The variety and widespread use of commercial susceptibility testing methods reflect the key role resistance detection plays in the responsibilities of clinical microbiology laboratories. In many instances, the commercial methods are variations of the conventional dilution or disk diffusion methods, and their accuracies have been evaluated by comparison of results with those obtained by conventional methods. Additionally, many of the media and environmental conditions standardized for conventional methods are maintained with the use of commercial systems. The goal of detecting resistance is the same for all commercial methods, but the principles and practices vary with respect to:

• Format in which bacteria and antimicrobial agents are brought together

 • Extent of automation for inoculation, incubation, interpretation, and reporting

• Method used for detection of bacterial growth inhibition

• Speed with which results are produced

 • Accuracy

Accuracy is an extremely important aspect of any susceptibility testing system and is addressed in more detail later in this chapter.

Broth Microdilution Methods. Several systems have been developed that provide microdilution panels already pre pared and formatted according to the guidelines for conventional broth microdilution methods (e.g., BBL Sceptor, BD Microbiology Systems, Cockeysville, Mary land; Sensititre, Trek Diagnostics Systems, Inc., Westlake, Ohio; MicroScan touch SCAN-SR, Dade Behring, Inc., West Sacramento, California). These systems enable lab oratories to perform broth microdilution without having to prepare their own panels.

The systems may differ to some extent regarding the volume in the test wells, how inocula are prepared and added, the availability of different supplements for the testing of fastidious bacteria, the types of antimicrobial agents and dilution schemes, and the format of medium and antimicrobial agents (e.g., dry-lyophilized or frozen). Furthermore, the degree of automation for inoculation of the panels and the devices available for reading results vary among the different products. In general, these commercial panels are designed to receive the standard inoculum and are incubated using conditions and durations recommended for conventional broth microdilution. They are growth-based systems that require overnight incubation, and CLSI interpretive criteria apply for interpretation of most results. Reading of these panels is frequently augmented by the availability of semiautomated reading devices.

Agar Dilution Derivations. One commercial system (Spiral Biotech Inc., Bethesda, Maryland) uses an instrument to apply antimicrobial agent to the surface of an already prepared agar plate in a concentric spiral fashion. Starting in the center of the plate, the instrument deposits the highest concentration of antibiotic and from that point drug application proceeds to the periphery of the plate. Diffusion of the drug in the agar establishes a concentration gradient from high (center of plate) to low (periphery of plate). Starting at the periphery of the plate, bacterial inocula are applied as a single streak perpendicular to the established gradient in a spoke wheel fashion. After incubation, the distance is measured between the point where growth is noted at the edge of the plate to the point where growth is inhibited toward the center of the plate (Figure 1). This value is used to calculate the MIC for the antimicrobial agent against each of the bacterial isolates on the plate.

Fig1. Growth patterns on a plate containing an antibiotic gradient (the concentration decreases from the center of the plate to the  periphery) applied by the Spiral Gradient instrument. The distance from the point where growth is noted at the edge of the plate to the point  where growth is inhibited toward the center of the plate is measured. This value is used in a formula to calculate the MIC of the antimicrobial  agent against each of the bacterial isolates streaked on the plate. (Courtesy Spiral Biotech, Inc., Bethesda, Md.)

Diffusion in Agar Derivations. One test has been developed that combines the convenience of disk diffusion with the ability to generate MIC data. The Etest (bioMéri eux, Durham, North Carolina) uses plastic strips; one side of the strip contains the antimicrobial agent concentration gradient, and the other contains a numeric scale that indicates the drug concentration (Figure 2). Mueller-Hinton plates are inoculated as for disk diffusion, and the strips are placed on the inoculum lawn. Several strips may be placed radially on the same plate so that multiple antimicrobials may be tested against a single isolate. After overnight incubation, the plate is examined and the number present at the point where the border of growth inhibition intersects the E-strip is taken as the MIC (Figure 2). The same MIC interpretive criteria used for dilution methods, as provided in CLSI guidelines, are used with the Etest value to assign an interpretive category of susceptible, intermediate, or resistant. This method provides a means of producing MIC data in situations in which the level of resistance can be clinically relevant (e.g., penicillin or cephalosporins against S. pneumoniae).

Fig2. The Etest® strip uses the principle of a predefined antibiotic gradient on a plastic strip to generate an MIC value. It is processed in the same way as the disk diffusion. A, Individual antibiotic strips are placed on an inoculated agar surface. B, After incubation,  the MIC is read where the growth/inhibition edge intersects the strip graduated with an MIC scale across 15 dilutions (arrow). Several antibiotic strips can be tested on a plate. (Courtesy bioMérieux*, Marcy l’Etoile, France.)

Another method (BIOMIC, Giles Scientific, Inc., New York, New York) combines the use of conventional disk diffusion methodology with video digital analysis to auto mate interpretation of inhibition zone sizes. Automated zone readings and interpretations are combined with computer software to produce MIC values and to allow for data manipulations and evaluations for detecting unusual resistance profiles and producing antibiogram reports.

Automated Antimicrobial Susceptibility Test Systems. The automated antimicrobial susceptibility test systems available for use in the United States include the Vitek Legacy and Vitek 2 systems (bioMérieux, Inc., Durham, North Carolina), the MicroScan WalkAway system (Dade International, Sacramento, California), and the Phoenix system (BD Microbiology Systems, Cockeysville, Mary land). These different systems vary with respect to the extent of automation of inoculum preparation and inoculation, the methods used to detect growth, and the algorithms used to interpret and assign MIC values and categorical findings (i.e., susceptible, intermediate, resistant).

For example, the Vitek 2 AST inoculum is automatically introduced by a filling tube into a miniaturized, plastic, 64-well, closed card containing specified concentrations of antibiotics (Figure 3). Cards are incubated in a temperature-controlled compartment. Optical readings are performed every 15 minutes to measure the amount of light transmitted through each well, including a growth control well. Algorithmic analysis of the growth kinetics in each well is performed by the system’s software to derive the MIC data. The MIC results are validated with the Advanced Expert System (AES) software, a category interpretation is assigned, and the organism’s antimicrobial resistance patterns are reported. Resistance detection is enhanced with the sophisticated AES software, which can recognize and report resistance patterns using MICs. In summary, this system facilitates standardized susceptibility testing in a closed environment with validated results and recognition of an organism’s antimicrobial resistance mechanism in 6 to 8 hours for most clinically relevant bacteria (Figure 4).

Fig3. The VITEK® 2 antimicrobial susceptibility test card  contains 64 wells with multiple concentrations of up to 22 antibiotics. The antibiotic is rehydrated when the organism suspension is  introduced into the card during the automated filling process.  (Courtesy bioMérieux*, Marcy l’Etoile, France.)

Fig4. The components of the VITEK® 2 system consist of the instrument housing; the sample processing and reader/incubator;  the computer workstation, which provides data analysis, storage, and epidemiology reports; the Smart Carrier Station, which is the direct  interface between the microbiologist on the bench and the instrument; and a bar code scanner to facilitate data entry.  (Courtesy bioMérieux*,  Marcy l’Etoile, France.)

Spectrophotometric analyzed panels require overnight incubation, and the growth patterns may be read manually as described for routine microdilution testing. Fluorometric analysis is based on the degradation of fluorogenic substrates by viable bacteria. The fluorogenic approach can provide susceptibility results in 3.5 to 5.5 hours. Either full dilution schemes or breakpoint panels are available. In addition to speed and facilitation of workflow, the automated systems provide increasingly powerful computer-based data management that can be used to evaluate the accuracy of results, manage larger databases, and interface with the pharmacy to improve and advance the utility of antimicrobial susceptibility testing data.

The Phoenix system provides a convenient, albeit manual, gravity-based inoculation process. Growth is monitored in an automated fashion based on a redox indicator system with results available in 8 to 12 hours. Supplemental testing (e.g., confirmatory extended spectrum beta-lactamase [ESBL] test for E. coli) is included in each panel, reducing the need for additional or repeat testing. Interpretation of results is augmented by a rules based data management expert system.

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Bactericidal Tests

Conventional Testing Methods: Agar Dilution

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Laboratory Diagnosis of Autoimmune Hemolytic Anemia

Blood Coagulation Tests

Western Blot Immunoassays

Indirect Fluorescent Antibody Tests and Other Immunomicroscopic Methods

Enzyme-Linked Immunosorbent Assays

Laboratory Diagnosis of Thyrotoxicosis

Laboratory Findings of Beta-Thalassemia Syndromes

Complement Fixation Assays

Flocculation Tests