Microbiology Primer: Direct sensitivity

	Unified Program in Infectious Diseases, Montefiore Medical Center and Albert Einstein College of Medicine.

Direct Sensitivity

The proper procedure for determining antibiotic suseptibility is MIC determination or agar disk diffusion (Kirby-Bauer), which use a defined and standardized inoculum of bacteria. However, clinical relevant information can be obtained by spreading 1 ml of the contents of a blood culture bottle onto a plate and placing antibiotic disks on the plate. Because of the variable inoculum, sensitivity to an antibiotic does not correlate 100% with the Kirby-Bauer method, but high level resistance is quite predictive. The advantage of this method is that it gives antibiotic suseptibility/resistance information a day or more earlier than would otherwise be possible.

Three examples:

Staphylococcus aureus (MRSA) Streptococcus pneumoniae (Pneumococcus) Pseudomonas aeruginosa
MRSA
Note the yellowish pigmentation of the bacterial lawn, and (see below) the lack of inhibition by the Oxacillin disk The brownish tint of the blood agar plate outside the zones of bacterial inhibition is caused by alpha-hemolysis. Note that in addition to antibiotics, an Optochin (P) disk shows inhibition, which helps to differentiate Pneumococcus from other alpha-hemolytic Strep. The greenish tint of the lawn and plate in general is caused by the diffusible pigment made by the Pseudomonas aeruginosa itself.

There are some details about disk diffusion suseptibility testing worth noting:

This particularly strain of Staph aureus is defined to be MRSA (Methicillin-resistant Staph aureus) because of its resistance to Penicillinase-resistant Penicillins. MRSA is a bit of a historical name, since sensitivity is determined by an Oxacillin (Ox) disk and intravenous therapy is most commonly Nafcillin, not Methicillin.
On the basis of this direct sensitivity, this particular isolate of Pneumococcus might have been suspected to have decreased sensitivity to Penicillin based on the small zone of inhibition around the Oxacillin disk. However, on formal testing after colony isolation, the zone of inhibition of Oxacillin was larger and the strain was Penicillin-sensitive. In this case, the direct sensitivity underestimated the sensitivity to Oxacillin/Penicillin.

Sometimes the boundary of the inhibition is not well-defined. There is a gradual transition from full growth to no growth. Sometimes the zone of inhibition has quite a sharp boundary.

Sometimes, there is a paradoxical increased density just outside the zone of inhibition. This is cause by a increase of colony size of the fewer bacteria that manage to grow there. The small zone of inhibition around this Ceftriaxone (CRO) disk is interpreted as resistant. However, a small population, possibly with even higher level resistance form "satellite" colonies within the zone of inhibition. Sometimes, this can be a clue to a mixed infection, since growth of the minor strain may only be apparent when the dominant strain is inhibited.

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id/micro/directsensi.htm/Created 21 Apr 1999/Last Modified 29 Apr 1999/Craig Werner/ werner@aecom.yu.edu

Staphylococcus aureus (MRSA)	Streptococcus pneumoniae (Pneumococcus)	Pseudomonas aeruginosa

Note the yellowish pigmentation of the bacterial lawn, and (see below) the lack of inhibition by the Oxacillin disk	The brownish tint of the blood agar plate outside the zones of bacterial inhibition is caused by alpha-hemolysis. Note that in addition to antibiotics, an Optochin (P) disk shows inhibition, which helps to differentiate Pneumococcus from other alpha-hemolytic Strep.	The greenish tint of the lawn and plate in general is caused by the diffusible pigment made by the Pseudomonas aeruginosa itself.


Sometimes the boundary of the inhibition is not well-defined. There is a gradual transition from full growth to no growth.	Sometimes the zone of inhibition has quite a sharp boundary.

Sometimes, there is a paradoxical increased density just outside the zone of inhibition. This is cause by a increase of colony size of the fewer bacteria that manage to grow there.	The small zone of inhibition around this Ceftriaxone (CRO) disk is interpreted as resistant. However, a small population, possibly with even higher level resistance form "satellite" colonies within the zone of inhibition. Sometimes, this can be a clue to a mixed infection, since growth of the minor strain may only be apparent when the dominant strain is inhibited.