Bactericidal tests are designed to determine the ability of antimicrobial agents to kill bacteria. The killing ability of most drugs is already known, and they are commonly classified as bacteriostatic or bactericidal agents. However, many variables, including the concentration of antimicrobial agent and the species of targeted organism, can influence this classification. For example, beta-lactams, such as penicillin, typically are bactericidal against most gram-positive cocci but are usually only bacteriostatic against enterococci. If bactericidal tests are clinically appropriate, they should be applied only to evaluate anti microbials typically considered to be bactericidal (e.g., beta-lactams and vancomycin) and not to agents known to be bacteriostatic (e.g., macrolides).

Key clinical situations in which achieving bactericidal activity is of greatest clinical importance include severe and life-threatening infections, infections in an immunocompromised patient, and infections in body sites where assistance from the patient’s own defenses is minimal (e.g., endocarditis or osteomyelitis). Based on research trials in animal models and clinical trials in humans, the most effective therapy for these types of infections is often already known. However, occasionally the laboratory may be asked to substantiate that bactericidal activity is being achieved or is achievable. The methods available for this include minimal bactericidal concentration (MBC) testing, time-kill studies, and serum-cidal testing. Regardless of the method used, the need to interpret the results cautiously, with the understanding of uncertain clinical correlation and the potential for substantial technical artifacts, cannot be overemphasized.

Minimal Bactericidal Concentration. The MBC test involves continuation of the procedure for conventional broth dilution testing. After incubation and determination of the antimicrobial agent’s MIC, an aliquot from each tube or well in the dilution series demonstrating inhibition of visible bacterial growth is subcultured to an enriched agar medium (usually sheep blood agar). After overnight incubation, the plates are examined and the CFUs determined. With the volume of the aliquot and the number of CFUs obtained, the number of viable cells per milliliter for each antimicrobial dilution can be calculated. This number is compared with the known CFU/mL in the original inoculum. The antimicrobial concentration resulting in a 99.9% reduction in CFU/mL compared with the organism concentration in the original inoculum is recorded as the MBC.

Although the clinical significance of MBC results is uncertain, applications of this information include considering whether treatment failure could be occurring as the result of an organism’s MBC exceeding the serum achievable level for the antimicrobial agent. Alternatively, if an antibiotic’s MBC is greater than or equal to 32 times higher than the MIC, the organism may be tolerant to the drug. Tolerance, a phenomenon most commonly associated with bacterial resistance to beta-lactam antibiotics, reflects an organism’s ability to be inhibited by an agent that is usually bactericidal. Although the physio logic basis of tolerance has been studied in several bacterial species, the actual clinical relevance of this phenomenon has not been well established.

Time-Kill Studies. Another approach to examining bactericidal activity involves exposing a bacterial isolate to a concentration of antibiotic in a broth medium and measuring the rate of killing over a specified period. By this time-kill analysis, samples are taken from the antibiotic-broth solution immediately after addition of the inoculum and at regular intervals afterward. Each time-sample is plated to agar plates; after incubation, CFU counts are performed as described for MBC testing. The number of viable bacteria from each sample is plotted over time to determine the rate of killing. Gener ally, a 1000-fold decrease in the number of viable bacteria in the antibiotic-containing broth after a 24-hour period, compared with the number of bacteria in the original inoculum, is interpreted as bactericidal activity. Although time-kill analysis is frequently used in the research environment to study the in vitro activity of antimicrobial agents, the labor intensity and technical specifications of the procedure preclude its use in most clinical microbiology laboratories to determine the proper treatment of a patient’s infection.

Serum Bactericidal (Schlichter Test). The serum bactericidal test (SBT) is analogous to the MIC-MBC test except the medium used is the patient’s serum containing the therapeutic antimicrobial agents the patient has been receiving. Using the patient’s serum to detect bacteriostatic and bactericidal activity also allows observation of the antibacterial impact of factors other than the antibiotics (e.g., antibodies and complement).

Two serum samples are required for each test. One is collected just before the patient is to receive the next antimicrobial dose; this is the trough specimen. The other sample is collected when the serum antimicrobial concentration is highest; this is the peak specimen. The appropriate time to collect the peak specimen varies with the pharmacokinetic properties of the antimicrobial agents and the route by which they are being administered. Peak levels for intravenously, intramuscularly, and orally administered agents are generally obtained 30 to 60 minutes, 60 minutes, and 90 minutes after administration, respectively. The trough and peak levels should be collected for the same dose and tested simultaneously.

Serial twofold dilutions of each specimen are pre pared and inoculated with the bacterial isolate from the patient (final inoculum of 5 × 10⁵ CFU/mL). Dilutions are incubated overnight. The highest dilution that inhibits visibly detectable growth is the serum-static titer (e.g., 1:8, 1:16, 1:32). Aliquots of known size are then taken from each dilution at or below the serum-static titer (i.e., dilutions that inhibited bacterial growth) and are plated on sheep blood agar plates. After incubation, the CFUs per plate are counted, and the serum dilution resulting in a 99.9% reduction in the CFU/mL, compared with the original inoculum, is recorded as the serum-cidal titer. For example, if a bacterial isolate showed a serum-static titer of 1:32, the tubes containing dilutions of 1:2, 1:4, 1:8, 1:16, and 1:32 would be subcultured. If the 1:8 dilution was the highest dilution to yield a 99.9% decrease in CFUs, the serum-cidal titer would be recorded as 1:8.

The SBT was originally developed to assist in the pre diction of the clinical efficacy of antimicrobial therapy for staphylococcal endocarditis. Peak serum-cidal titers of 1:32 to 1:64 or greater have been thought to correlate with a positive clinical outcome. However, even though the test is performed on the patient’s serum, many differences go unaccounted for between the in vitro test environment and the in vivo site of infection. Therefore, although the test is used to evaluate whether effective bactericidal concentrations are being achieved, the predictive clinical value for staphylococcal endocarditis or any other infection caused by other bacteria is still uncertain.

Details regarding the performance of these bactericidal tests are provided in the CLSI document M26-A, “Methods for Determining Bactericidal Activity of Antimicrobial Agents.”

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