Bacteria can sometimes adapt to the antibiotics used to kill them. This adaptation, which can involve structural changes or the production of enzymes that render the antibiotic useless, can make the particular bacterial species resistant to the particular antibiotic. Furthermore, a given bacterial species will usually display a spectrum of susceptibilities to antibiotics, with some antibiotics being very effective and others totally ineffective. For another bacterial species, the pattern of antibiotic sensitivity and resistance will be different. Thus, for diagnosis of an infection and for clinical decisions regarding the best treatment, tests of an organism's response to antibiotics are essential.
A standard method of testing for antibiotic resistance involves growth of the target bacteria in the presence of various concentrations of the antibiotic of interest. Typically, this test is performed in a specially designed plastic dish that can be filled with agar (a Petri plate). Contamination of the agar, which would spoil the test results, is guaranteed by the sterility of the plate and the lid that fits over the agar-containing dish. The type of agar used is essential for the validity of the tests results. Typically, Iso-Sensitest agar is used.
The hardened agar surface receives a suspension of the test bacteria, which is then spread out evenly over the surface of the agar. The intention is to form a so-called lawn of organisms as growth occurs. Also on the agar surface are discs of an absorbent material. A plate is large enough to house six discs. Each disc has been soaked in a known and different concentration of the same or of different antibiotics.
As growth of the bacteria occurs, antibiotic diffuses out from each disc into the agar. If the concentration of the antibiotic is lethal, no growth of the bacteria will occur. Finally, the diffusing antibiotic will be below lethal concentration, so that growth of bacteria can occur. The result is a ring of no growth around a disc. From comparison with known standards, the diameter of the growth inhibition ring will indicate whether the bacteria are resistant to the antibiotic.
Automated plate readers are available that will scan the plates, measure the diameter of the growth inhibition zones and consult a standard database to indicate the antibiotic resistance or susceptibility of the sample bacteria.
In the past 15 years, the use of fluorescent indicators has become popular. A myriad of compounds are available that will fluoresce under illumination of specific wavelengths. Among the uses for the fluorescent compounds is the viability of a bacterium. For example, living bacteria will fluoresce in the presence of acridine orange, while dead bacteria will not. These probes combined with the optical technique of confocal laser microscopy, now enables populations of cells to be viewed without disrupting them to see if they fluoresce or not in the presence of an antibiotic of interest.
The ability of living bacteria to fluoresce can also be exploited by another machine called a flow cytometer. This machine operates essentially by forcing a suspension of bacteria (or other cells) through an opening so that only one bacterium at a time passes by a sensor. The sensor monitors each passing bacterium and can sort these into categories, in this case, fluorescing (living) from non-fluorescing (dead). The entire process can be completely quickly. This provides an almost "real-time" assessment of the proportion of a population that has been killed by an antibiotic. If the proportion of dead bacteria is low, resistance is indicated.
All the assessments of antibiotic effectiveness need to be done in a controlled manner. This necessitates the use of standard test types of bacteria (strains that are known to be resistant to the particular antibiotic as well as other strains that are known to be sensitive to the antibiotic). The concentration of the bacteria used is also important. Too many bacteria can "dilute" out the antibiotic, producing a false indication of resistance. Controls need to be included to verify that the experiment was not subject to contamination, otherwise the possibility that a finding of resistance was due to a contaminating bacteria could not be discounted.
In clinical settings, a finding of resistance would prompt the search for another antibiotic. Often, identification of the bacteria will suggest, from previous documented tests of others, an antibiotic to which the organism will be susceptible. But, increasingly, formerly effective antibiotics are losing their potency as bacteria acquire resistance to them. Thus, tests of antibiotic resistance grow in importance.
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