The expression “phenotypic” is from the word phenotype, which means “the observable characteristics of an organism”. It describes the physical features of an organism in terms of its morphology, motility, colour and shape amongst other noticeable attributes that are typical to a particular organism. Thus, the phenotypic detection and characterization of antimicrobial resistant genes in pathogenic bacteria describes the general physical reaction or observable traits of microbes to a variety of antimicrobial agents such as antibiotics, disinfectants and disinfectants. In phenotypic detection, pathogenic strains of microorganisms are directly tested in vitro for their susceptibility or resistance to a range of antimicrobial agents (especially antibiotics) that are usually used for their control.

The result obtained from a phenotypic detection and characterization experiment generally gives a picture of the performance of the test drugs in vivo (i.e. when used for the practical treatment of a sick host). Phenotypic detection methods provides prompt and reliable drug susceptibility results that give the microbiologist a picture of the prevailing resistant profile of the test pathogen, and this guides physicians on the choice of antimicrobial therapy to initiate in the patient. Phenotypic detection and characterization techniques used in the microbiology laboratory to classify resistant genes of pathogenic bacteria are easy to perform and report that their genotypic counterparts. Some phenotypic detection methods (e.g. the VITEK system) are automated and can be used to perform computerized or automated antimicrobial susceptibility testing in the microbiology laboratory.

MicroScan and Phoenix systems are other available automated antimicrobial susceptibility methods that can be used in the microbiology laboratory to promptly evaluate the susceptibility profile of pathogenic bacteria based on their MIC values. Phenotypic decoction and characterization techniques for the identification of resistance genes in pathogenic bacteria are generally less expensive and straightforward to perform. Most importantly, they can be used to monitor the progress and response of a diseased patient to antimicrobial therapy.    One of the extensively used and generally accepted methods of characterizing the resistant genes of pathogenic bacteria by phenotypic methods in the microbiology laboratory is the Kirby-Bauer disk diffusion technique.

The Kirby-Bauer disk diffusion technique is one of the oldest approaches to the phenotypic antimicrobial susceptibility testing of pathogenic bacteria, and it still remains one of the most widely used antimicrobial susceptibility testing methods in the routine susceptibility studies of pathogenic microorganisms. It is most suitable for testing the majority of bacterial pathogens that are commonly encountered in the microbiology laboratory; and it is usually carried out using a template to avoid any form of bias when performing the test (Figure 1). Briefly, when a paper disk impregnated with a known concentration of a drug is placed on a agar plate, moisture from the agar plate is immediately absorbed into the drug laden disk. The drug in the disk begins to diffuse into the surrounding area of the agar which has been previously inoculated with the test bacteria and incubated at the appropriate temperature. 

Figure 1. Template for placement of antibiotic disks on agar plate of 90 mm. Note: When conducting antimicrobial susceptibility testing (AST) in the microbiology laboratory, the distance between antibiotic disks should be 25 mm (2.5 cm) i.e. disks should be placed at a distance of 25 mm apart.

Bacterial growth occurs in the presence of an antibiotic when the bacteria reaches a critical mass and can overpower the inhibitory effects of the drug in the paper disk. The point at which critical mass of the bacteria is reached is demonstrated by a sharply marginated circle of bacterial growth around the paper disk (Figure 2)

Figure 2. Petri dish plate showing zone of inhibition (IZD) caused by antibiotic impregnated disks by Modified Kirby-Bauer disk diffusion technique. The organism is a clinical isolate of Escherichia coli obtained from the Microbiology Laboratory Department of University of Nigeria Teaching Hospital (UNTH), Ituku-Ozalla, Enugu State, Nigeria; and the microbe is susceptible to gentamicin (CN), ofloxacin (OFX), ciprofloxacin (CIP), and sulphamethoxazole-trimethoprim (SXT) at a measured IZD of 8 mm. Arrow shows the zone of growth inhibition that is to be measured using a meter rule or caliper.

When the test organism is resistant to the antimicrobial agents used, a smaller zone of inhibition is usually produced (Figure 3). The marginated circle of bacterial growth produced around the antimicrobial disk is often referred to as the inhibition zone of the drug and it is measured to the nearest millimeter using a meter rule.

Figure 3. Petri dish showing zone of inhibition (IZD) caused by antibiotic impregnated disks. The organism is a clinical isolate of Klebsiella pneumoniae from the Microbiology Laboratory Department of University of Nigeria Teaching Hospital (UNTH), Ituku-Ozalla, Enugu State, Nigeria. The isolate is completely resistant to some of the antibiotics tested including ofloxacin (OFX), ciprofloxacin (CIP), and sulphamethoxazole-trimethoprim (SXT), and they are seen to be growing up to antibiotic disks. Susceptibility was recorded for gentamicin (CN), at an IZD of 3 mm (arrowhead).

The zone of inhibition is usually compared to standard inhibition zone diameters (antibiotic breakpoints) as per the criteria of the Clinical Laboratory Standard Institute, CLSI (formerly known as the National Committee for Clinical Laboratory Standard, NCCLS. Observed inhibition zone diameters are normally reported as sensitive, intermediate or resistant in line with the CLSI criteria. Generally, the size of the IZD of the drug against the test pathogenic bacteria is used to evaluate the in vivo susceptibility of the organisms to the tested drug. In addition to the inhibition zone diameter (IZD) of a drug to pathogenic bacteria, the phenotypic decoction and characterization of antibiotic resistance gene also gives a clue of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the tested drug.  


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