Antibiogram

An antibiogram is a laboratory test used to determine the sensitivity pattern of a given microorganism to a range of antibiotics. The advantages of antibiogram use and the techniques involved in running these tests are well known. I would like to point out a few details regarding the management and interpretation of these tests and how they are used in conjunction with poultry pathology and clinical evaluation.

The answer has to do with medicating high-density animal populations (many thousands of birds in a single poultry house) and to the obvious inter-relationship between the birds and their environment. Bacteria, whether pathogenic or not, are also an integral part of this environment. Their presence, spread and permanence depend on opportunistic changes occurring in the eco-system's equilibrium.

Isolation, identification and antibiogram of a pathogenic agent are normally carried out when a bacterial disease has produced a problem on the farm, expressed as economic losses in the form of mortality or failing to achieve set production targets in terms of body weight and feed conversion rate (FCR). The bacteria most frequently involved are E.coli, Salmonella, Pasteurella, Haemophilus and occasionally gram-positives. These microbes are isolated from specific pre- or post-mortem lesions. Antibiograms are then run to determine whether:

Antibiograms are also used to:

Once the efficacy of the initial therapy used on the farm has been evaluated one must consider the management not only of the infected flock but also those flocks that will subsequently share the same ecosystem. This is even more important when the infective problem is found to be repetitive in the same farm, or poultry house. Colibacillosis (and derived complications) is one of the more classic examples. An antibiogram should be run for every E.coli sample isolated from the affected farm, each time the problem appears and after placement of every new flock. This method will help verify the bacterial response or resistance to the antibiotic of choice. In many poultry operations the same litter is used and re-used for subsequent flocks. Bacterial sensitivity should therefore be carefully noted and included in the clinical-microbiological records kept for every farm.

In summary one single, isolated antibiogram is of present and temporary use only. Periodic monitoring of the micro-ecology will keep us informed about the use of appropriate antimicrobials and will also indicate the appropriate time for complementary prophylactic programs involving change of litter, disinfectants, etc.

The correct interpretation of the antibiogram will be of interest to poultry pathologists and laboratory technicians alike. Standardized methods are established and can be found in the WHO manuals. It is this technical side of the test that I will describe in the last part of this paper.

One of the most frequently asked questions relates to in-vitro (in the lab)/in-vivo (in the field) relationships. Why is it that satisfactory in vitro or laboratory test results can translate into poor clinical efficacy on a farm? In these situations, thought should be given to:

a) Whether or not the diagnosis correct?
b) Whether the problem is really caused by the isolated bacteria?
c) Whether the product was administered correctly, at the right dose level, for the correct number of days?
d) Whether the activity level of the drug coincides with label specifications?

e) Whether poor quality water interfered with the antibiotic and reduced the availability?

The basic idea of diffusion assays is as follows: the tested antibiotics are impregnated in paper discs which are placed on plate of agar medium inoculated with the bacteria in question. Following diffusion of the compounds through the agar, a "halo" or zone of inhibition forms where a concentration of the specific diffused antibiotic is sufficient to inhibit that microbial growth.

Based on this reasoning, the diffusion method is sometimes mistakenly interpreted as a quantitative method. The more potent an antimicrobial compound, the less concentrated it need be, and consequently at points further from the disc with consequently lower concentrations, microbial growth will still be inhibited. Therefore, it is often assumed that the larger the diameter of the zone of inhibition, the more potent the antimicrobial. A number of factors however, may interfere with this interpretation. Firstly, the concentration of the antibiotic in the disc must be taken into account. The higher the concentration in the disc, the more concentrated the compound will be at a given distance from the disc itself. Also, the length of time allowed for the process to occur can greatly influence the diameter of the zone of inhibition as the longer diffusion is allowed to take place the higher the concentrations at any given point in the gradient will be. Related to this, the diffusibility of the antimicrobial compound through the agar can greatly impact on the observed zone of inhibition, such that a very potent inhibitor may produce a relatively small "halo" simply because it is unable to diffuse adequately through the medium. In addition, the extent of the growth of the microbe, in relation to the degree of diffusion, can influence the resulting zone of inhibition, such that the timing of both factors, microbial growth and diffusion, interplay.

Because of these complexities in the diffusion method, it is perhaps best to consider the results of the diffusion assay as qualitative only, expressed as: Sensitive, Intermediate and Resistant.

Samples from the problem farm (organs or swabs) are cultured on agar plate. Isolated colonies of each type of organism that may play a pathogenic role should be selected from primary agar plates and tested for susceptibility. Identification procedures are often performed at the same time. Mixtures of different types of organisms should not be tested on the same susceptibility test plate.  This is because when the nature of the infection is not clear and the specimen contains mixed growth or normal flora, the organisms probably bear little or no relationship to the infection process being treated. 

The practice of conducting susceptibility tests directly with clinical material should be avoided except in clinical emergencies when direct gram staining suggests the presence of a single pathogen.

The selection of the most appropriate antibiotics to test is a decision best made by each clinical laboratory in consultation with the field veterinarian. To avoid unnecessary duplication, only one antibiotic from each family should be used. Agents of the same family have similar clinical efficacy, show nearly the same spectrum of activity and have similar cross-resistance and cross-susceptibility.

At least three to five well-isolated colonies of the same morphological type bacteria are selected from a primary agar plate culture and transferred into a tube containing 4-5 ml of broth medium. This is incubated at 35°C for 2-6 hours and then applied with a sterile cotton swab on a dried surface of a Mueller-Hinton agar plate. Alternatively the inoculums can be prepared by making a direct broth or saline suspension of isolated colonies selected from a primary agar plate.

The predetermined battery of anti-microbial disks is dispensed onto the surface of the inoculated agar plate. Each disk must be pressed down to ensure complete contact with the agar surface. The disk must be distributed evenly no closer than 24mm from center to center. The plates are inverted and placed in an incubator at 35°C.

After 16-18 hours of incubation, each plate is examined. The resulting zones of inhibition (halo) will be uniformly circular and there will be a confluent lawn of growth. The diameters of the zone of complete inhibition are measured, including the diameter of the disk. The measurement can be done using a sliding caliber, ruler or compass as demonstrated in Figure One. The zone margin should be taken as the area showing no obvious, visible growth that can be detected by the naked eye.

For the accurate interpretation of the measurement of the zone of inhibition, we use a table that categorizes the levels of susceptibility of organisms to various antimicrobials agents. These categories were developed by:

Susceptible

This category implies that an infection due to this bacterium may be appropriately treated with the dosage of antibiotic recommended for that type of infection and infecting species, unless otherwise contraindicated.

Intermediate

This category implies that an infection due to this bacteria, may be treated when possible with higher than normal dosage of this antibiotic, or when the drug is physiologically concentrated in certain body sites, like Quinolones in urine.

Resistant

Resistant strain will probably not respond to any treatment, as they are not inhibited by the usually achievable systemic concentrations of the antibiotic when normal dosage schedules are used. They may also have MICs that fall within the range where specific, microbial resistance mechanisms are likely and clinical efficacy has not been reliable in treatment studies.

The above, is only a general description of the procedures used for the performance of susceptibility test of non-fastidious bacteria. For more detailed information, look at the WHO's technical manual.