Antibiotic Resistant Test PDF

Veterinary Microbiology and Mycology Practical part l su scept ibilit y Antibacteria testing by Dr. Nasr Jalboush  Tests to determine the most suitable antibiotic for the effective treatment in a given disease can be cond...

Veterinary Microbiology and Mycology Practical part l su scept ibilit y Antibacteria testing by Dr. Nasr Jalboush  Tests to determine the most suitable antibiotic for the effective treatment in a given disease can be conducted on isolates from clinical cases. These tests which are carried out in vitro cannot allow for the various factors which may affect antibacterial activity in vivo. The results obtained following treatment may not reflect the susceptibility pattern of an isolate as determined in the laboratory. A number of antibacterial susceptibility tests are available including: – broth dilution, – disc diffusion, – agar gradient (E-tests)  Bacteria demonstrate two kinds of resistance to antibiotics: – Intrinsic resistance means that the species was resistant to an antibiotic even before its introduction. – Acquired resistance means that the species was originally susceptible to an antibiotic, but later became resistant. Bacteria can acquire antibiotic resistance either by: – mutation or; – through exchange of genetic material among same or closely related species. The sudden acquisition of resistance to antibiotics poses difficulties in treating infections.  Resistance to several different antibiotics at the same time is even more significant problem. It is because of the acquired resistance that bacterial isolates must be subjected to antibiotic susceptibility testing. Bacteria showing reduced susceptibility or resistance to an antibiotic implies that it should not be used on the patient. Quantitative Methods: In these tests, the minimum amount of antibiotic that inhibits the visible growth of an isolate or minimum inhibitory concentration (MIC) is determined. Bacterial isolate is subjected to various dilutions of antibiotics. These tests can be performed on broth or agar. 1. Broth dilution methods a. Macrobroth dilution MIC tests b. Microbroth dilution MIC tests 2. Agar dilution methods (E-tests) Determination of the minimum inhibitory concentration The MIC of an antibacterial agent for a specific bacterium can be determined in vitro. The MIC is the highest dilution of an antibacterial agent which inhibits growth of an isolate. The minimum bactericidal concentration (MBC) is the highest dilution of a drug which can kill a particular bacterium. Macrobroth dilution tests: A serial two-fold dilution of antibiotic are made in test tubes from 0 to maximum concentration that is achieved in vivo without toxic effect on patient. The inoculum density of bacterial isolate to be tested is standardized with 0.5 McFarland turbidity standard. The suspension should have a final inoculum of 5 x 10 5 cfu/ml. 1ml of bacterial suspension is added to rows of antibiotic solution and incubated at 37˚C overnight. The lowest concentration of antibiotic that completely inhibits visual growth of bacteria (no turbidity) is recorded as MIC. McFarland standards (left to right) 0.5, 1.0, 2.0, 3.0, positioned in front of a Wickerham card. McFarland standards are used to prepare bacterial suspensions to a specified turbidity. Microbroth dilution tests: A polystyrene tray containing 80 wells is filled with about 0.05 to 0.1 ml total broth volume of serial two-fold dilutions of different antibiotics. The inoculum suspension and standardization is done according to McFarland standard. The bacterial inoculum is then inoculated into the wells and incubated at 37˚C overnight. MIC is determined as in macrobroth dilution test.  E-test Plastic strips with a predefined gradient of – One antibiotic – One antifungal Only one manufacturer One strip per antibiotic Wide range of antibiotics Easy to use Storage at -20°C Short shelf life, expensive Reading E-tests Ciprofloxacin for Yersinia pestis Resistant > 4 ug/ml Intermediate 1-4 ug/ml Susceptible < 1 Upper reading Qualitative Methods: These tests categorize a bacterial isolate as sensitive, intermediate or resistant to a particular antibiotic. Disk diffusion test: In this method the standardized bacterial isolate is spread on an agar plate and then paper disc containing specific concentration of antibiotics are placed and incubated at 37˚C overnight. If the isolate is susceptible to the antibiotic, it does not grow around the disk thus forming a zone of inhibition. Strains resistant to an antibiotic grow up to the margin of disk. The diameter of zone of inhibition must be measured and result read from the Kirby Bauer chart as sensitive, intermediate or resistant.  The diameter of each zone of inhibition is measured in millimetres and the results compared with standards for interpretation of the zone size. Susceptibility to an antibacterial drug indicates that the infection caused by the bacterium may respond to treatment if the drug reaches therapeutic levels in the affected tissues.  Host factors affecting treatment Diffusion in tissues Serum protein binding Drug interactions Immune system Multiple simultaneous infections Virulence of organism Site and severity of infection  Common interpretation problems Results depends on the technique used Many factors influence results – Lack of standardization of the inoculums – Thickness and quality of the culture media – Quality and conservation of the disks – Wuality control with standardized strains – Condition and duration of incubation Common interpretation problems An agar gel that is too thick leads to smaller zones Source: http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/  Common interpretation problems Problem with the size of the inoculums Solution: Use McFarland 0.5 photometer Scale -> same tubes  Common interpretation problems Contamination with another organism  Common interpretation problems Bad manipulation Inoculation of the Muller Hinton – Swabbing – Not by flooding