Antimicrobial sensitivity testing in milk samples

Agar disc difussion test

There are two international organisations that set up guidelines and interpretive breakpoints for bacteriology and susceptibility testing.
The CLSI or Clinical and Laboratory Standards Institute is a non-profit organisation with a mission to develop clinical and laboratory practices and promote their use worldwide. Their laboratory testing standards are based on input from and consensus among the industry, governments and healthcare professionals. The CLSI has a Veterinary Subcommittee that approves clinical interpretive breakpoints: this means that the breakpoints of sensitivity discs are related to the actual clinical cure for a certain disease.

The EUCAST or European Committee on antimicrobial susceptibility testing is jointly organised by institutions linked to the European Union such as EMA (European Medicines Agency). EUCAST has formed a Veterinary Committee on Antimicrobial Susceptibility Testing (VetCAST). This committee is aimed at dealing with antimicrobial susceptibility testing of bacterial pathogens of animal origin and animal bacteria with zoonotic potential. VetCAST plans to define clinical MIC breakpoints for new veterinary antimicrobial agents, review breakpoints for generic drugs and give advice on the bacterial spectrum of veterinary antimicrobial agents.
The information below and in the pdf file is based on the CLSI guidelines, unless otherwise specified.

Possible testing methods

Agar disc diffusion

The agar disc diffusion test is the most widely used and versatile method for antimicrobial susceptibility testing (AST) of a majority of bacterial pathogens. The methodology for this test is described further in the manual (see pdf).
When performed according to recommendations, with careful standardisation and the use of quality controls, disc diffusion is a reproducible and accurate method for AST.

Broth microdilution

The broth microdilution test is not further described in this article. Disc diffusion zone diameters and MIC breakpoints from broth microdilution are correlated. However,
the zone diameters may not correspond precisely to the listed MIC breakpoints due to differences in the methodologies. Therefore, extrapolation of inhibition zones to MIC values is not possible.

Disc diffusion

Materials needed

Media

  • Mueller-Hinton agar (MHA) without supplements for Enterobacteriaceae, staphylococci and enterococci.
  • Mueller-Hinton agar with blood (MH-F) for streptococci.

Paper discs

  • Antimicrobial paper discs for each antimicrobial to be tested, with the correct dose for the bacterial species to be tested.
  • Discs can be dispensed with single dispensers or multi dispensers.
  • Discs have to be stored in accordance with the guidelines to ensure correct results.

Sterile inoculating loop (10 μl size)

  • Disposable plastic inoculating loops or wired inoculating loops (with a Bunsen burner for sterilisation) are used for inoculation of the plates with one loop full of milk.
  • In case mixed cultures grow from milk samples, single colonies can be picked with a sterile inoculating loop to subculture pure colonies.

Instructions

Obtain single identified colonies

With milk obtained aseptically from a mastitic quarter, streak to obtain single colonies on blood agar. One loop (10 μl) of milk is used to inoculate each plate. When necessary, subculture to obtain pure colonies before preparation of the disc diffusion test.
Make an identification of the pure isolate(s). Since breakpoints vary for different species, sensitivity testing without correct identification has very little value.

Preparation of the inoculum

Suspend 3-5 similar colonies from the agar plate using a sterile loop or cotton swab and suspend in 4-5 ml of sterile 0.9% saline.
The density of the colony suspension (inoculum) should be equivalent to 0.5 McFarland and is preferably measured with a photometric device. Alternatively, a 0.5 McFarland standard can be used to visually compare the inoculum.
The density can be adjusted by adding more organisms if the density is less than 0.5, or adding more saline if the density is more than 0.5 McFarland.

Application of the inoculum

The inoculum should be used within 15 minutes of preparation.
A sterile cotton swab is immersed in the inoculum and excess fluid removed by turning the swab against the inside of the tube to avoid over-inoculation of the plates.
The inoculum is spread evenly over the entire surface of the plate by swabbing in three directions.

Application of discs

Apply the discs firmly on the agar within 15 minutes of preparing the inoculum (otherwise the bacteria start to grow before the discs have been applied, altering the sizes of the inhibition zones).
The number of discs should be limited so that overlapping of zones is avoided. Use no more than six discs on a standard 90 mm plate, eight discs on a 100 mm plate and 12 on a 150 mm circular plate.

Incubation of plates

Invert the plates and incubate them within 15 minutes of disc application. Over-stacking of plates in the incubator may influence the distribution of heat.
Incubate the plates for 16-20 hours at 35 ± 2 °C before reading.
Incubate Enterobacteriaceae, staphylococci and enterococci in ambient air and streptococci in 4-6 % CO2 enriched air.

Examination of plates

Examine the growth on the plates. A correct inoculum results in confluent, even growth, with circular inhibition zones. If individual colonies can be seen, the inoculum is too light and the test must be repeated.

Reading the zones

Zone edges should be read at the point of complete inhibition as judged by the naked eye with the plate held at about 30 cm from the eye.
Measure the zone diameter with a ruler, calliper or an automated zone reader.
Read MHA plates from the back against a dark background illuminated with reflected light.
Read MH-F plates from the front with the lid removed illuminated with reflected light. The plates have to be tilted in the light in order to correctly distinguish the outer zones of inhibition.
If distinct colonies are observed within an inhibition zone, subculture the colonies, check for purity and repeat the test if necessary. When the colony found within the diffusion zone is not a contamination, the test has to be reported as resistant.

Find more about antimicrobial susceptibility testing on milk samples, download the pdf file!

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