volume of the broth. MIC of an antibiotic using the broth dilution method is determined by using the following procedure  Preparation of antibiotic stock solution Preparation of antibiotic dilution range Preparation of agar dilution plates Preparation of inoculum Inoculation Incubation Reading and interpreting results

35. Preparation of antibiotic dilution range Use sterile 13- x 100-mm test tubes to conduct the test. If the tubes are to be saved for later use, be sure they can be frozen. Close the tubes with loose screw caps, plastic or metal closure caps, or cotton plugs. Prepare the final two-fold (or other) dilutions of antimicrobial agent volumetrically in the broth. A minimum final volume of 1 mL of each dilution is needed for the test . For microdilution , only 0.1 ml is dispensed into every 96 wells of a standard tray.

36. 2. Preparation of inoculum Prepare the inoculum by making a direct broth suspension of isolated colonies selected from an 18- to 24-hour agar plate (use a non-selective medium, such as  blood agar ). Adjust the suspension to achieve turbidity equivalent to a  0.5 McFarland turbidity standard . This results in a suspension containing approximately 1 to 2 x 10^8 colony forming units (CFU)/mL for Escherichia coli  ATCC®a 25922. Compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines. Optimally within 15 minutes of preparation, dilute the adjusted inoculum suspension in broth so, after inoculation, each tube contains approximately 5 x 10^5 CFU/ mL.  Note: This can be accomplished by diluting the 0.5 McFarland suspension 1:150, resulting in a tube containing approximately 1 x 10^6 CFU/ mL. The subsequent 1:2 dilution in step 3 brings the final inoculum to 5 x 10^5 CFU/ mL. 3. Inoculation Within 15 minutes after the inoculum has been standardized as described above, add 1 mL of the adjusted inoculum to each tube containing 1 mL of antimicrobial agent in the dilution series (and a positive control tube containing only broth), and mix. This results in a 1:2 dilution of each antimicrobial concentration and a 1:2 dilution of the inoculums.

37. Contd …. 4. Incubation Incubate the inoculated tubes at 35 ± 2 ºC for 16 to 20 hours in an ambient air incubator. To maintain the same incubation temperature for all cultures, do not stack microdilution trays more than four high . 5. Interpretation Compare the amount of growth in the wells or tubes containing the antimicrobial agent with the growth in the growth-control wells or tubes (no antimicrobial agent) used in each set of tests when determining the growth endpoints. For a test to be considered valid, acceptable growth (≥ 2 mm button or definite turbidity) must occur in the growth-control well.

38. Antimicrobial Synergy Testing/Checkerboard Assay The antibacterial synergy test/checkerboard test is an experimental method used to evaluate the interaction of two antibacterial test products. In this assay, the MIC and MBC values of the test compound are used alone, and combined with the MIC and MBC values of each bacterial strain evaluated to calculate the accumulation. Synergy measurements by checkerboard analysis can be used to determine the change in antibacterial efficacy of a combination of antibiotics relative to their individual activity. The comparison is then calculated as a fractional inhibitory concentration (FIC) index value. FIC index value shows the combination of antibiotics with the greatest change compared to the MIC of a single antibiotic. The figure below shows an example of a checkerboard test method in which the synergistic activity of kanamycin and ampicillin is determined.

39. P rocedure Before the test, a stock solution of each drug and two consecutive dilutions were prepared, leading to at least double the  MIC . A total of 50μl of Mueller-Hinton broth was dispensed into each well of the microdilution plate. The first antibiotic in the combination is diluted sequentially along the ordinate, while the second drug is diluted along the abscissa. An inoculum equal to 0.5 McFarland turbidity standard was prepared from each Pseudomonas aeruginosa isolate in Mueller-Hinton broth. Inoculate 100μl of 5 x 10 5  CFU/ml bacterial inoculum in each microtiter well, and incubate the plate at 35°C for 48 hours under aerobic conditions. The resulting checkerboard contains each combination of the two antibiotics, and the test tube with the highest concentration of each antibiotic in the opposite corner. In order to quantify the interaction between the tested antibiotics (FIC index), use the following formula to calculate: A /MIC A + B/MIC B = FIC A + FIC B= FIC Index Where A and B are the MICs of each antibiotic combined (in one well), and MIC A and MIC B are the MICs of each drug, respectively. Then, the FIC Index value is used to classify and judge the interaction of the two tested antibiotics. When the FIC value is less than 0.5, the two antibiotics have a synergistic effect; when FIC> 4, the two antibiotics are antagonistic; when the FIC is 0.5-4, the two are shown as additive or indifference.

40. Antimicrobial Synergy Testing Services Standard bacteria culturing Antimicrobial synergy testing Statistics analysis of experimental results Advantages Ensure high efficiency and quality for antimicrobial synergy detection services Competitive price in the market of antimicrobial synergy testing services Ensure 24/7 online service Timely result feedback