Antimicrobial Drug Sensitivity

testing and therapeutic use in

Veterinary Practice
http://www.slideshare.net/singh_br1762/antimicrobial-drug-sensitivity-
testing-and-therapeutic-use-in-veterinary-practice
https://www.researchgate.net/publication/264165875_Antimicrobial_Drug_Se
nsitivity_testing_and_therapeutic_use_in_Veterinary_Practice

BR Singh
Division of Epidemiology, ICAR-
IVRI, Izatnagar-243122, India
• 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,"
 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.
 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.
 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%
 Antibiotics and the Laboratory
• If there was no antibiotic resistance, there
would be no need for a Bacteriology lab!

• Infections could be treated “syndromically”

Antibiotic Sensitivity Testing

Disc testing

Antibiotic Breakpoint
Assays testing

Lab Aspects
Of Antimicrobial
Detecting Chemotherapy
resistance MIC tests
mechanisms

Molecular Automated
methods methods
 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?
 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?
 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…
 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.
 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.
 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.
 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.
 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
 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.
 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.
 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.
Vitek modules
 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.
 Why & how do we assay
antimicrobial serum levels?
• We assay to ensure
adequate therapeutic levels &
to avoid the accumulation of Therapeutic
toxic levels. failure
• Most antimicrobial agents
have a large therapeutic Therapeutic range
index & are given in large
doses without causing harm.
• The aminoglycosides, the
glycopeptides & some
Therapeutic
antifungals have a narrow
failure
therapeutic range which can
be close to the toxic range.
 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)
 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)
 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.
 Contraindicated in other animals

Swines Sulfamethazine (due to residue in meat), monensin,

salinomycin, arsanilic acid (cuases muscle tremors,
listlessness)
Poultry Furazolidones, Nitrofurans, Apramycin, dimetridazole,
birds Metronidazole (Not approved for food animals)
Guinea pigs Penicillins, tetracyclines, clindamycin or lincomycin
Rabbit causes Enteritis, profuse diarrhoea and death in 4-5 days,
Hamsters enterotoxemia due to Clostridial-infection. In rabbits
diarrhoea due to Clostridium spiroformae is common
after antibiotic use.
Mice & Dihydro-streptomycin (Cardiovascular effect death
gerbil within minutes)
Pet birds Tetracyclines during egg production and breeding season
results in to soft shelled eggs and rickets in chicks
 What to use?
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
 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.
7.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.
8. Take time to while prescribing antimicrobials.
9. 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.
1. Keep an eye on guidelines reviewed regularly about antimicrobial drug use.
2.Report treatment failures. This is a vital part of monitoring for developing and
emerging resistance.