Learning Objective 7
Learning Objective 7
1. DILUTION METHODS
The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial agents in a broth environment. Microdilution testing uses about 0.05 to 0.1 ml total broth volume and can be conveniently performed in a microtiter format. Macrodilution testing uses broth volumes at about 1.0 ml in standard test tubes.
1. DILUTION METHODS
For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration or MIC. The MIC is thus the minumum concentration of the antibiotic that will inhibit this particular isolate. The test is only valid if the positive control shows growth and the negative control shows no growth.
1. DILUTION METHODS
A procedure similar to broth dilution is agar dilution. Agar dilution method follows the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited.
A growth medium, usually Mueller-Hinton agar, is first evenly seeded throughout the plate with the isolate of interest that has been diluted at a standard concentration (approximately 1 to 2 x 108 colony forming units per ml). Commercially prepared disks, each of which are preimpregnated with a standard concentration of a particular antibiotic, are then evenly dispensed and lightly pressed onto the agar surface.
3. E-TEST
E-test is a commercially available test that utilizes a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic. The strip also displays a numerical scale that corresponds to the antibiotic concentration contained therein. This method provides for a convenient quantitative test of antibiotic resistance of a clinical isolate. However, a separate strip is needed for each antibiotic, and therefore the cost of this method can be high.
5. MECHANISM-SPECIFIC TESTS
Resistance may also be established through tests that directly detect the presence of a particularresistance mechanism. For example, beta lactamase detection can be accomplished using an assay such as the chromogenic cephalosporinase test (Cefinase disk by BD Microbiology Systems, Cockeysville, MD and BBL DrySlide Nitrocefin, Becton Dickinson, Sparks, MD) and detection for chloramphenicol modifying enzyme chloramphenicol acetyltransferase (CAT) may utilize commercial colorimetric assays such as a CAT reagent kit (Remel, Lenexa, Kansas).
6. GENOTYPIC METHODS
Since resistance traits are genetically encoded, we can sometimes test for the specific genes that confer antibiotic resistance. However, although nucleic acid-based detections systems are generally rapid and sensitive, it is important to remember that the presence of a resistance gene does not necessarily equate to treatment failure, because resistance is also dependent on the mode and level of expression of these genes11.
Some of the most common molecular techniques utilized for antimicrobial resistance detection are as follows
Polymerase chain reaction (PCR) is one of the most commonly used molecular techniques for detecting certain DNA sequences of interest. This involves several cycles of denaturation of sample DNA, annealing of specific primers to the target sequence (if present), and the extension of this sequence as facilitated by a thermostable polymerase leading to replication of a duplicate DNA sequence, in an exponential manner, to a point which will be visibly detectable by gel electrophoresis with the aid of a DNAintercalating chemical which fluoresces under UV light.
DNA hybridization. This is based on the fact that the DNA pyrimidines (cytosine and thymidine) specifically pair up with purines (guanine and adenine; or uracil for RNA). Therefore, a labeled probe with a known specific sequence can pair up with opened or denatured DNA from the testsample, as long as their sequences complement each other. If this hybridization occurs, the probe labels this with a detectable radioactive isotope, antigenic substrate, enzyme or chemiluminescent compound. Whereas if no target sequence is present or the isolate does not have the specific gene of interest, no attachment of probes will occur, and therefore no signals will be detected. Modifications of PCR and DNA hybridization. With these basic principles, several modifications have been introduced which further improve the sensitivity and specificity of these standard procedures. Examples of such development were the use of 5fluorescence-labeled oligonucleotides, the development of molecular beacons, development of DNA arrays and DNA chips, among many others.