Antimicrobial drug sensitivity testing and therapeutic use in veterinary practice


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Antimicrobial drug sensitivity testing and therapeutic use in veterinary practice

  1. 1. Antimicrobial Drug Sensitivity testing and therapeutic use in Veterinary Practice testing-and-therapeutic-use-in-veterinary-practice nsitivity_testing_and_therapeutic_use_in_Veterinary_Practice BR Singh Division of Epidemiology, ICAR- IVRI, Izatnagar-243122, India
  2. 2. • Microbial abundance to be 2.9·1029 cells [corresponding to 4.1 petagram (Pg= 1015g, a billion tonnes) ∼0.6% of Earth’s total living biomass]. (Kallmeyer et al., 2012) • 107 to 109 different species of bacteria (Schloss and Handelsman, 2004). • A 'reference man' (one who is 70 kilograms, 20–30 years old and 1.7 metres tall) contains on average about 30 trillion human cells and 39 trillion bacteria, (Milo and Sender, 2016). • The term antibiotic was coined by Selman Waksman in 1942 to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. • The term antibiotic, meaning “substances against life,"
  3. 3. Around 1014 bacteria/ gm of faecal matter "More recent estimates, he noted, put the total number of human cells at anywhere from 15 trillion to 724 trillion, and the number of gut microbes at anywhere between 30 trillion and 400 trillion. Which gives a ratio that can best be expressed as ¯_(ツ)_/¯." 10 nonillion virus-like particles in the world. That’s 10 quintillion (1*1031) for every person.
  4. 4. Rules of Antibiotic use (Marino, 2007, The ICU Book) • Try not to use them, • Try not to use too many of them. • Always follow rule 1 and 2.
  5. 5. What you use? Drug % Sensitivity Cefoxitin 64% Amoxyrum 70% Tetracycline 75% Ampicillin +sulbactam 80% Ceftriaxone 84% Ceftriaxone+tazatobactam 94% Cipro/ Enerofloxacin 80% Chloramphenicol 87% Gentamicin 88% Cotrimoxazole 75% Amikacin 76%
  6. 6. Antibiotics and the Laboratory • If there was no antibiotic resistance, there would be no need for a Bacteriology lab! • Infections could be treated “syndromically”
  7. 7. Antibiotic Sensitivity Testing Antibiotic Assays Detecting resistance mechanisms Molecular methods Automated methods MIC tests Breakpoint testing Disc testing Lab Aspects Of Antimicrobial Chemotherapy
  8. 8. Questions • (1) Why do we test antimicrobial susceptibility? • (2) How do we perform antimicrobial susceptibility tests? • (3) How can we detect resistance mechanisms? • (4) Why & how do we assay antimicrobial serum levels?
  9. 9. Deciding on whether to use an antibiotic: • Clinical Practice: – Day 1: guess the disease, guess the bug, guess the sensitivity pattern and administer the treatment. • Standard Practice: – Day 1 Clinical assessment – Day 2 Positive microbiology – Day 3 Antibiotic sensitivity tests – BUT TOO LATE?
  10. 10. But remember… • Other factors are very important when we choose an antibiotic • The history of previous antimicrobial use. • Will it get to where the infection is? • Bioavailability • Cost • Toxicity • Likelihood of development of resistance. Etc…
  11. 11. Why do we test antimicrobial susceptibility? • To direct & predict antimicrobial chemotherapy. • To review & monitor epidemiological trends. • To set national & local antibiotic policies. • To test the activity of a new antimicrobial agent. • To presumptively identify isolates.
  12. 12. How do we perform antimicrobial susceptibility tests? • We can use a number of methods including:- • Disc susceptibility tests - Kirby-Bauer - Stokes’ - BSAC. • Agar Breakpoint method. • Minimum Inhibitory Concentration (MIC) – Tube MIC or E-tests. • Automated methods – Vitek. • Molecular methods – PCR.
  13. 13. Disc Susceptibility Tests • Developed in the USA in 1966. • Based on NCCLS ( National Committee for Clinical Laboratory Standards ) data. • Use Mueller-Hinton agar. • Use standard 0.5 McFarland (BaSO4) inoculum. • Streak inoculum in 3 directions or rotary plate. • Use standard NCCLS tables to interpret zone sizes as S, I or R.
  14. 14. Stokes’ Comparative Method • Developed in the U.K (1972). • A variety of media can be used including Iso- sensitest agar (ISA), ISA & 5% horse blood & Chocolate ISA. • Use NCTC (National Collection of Type Cultures) controls e.g. NCTC 6571 Staph aureus, NCTC 10602 Ps. aeruginosa, NCTC 10418 E.coli. • Using a rotary plater apply the control suspension on the outer edge & the test suspension in the centre, leaving a gap for the discs. • Interpretation based on comparison between zones seen with the test organism & those of the known sensitive control. • Sensitive = zone radius of test, larger, equal, or not more than 3mm smaller than the control. • Intermediate = zone radius more than 3mm, but smaller than the control by more than 3mm. • Resistant = zone radius of 3mm or less.
  15. 15. BSAC Method • Developed in the U.K in 1998 by BSAC working party, through statistical regression analysis of zone diameter & MIC data, on hundreds of strains. • Use ISA and/or ISA & 5% horse blood & 20mg/l NAD. • Use standard 0.5 McFarland inoculum • Use dilution protocol of 0.5 McFarland standard as specified by BSAC. • Neat 0.5 Neisseria gonorrhoeae. • 1:10 dilution of 0.5 Staphylococci. • 1:10 dilution of 0.5 for Streptococci. • 1:100 dilution of 0.5 for Coliforms. • 1:100 dilution of 0.5 for Haemophilii
  16. 16. Tube MIC • Set up a series of antibiotic doubling dilutions in tubes containing liquid media (ISA or ISA with lysed blood). • For example 128mg/l, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0. • Set up tubes for test organism & NCTC control organism. • Add standard organism inoculum to each tube. • Include an antibiotic free tube i.e organism only. • Include an organism free tube i.e antibiotic only. • Incubate tubes overnight. • Examine for presence of growth by shaking each tube & observing turbidity. • Check antibiotic free tube has growth. • Check organism free tube has no growth. • Check the NCTC control gives the recommended MIC. • The MIC is the first tube dilution without visible growth. • The tube MIC is very labour intensive, difficult to get right & prone to error.
  17. 17. E-tests • A commercial alternative to tube MIC. • Consists of a plastic strip 6cm by 0.5cm in size. • Exponential gradient of antimicrobial dried on one side. • MIC scale printed on the other side. • The range corresponds to fifteen 2-fold dilutions. • Follow manufacturers’ instructions for inoculum preparation, media recommendations & incubation conditions. • MIC interpretation made where growth of inhibition ellipses the strip. • Most E-test require examination with a hand-lens to look for minute colonies intersecting the strip.
  18. 18. Automated Methods • Three main methods are in use in the U.K. • These include the Vitek (Biomerieux), the Phoenix (Becton-Dickinson) & the Mastascan Elite (Mast). • Not in clinical use in India. • The Vitek I was originally designed by NASA for use as an on-board space exploration test system. • It is based on the use of small thin plastic cards each containing many wells linked by capillaries. • These cards are available as susceptibility & identification cards.
  19. 19. Vitek modules
  20. 20. Vitek advantages / disadvantages • Can give rapid “expert” results for identification & susceptibility. • It can be directly linked to the LIMS. • It decreases the incidence of operator error & can be operated by an MLA. • It is very expensive, approx £7 for an id & sens. • It needs regular software updates & expert analysis by a BMS.
  21. 21. Why & how do we assay antimicrobial serum levels? • We assay to ensure adequate therapeutic levels & to avoid the accumulation of toxic levels. • Most antimicrobial agents have a large therapeutic index & are given in large doses without causing harm. • The aminoglycosides, the glycopeptides & some antifungals have a narrow therapeutic range which can be close to the toxic range. Therapeutic failure Therapeutic range Therapeutic failure
  22. 22. Antibiotics contraindicated in Horses Tetracyclines, Chloramphenicol, Lincomicin, Clindamycin, Tylosin, Tiamulin, Spiramycin, Ciprofloxacin, Norfloxacin, Enerofloxacin, Tilmicosin, Erythromycin (in adults), cefotaxime, ceftriaxone, ceftiofur, cefoxitin, latmoxef (moxalactam), Imipenem, Neomycin, gentamicin in foals (by injection also), Amphotericin B (should not be used locally)
  23. 23. Antibiotics contraindicated in Dogs and Cats Tobramycin, spectinomycin, Gentamicin (neuromuscular & renal toxicity), phenylbutazone, chloramphenicol and griseofulvin, cefachlor/ cefadroxil/ cephalexin/ cepharadine (cause diarrhoea and vomiting), enerofloxacin/ ciprofloxacin/ norfloxacin (may induce iarrhoea and vomiting, nephrotocity, must be avoided in pregnant and neonates), erythromycin/ clindamycin/ lincomycin (cuases vomiting, diarrhoea and hepatotoxicity), metronidazole (avoid in early pregnancy, anorexia, vomiting, neuro problems), nitrofurans and rifampin (hepatotoxic, discolour tears and urine, avoid during pregnancy), ketoconazole, itraconazole (anorexia, depression, dirrhoea, vomiting, coat change, in cats fever, anemia)
  24. 24. Antimicrobial contraindicated in Ruminants Antibiotics should not be used orally and those antibiotics have hepatobiliary excretion should be avoided by injections too. Tetracyclines, sulfonamides and coccidiostats can be used orally. Monensin, a coccidiostat, has often problem with uniform mixing in feed thus poisoning may result.
  25. 25. Swines Sulfamethazine (due to residue in meat), monensin, salinomycin, arsanilic acid (cuases muscle tremors, listlessness) Poultry birds Furazolidones, Nitrofurans, Apramycin, dimetridazole, Metronidazole (Not approved for food animals) Guinea pigs Rabbit Hamsters Penicillins, tetracyclines, clindamycin or lincomycin causes Enteritis, profuse diarrhoea and death in 4-5 days, enterotoxemia due to Clostridial-infection. In rabbits diarrhoea due to Clostridium spiroformae is common after antibiotic use. Mice & gerbil Dihydro-streptomycin (Cardiovascular effect death within minutes) Pet birds Tetracyclines during egg production and breeding season results in to soft shelled eggs and rickets in chicks Contraindicated in other animals
  26. 26. Disease/ Pathogen Drugs of choice Brucellosis Tetracyclines, chloramphenicol, gentamicin Edwardsiellosis Azithromycin, aztreonam, ceftriaxone, fluoroquinolones, chloramphenicol Escherichia coli Nitrofurantoin, gentamicin, ceftriaxone+tazatobactam, chloramphenicol, cefoperazone+sulbactam Klebsiella pneumoniae Fluoroquinolones, gentamicin, imipenem, meropenem, cefoperazone+sulbactam, ceftriaxone+tazatobactam, aztreonam Moraxella bovis Strepto-penicillin, and most of the common antibiotics Pasturellosis Tetracycline, fluoroquinolones, amoxycillin, gentamicin, ceftriaxone Salmonellosis Chloramphenicol, gentamicin, ceftriaxone+tazatobactam, cotrimoxazole, imipenem Staphylococcal infections Tetracycline, nitrofurantoin, chloramphenicol, amoxyclav Streptococcal infections Clindamycin, gentamicin, amoxyclav, vancomycin, Pseudomonas infections No, use only after testing What to use?
  27. 27. Guidelines to good antimicrobial stewardship 1. Arm yourselves with a working knowledge of commonly used antimicrobials. 2. If possible, a narrow spectrum should be chosen 3. Prophylactic use should generally be avoided except in case of surgery. 4. If the use is justified, the correct dose, dose frequency and duration of treatment should be used. 5. Topical or local use should always be considered. 6. Use only rational combinations of antimicrobials if considered necessary. 6. Routine use of fluoroquinolones, third- and fourth-generation cephalosporins and amikacin should be absolutely avoided. Similarly , ‘last resort’ antimicrobials, such as imipenem and vancomycin, should not be used for veterinary patients. 7. Take time to while prescribing antimicrobials. 8. Create a table of first-, second- and third-choice antimicrobials for your practice. First-choice antimicrobials would comprise agents appropriate for initial treatment, not necessarily based on culture and sensitivity information. Second-choice- prescribed based on culture and sensitivity data, Third-choice antimicrobials should only be prescribed for serious and life- threatening infections, based on culture and sensitivity data, and only where no first- or second-choice agents are appropriate. 9. Keep an eye on guidelines reviewed regularly about antimicrobial drug use. 10. Report treatment failures. This is a vital part of monitoring for developing and emerging resistance.