Determination of resistance/susceptibility

  1. Incubate vegetative cells of each P. larvae strain to be tested on MYPGP agar for 48 h at 36 °C ± 1.
  2. Suspend the cells directly from MYPGP in screw capped tubes containing sterile distilled water or sterile saline.
  3. Adjust the bacterial suspension until the OD620 is in the range of 0.4.  Alternatively, individual colonies of each strain can be incubated at 36°C ± 1 in 2 ml aliquots of MYPGP broth for 26 to 75 h until the OD is in the range of 0.5. Optimally, use the adjusted suspension within 15 min of preparation and always within 60 min.
  4. Vortex for 3 minutes.
  5. Dip a sterile cotton swab in the bacterial suspension and remove excess fluid on the swab by turning it against the inside of the tube.
  6. Spread the inoculum evenly over the entire surface of the plate prepared in  by inoculating in three directions.
  7. Place antibiotic discs (pre-warmed to RT) on the plate.
    Discs should be in firm, even contact with the surface of the medium. At least three replications for each bacterial strain is recommended. It is possible to apply more than one disc per plate; in this case, discs should be spaced so that zones of inhibition in susceptible isolates do not overlap. Overlapping will impede the measurement of zone diameters.
  8. Apply the “15-15-15 rule”: Use the inoculum within 15 min of preparation and never beyond 60 min. Apply discs within 15 min of inoculating plates. Start incubation within 15 min of application of discs.
  9. Incubate the plates inverted at 36°C ± 1 during 72 h.
  10. Measure the resulting inhibition zone (clear area without bacterial growth including the disc) by using a calliper or an automated zone reading. Read the plates from the back against a black background illuminated with reflected light.
  11. A correct inoculum and satisfactorily spread plates will result in a confluent lawn of growth in the absence of antibiotic. It is important that there is an even lawn of growth to achieve uniformly circular inhibition zones. If individual colonies can be seen, the inoculum is too light the test must be repeated. In case of distinct colonies within zones, subculture the colonies, check purity and repeat the test if necessary.
  12. Interpretation: for tetracyclines, an inhibition zone of less than 20 mm in diameter (including the disc) is considered as the separation point between resistant and susceptible strains as follows:  ”resistant” ≤14 mm; ”intermediate”:  between 15-19 mm and ”susceptible”: ≥20 mm (Alippi et al., 2007).
  13. It is strongly recommended to use reference standard strains according to the indications of NCCLS or EUCAST with the only difference that MYPGP agar (see section 3.1. for recipe) should be used as basal medium.