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Dr.T.V.Rao MD Dr.T.V.Rao MD 1 Uses of Antibiotic Sensitivity Testing test: A laboratory Antibiotic sensitivity test which determines how effective antibiotic therapy is against a bacterial infections. Antibiotic sensitivity testing will control the use of Antibiotics in clinical practice Testing will assist the clinicians in the choice of drugs for the treatment of infections. Dr.T.V.Rao MD 2 What is the goal of Antibiotic Sensitivity testing? The goal of antimicrobial susceptibility testing is to predict the in vivo success or failure of antibiotic therapy. Tests are performed in vitro, and measure the growth response of an isolated organism to a particular drug or drugs. The tests are performed under standardized conditions so that the results are reproducible. The test results should be used to guide antibiotic choice. The results of antimicrobial susceptibility testing should be combined with clinical information and experience when selecting the most appropriate antibiotic for our patients. Dr.T.V.Rao MD 3 Components of Antibiotic Sensitivity Testing 1.The identification of relevant pathogens in exudates and body fluids collected from patients 2. Sensitivity tests done to determine the degree of sensitivity or resistance of pathogens isolated from patient to an appropriate range of antimicrobial drugs 3. Assay of the concentration of an administered drug in the blood or body fluid of patient required to control the schedule of Dr.T.V.Rao MD 4 dosage. Why Need Continues for Testing Antibiotic Sensitivity Bacteria have the ability to develop resistance following repeated or subclinical (insufficient) doses, so more advanced antibiotics and synthetic antimicrobials are continually required to overcome them. Antibiotic sensitivity testing is essential part of Medical Care Dr.T.V.Rao MD 5 Introduction Susceptibility test, main purposes: As a guide for treatment Sensitivity of a given m.o. to known conc. of drugs Its concentration in body fluids or tissues As an epidemiological tool The emergence of resistant strains of major pathogens (e. g. Shigella, Salmonella typhi) Continued surveillance of the susceptibility pattern of the prevalent strains (e. g. Staphylococci, Gram- negative bacilli) Dr.T.V.Rao MD 6 Introduction Methods for antimicrobial susceptibility testing Indirect method cultured plate from pure culture Direct method Pathological specimen e.g. urine, a positive blood culture, or a swab of pus Dr.T.V.Rao MD 7 What Does the Laboratory Need to Know about Antimicrobial Susceptibility Testing (AST) ? Which organisms to test? What methods to use? What antibiotics to test? How to report results? Dr.T.V.Rao MD 8 Routine Susceptibility Tests Disk diffusion (Kirby Bauer) Broth micro- dilution MIC NCCLS reference method Etest Dr.T.V.Rao MD 9 Preparing for Testing Inoculum preparation - Number of test organisms can be determined using different methods: Direct count (Microscopic examination) The optical density (OD) at 600 nm (Spectrophotometry) Plate count: making dilution first Turbidity standard (McFarland) routinely performed. Dr.T.V.Rao MD 10 Choosing the Appropriate Antibiotic Drugs for routine susceptibility tests: Set 1: the drugs that are available in most hospitals and for which routine testing should be carried out for every strain Set 2: the drugs that are tested only: at the special request of the physician or when the causative organism is resistant to the first- choice drugs or when other reasons (allergy to a drug, or its unavailability) make further testing justified Dr.T.V.Rao MD 11 Table 1: Basic sets of drugs for routine susceptibility tests (http://w3.whosea.org/) Set 1 Set 2 Staphylococcus Benzyl penicillin Gentamicin Oxacillin Amikacin Erythromycin Co-trimoxazole Tetracycline Clindamycin Chloramphenicol Intestinal Ampicillin Norfloxacin Chloramphenicol Co-trimoxazole Nalidixic acid Tetracycline Enterobacteriaceae Sulfonamide Norfloxacin Urinary Trimethoprim Chloramphenicol Co-trimoxazole Gentamicin Ampicillin Nitrofurantoin Nalidixic acid Tetracycline Blood and tissues Ampicillin Cefuroxime Chloramphenicol Ceftriaxone Cotrimoxazole Ciprofloxacin Tetracycline Piperacillin Gentamicin Amikacin Pseudomonas aeruginosa Piperacillin Amikacin Gentamicin Tobramycin Dr.T.V.Rao MD 12 Antimicrobial Susceptibility Testing Diffusion method Put a filter disc, or a porous cup/a bottomless cylinder containing measured quantity of drugs on the a solid medium that has been seeded with test bacteria Dilution method vary amount of antimicrobial substances incorporated into liquid or solid media followed by inoculation of test bacteria Dr.T.V.Rao MD 13 Susceptibility Testing Methods Incubate plate Inoculate Place disks 18-24 hr, 35 C MH plate on agar plate Measure and record zone of inhibition around each disk Diffusion Method Disc diffusion method : The Kirby-Bauer test Antibiotic-impregnated filter disc* Susceptibility test against more than one antibiotics by measuring size of “inhibition zone ” 1949: Bondi and colleagues paper disks 1966: Kirby, Bauer, Sherris, and Tuck filter paper disks Demonstrated that the qualitative results of filter disk diffusion assay correlated well with quantitative results from MIC tests Dr.T.V.Rao MD 15 Disc Diffusion Method Procedure (Modified Kirby-Bauer method: National Committee for Clinical Laboratory Standards. NCCLS) Prepare approximately. 108 CFU/ml bacterial inoculum in a saline or tryptic soy broth tube (TSB) or Mueller-Hinton broth (5 ml) Pick 3-5 isolated colonies from plate Adjust the turbidity to the same as the McFarland No. 0.5 standard.* Streak the swab on the surface of the Mueller-Hinton agar (3 times in 3 quadrants) Leave 5-10 min to dry the surface of agar Dr.T.V.Rao MD 16 Examining purity of plate Select the Colonies from Pure Isolates Reflect Transmitted light ed light Dr.T.V.Rao MD 17 Disk Diffusion Test Prepare inoculum suspension Prepare inoculum Select colonies suspension Dr.T.V.Rao MD 18 Prepare the Material for Inoculation Standardize inoculum Suspension as per Mac farland Mix well standard Dr.T.V.Rao MD 19 Swab the plate with optimal sample Remove sample Swab plate Dr.T.V.Rao MD 20 Select the Disks and Apply Select disks Dr.T.V.Rao MD 21 Incubate Overnight Dr.T.V.Rao MD 22 Disc Diffusion Method Place the appropriate drug- impregnated disc on the surface of the inoculated agar plate Invert the plates and incubate them at 35 oC, o/n (18-24 h) Measure the diameters of inhibition zone in mm Dr.T.V.Rao MD 23 Read the Results with Precision Transmitted Light Dr.T.V.Rao MD 24 Disc Diffusion Method Measurement of the diameters of inhibition zone Measure from the edge where the growth stats, BUT there are three exceptions With sulfonamides and co-trimoxazole, ignore slight growth within the zone Certain Proteus spp. may swarm into the area of inhibition When beta-lactamase producing Streptococci are tested, zone of inhibition are produced with a heaped-up, clearly defined edge, regardless of the size of the inhibition zone, they should be reported as resistant Dr.T.V.Rao MD 25 Look at the Charts for establishing the zones of Sensitivity 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. Dr.T.V.Rao MD 26 Disc Diffusion Method Reporting the Results Interpretation of results By comparing with the diameters with “standard tables” Susceptible Intermediate susceptible Low toxic antibiotics: Moderate susceptible High toxic antibiotics: buffer zone btw resistant and susceptible Resistant Dr.T.V.Rao MD 27 Factors Affecting Size of Zone of Inhibition Larger zones with light Inoculum density inoculum and vice versa If after application of disc, the Timing of disc application plate is kept for longer time at room temperature, small zones may form Temperature of incubation Larger zones are seen with temperatures < 35 oC Incubation time Ideal 16-18 hours; less time does not give reliable results Dr.T.V.Rao MD 28 Factors Affecting Size of Zone of Inhibition Size of the plate Smaller plates accommodate less number of discs Depth of the agar medium (4 mm) Thin media yield excessively large inhibition zones and vice versa Proper spacing of Avoids overlapping of the discs (2.5 cm) zones Dr.T.V.Rao MD 29 Factors Affecting Size of Zone of Inhibition Potency of antibiotic Deterioration in contents leads discs to reduced size Composition of Affects rate of growth, medium diffusion of antibiotics and activity of antibiotics Acidic pH of medium Tetracycline, novobiocin, methicillin zones are larger Aminoglycosides, Alkaline pH of erythromycin zones are larger medium Subjective errors in Reading of zones determining the clear edge Dr.T.V.Rao MD 30 Quality Assurance in Antibiotic Susceptibility Testing Visit - WHO-Regional Office for South East Asia website Medium: Mueller-Hinton agar plates Enterococcus faecalis (ATCC 29212 or 33l86) and a disc of co-trimoxazole 20 mm in diameter of the inhibition zone Procedure: Modified Kirby-Bauer method recommended by National Committee on Clinical Laboratory Services (NCCLS) Susceptibility test with quality control strains Dr.T.V.Rao MD 31 Quality Assurance in Antibiotic Susceptibility Testing with Control strains Susceptibility test with quality control strains for every new batch of Mueller-Hinton agar Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922) Pseudomonas aeruginosa (ATCC 2785 ) Dr.T.V.Rao MD 32 Quality Assurance in Antibiotic Susceptibility Test Salient features of quality control Use antibiotic discs of 6 mm diameter Use correct content of antimicrobial agent per disc Store supply of antimicrobial discs at -20 oC Use Mueller-Hinton medium for antibiotic sensitivity determination Use appropriate control cultures Use standard methodology for the test Dr.T.V.Rao MD 33 Need for Modified Methods Modified Methods in Disc diffusion for Antibiotic sensitivity testing to be used for detections of following bacterial isolates 1 MRSA 2 ESBL 3 Enterobacteriaceae and Gram negative bacteria and Carbapenems resistant using Modified Hodge test Dr.T.V.Rao MD 34 Dilution Method Concentration (MIC) Minimum Inhibition The lowest concentration of antimicrobial agent that inhibits bacterial growth/ multiplication Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC) The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive 35 Antimicrobial susceptibility testing using micro-broth dilutions ug/ml 64 32 16 8 4 2 • • • • • • • • • • • • • 96 well microtiter plate Broth Dilution Method Procedure Making dilutions (2-fold) of antibiotic in broth Mueller-Hinton, Tryptic Soy Broth Inoculation of bacterial inoculum, incubation, overnight Controls: no inoculum, no antibiotic Turbidity visualization MIC Sub culturing of non-turbid tubes, overnight Growth (bacterial count) MBC 37 Creating Dilutions Dr.T.V.Rao MD 38 Broth Dilution Method Day 1 128 64 32 16 8 4 2 C1 C2 Add 1 ml of test bacteria (1*106 CFU/ml) to tubes containing 1 ml broth and concentration of antibiotic (mg/l) 64 32 16 8 4 2 1 C1 C2 Controls: C1 = No antibiotic, check Bacterial conc.= 5*105 CFU/ml viability on agar plates immediately Incubate 35 oC, o/n C2 = No test bacteria 39 Broth Dilution Method Day 2 64 32 16 8 4 2 1 C1 C2 Record visual turbidity Subculture non-turbid tubes to agar plates (use 0.01 ml standard loop) 0.01 ml (spread plate), Incubate 35 oC, o/n MIC = 16 mg/l Day 3 Determine CFU on plates: At 16 mg/ = 700 CFU/ml > 0.1% of 5*105 CFU/ml 64 32 16 MBC = 32 mg/l 40 Broth Dilution Method 100% of original bacterial conc. = 5*105 CFU/ml 0.1% = [(5*105)*0.1]/100 CFU/ml = 500 CFU/ml The bacteria count should be less than 5 CFU on agar plate subcultured with 0.01 ml 500*0.01 = 5 CFU 41 Broth Dilution Method are Technically Difficult Disadvantages : Solutions?? Only one Agar dilution antibiotic & one method organism can be Disc diffusion method tested each time Micro broth dilution Time-consuming method Dr.T.V.Rao MD 42 Micro broth Dilution Method Micro dilution plates: “Micro dilution/ Micro broth dilutions” 96 wells/ plate: simultaneously performed with many tests organisms/ specimens, less reagent required Manually prepared Commercially prepared Frozen or Dried/ lyophilized Consistent performance but high cost May suffer from degradation of antibiotic during shipping and storage 43 Agar Dilution Method Procedure Making dilutions of antimicrobial agent in melted media and pouring plates One concentration of antibiotic/ plate Possible for several different strains/plate 64 uGu/ml 32 ug/ml 16 ug/ml 44 Agar Dilution Method Procedure Inoculation of bacterial inoculum (McFarland No. 0.5) Using a replicating inoculator device called “A Steers- Foltz replicator” Delivers 0.001 ml of bacterial inoculum Incubation Spot of growth MIC 32 ug/ml 45 Minimal inhibitory concentration The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium Interpret: Susceptible Intermediate Resistant Dr.T.V.Rao MD 46 Clinical Conditions when MICs are Useful Endocarditis Meningitis Septicemia Osteomyelitis Immunosuppressed patients (HIV, cancer, etc.) Prosthetic devices Patients not responding despite “S” Reports Dr.T.V.Rao MD 47 Inoculum Preparation MIC Testing (NCCLS Reference Method) Standardize inoculum suspension Final inoculum concentration 3 – 5 x 105 CFU/ml (3 – 5 x 104 CFU/well) Dr.T.V.Rao MD 48 Select Micro titration plate and prepare optimal inoculum Prepare inoculum suspension Micro dilution MIC tray Dr.T.V.Rao MD 49 Dilute & mix inoculum suspension Dr.T.V.Rao MD 50 Pour inoculum into reservoir and inoculate MIC tray Dr.T.V.Rao MD 51 Incubate overnight Do not forget to check the purity of Inoculum Inoculate purity plate Dr.T.V.Rao MD 52 Optimal Use of Purity Plates Sub final test suspension to non-selective medium (after inoculating MIC test) Streak for isolation (avoid several specimens per plate - may not reveal contaminants if no isolated colonies) Examine before reading MIC (usually at 16-20 h) Re-incubate if Antibiograms questionable Read MICs - + 0.5 1 2 4 8 16 32 64 >6 >6 4 4 The gradient technique, Etest® Etest is a well established AST method in microbiology laboratories around the world. The Etest technique comprises a predefined gradient of antibiotic concentrations on a plastic strip, and can be used to determine the Minimum Inhibitory Concentration (MIC) of antibiotics, antifungal agents and antimycobacterial agents. Dr.T.V.Rao MD 55 E test – MIC Reports are helpful in Critical management decisions Quantitative MIC data is a prerequisite for the management of critical infections, including sepsis, especially among critical care patients. Etest is particularly valuable in such situations, when on-scale MICs are needed for treatment Dr.T.V.Rao MD 56 decisions. Antimicrobial Gradient Testing E-test® Read plates after recommended Incubation Read MIC where elipse intersects scale MIC of the Bacteria can be read Directly Dr.T.V.Rao MD 58 MIC on a strip abbiodisk.com Serum Susceptibility Tests To determine drug concentration in the patient’s serum = MIC*SIT The Serum Inhibitory Titer (SIT) The highest dilution of patient’s serum that inhibit bacteria To determine the ability of drug in the patient’s serum to kill bacteria The Serum Bactericidal Level (SBL) The lowest dilution of patient’s serum that kills bacteria Technically Demanding 5-Jan-06 Chiang Mai University 60 Antibiotic Sensitivity testing can be done with automation Dr.T.V.Rao MD 61 VITEK 2 Automates Reporting of Resistance Integrated in the VITEK 2 system is the Advanced Expert System (AES™), a software which validates and interprets susceptibility test results, and detects antibiotic resistance mechanisms. The AES Expert System is the most developed software system in this field, and is capable of identifying even emerging and low-level resistance. Dr.T.V.Rao MD 62 What is the Role of Microbiology Departments a staff member Each laboratory should have with the time, interest, and expertise to provide leadership in antibiotic testing and resistance. This person would read relevant publications, network with other laboratories, and evaluate potentially useful tests to detect new forms of resistance before new CLSI-recommended tests become available” - Ken Thomson, Emerging Infect. Dis., 2001 Dr.T.V.Rao MD 63 References 1Usanee Anukool (Ph.D.) Clinical Microbiology,AMS, Chiang Mai University 2National Committee For Clinical Laboratory Standards. 1998. NCCLS document M100 - S8 . Performance Standards for Antimicrobial Susceptibility Testing. 8th edition, NCCLS, Waynae, Pa. Dr.T.V.Rao MD 64 Created by Dr.T.V.Rao MD for ‘e’ learning resources for Microbiologists in Developing World Email firstname.lastname@example.org Dr.T.V.Rao MD 65
"ANTIBIOTIC SENSITIVITY TESTING IN ENTEROBACTERIACE"