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Chapter 5.10 : Tests to Assess Bactericidal Activity

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Abstract:

The minimum bactericidal concentration (MBC) test can be used to assess the ability of an antimicrobial agent to kill a bacterial isolate. MBC tests are performed after consultation in very select clinical situations when it is necessary to deter-mine the bactericidal activity of an anti-microbial agent against a bacterial isolate. These situations include those in which immune mechanisms offer little help in eradicating the infecting organisms, such as in patients with endocarditis or osteomyelitis or in immunosuppressed patients, particularly those with neutropenia. Bactericidal tests provide a rough prediction of bacterial eradication, but other factors may also impact the bacteriologic responses of patients. MBC testing is an accepted parameter in the evaluation of new antimicrobial agents and is frequently used as a research tool.

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Preparation of inoculum for reaction tubes in time-kill assay.

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Figure 5.10.2–A1

Dilution scheme for bacterial colony counts.

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Figure 5.10.2–A2

Plate configuration for inoculating 0.25 ml of each dilution from reaction tube to agar plate for colony counts. (Adapted and reprinted with permission from G. M. Eliopoulos and R. C. Moellering. 1996. Antimicrobial combinations, p. 330–396, Fig. 9.5 [p. 339]. V. Lorian [ed.], , 4th ed. The Williams & Wilkins Co., Baltimore, Md.)

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Sample graph for time-kill assay of with vancomycin. Symbols: □, growth control; ■, vancomycin at 0.25 µg/ml; △, vancomycin at 0.5 µg/ml; ▲, vancomycin at 1 µg/ml.

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Sample graph of time-kill assay of with piperacillin and amikacin. Symbols: □, growth control; ■, piperacillin (32 µg/ml); △, amikacin (8 µg/ml); ▲, piperacillin (32 lg/ml) and amikacin (8 µg/ml).

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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References

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1. Eagle, H.,, and A. D. Musselman. 1948. The rate of bacterial action of penicillin in vitro as a function of its concentration, and its paradoxically reduced activity at high concentrations against certain organisms. J. Exp. Med. 88:99131.
2. Gunnison, J. B.,, M. A. Fraher,, and E. Jawetz. 1963. Persistence of Staphylococcus aureus in penicillin in vitro. J. Gen. Microbiol. 34:335349.
3. Handwerger, S.,, and A. Tomasz. 1985. Antibiotic tolerance among clinical isolates of bacteria. Rev. Infect. Dis. 7:368386.
4.NCCLS. 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved guideline M26-A. NCCLS, Wayne, Pa.
5.NCCLS. 2003. Methods for Dilution Susceptibility Tests for Bacteria That Grow Aerobically, 6th ed. Approved standard M7-A6. NCCLS, Wayne, Pa.
6.NCCLS. 2001. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 5th ed. Approved standard M11-A5. NCCLS, Wayne, Pa.
7. Pearson, R. D.,, R. T. Steigbigel,, H. T. Davis,, and S. W. Chapman. 1980. Method for reliable determination of minimal lethal antibiotic concentrations. Antimicrob. Agents Chemother. 18:699708.
8. Reimer, L. G.,, C. W. Stratton,, and L. B. Reller. 1981. Minimum inhibitory and bactericidal concentrations of 44 antimicrobial agents against three standard control strains in broth with and without human serum. Antimicrob. Agents Chemother. 19:10501055.
9. Shanholtzer, C. J.,, L. R. Peterson,, M. L. Mohn,, J. A. Moody,, and D. N. Gerding. 1984. MBCs for Staphylococcus aureus as determined by macrodilution and microdilution techniques. Antimicrob. Agents Chemother. 26:214219.
10. Woolfrey, B. F.,, R. T. Lally,, and M. N. Ederer. 1985. Evaluation of oxacillin tolerance in Staphylococcus aureus isolates by a novel method. Antimicrob. Agents Chemother. 28:381388.
11. Gerding, D. N.,, J. Foxworth,, C. E. Hughes,, D. M. Bamberger,, and T. A. Larson,. 1996. Extravascular antimicrobial distribution and the respective blood concentrations in humans, p. 835899. In V. Lorian (ed.), Antibiotics in Laboratory Medicine, 4th ed.The Williams & Wilkins Co., Baltimore, Md.
12. Taylor, P. C.,, F. D. Schoenknecht,, J. C. Sherris,, and E. C. Linner. 1983. Determination of minimal bactericidal concentrations of oxacillin for Staphylococcus aureus: influence and significance of technical factors. Antimicrob. Agents Chemother. 23:142150.
1.NCCLS. 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved guideline M26-A. NCCLS, Wayne, Pa.
2. Woolfrey, B. F.,, R. T. Lally,, and M. N. Ederer. 1985. Evaluation of oxacillin tolerance in Staphylococcus aureus isolates by a novel method. Antimicrob. Agents Chemother. 28:381388.
1. Eliopoulos, G. M.,, and C. T. Eliopoulos. 1988. Antibiotic combinations: should they be tested? Clin. Microbiol. Rev.1:139156.
1. Eliopoulos, G. M.,, and C. T. Eliopoulos. 1988. Antibiotic combinations: should they be tested? Clin. Microbiol. Rev. 1:139156.
2. Eliopoulos, G. M.,, and R. C. Moellering, Jr., 1996>. Antimicrobial combinations, p. 330396. In V. Lorian(ed.), Antibiotics in Laboratory Medicine, 4th ed. The Williams & Wilkins Co., Baltimore, Md.
3.NCCLS. 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved guideline M26-A. NCCLS, Wayne, Pa.

Tables

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Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Untitled

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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MBC testing conditions for various bacteria

Inhibitory portion of test is to be incubated for 24 h. This table applies to broth macrodilution and microdilution.

Specified broth appropriate for growing inoculum to log phase and for time-kill assays. HTM, Haemophilus test medium; LHB, lysed horse blood; THB, Todd-Hewitt broth.

All incubations were at 35°C.

Add 2% NaCl for penicillinase-resistant penicillins (oxacillin, methicillin, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin).

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
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Rejection value and calculated sensitivity and specificity for each initial concentration based on duplicate 0.01-ml samples

When the sum of colonies from duplicate samples was equal to or less than the rejection value, the antimicrobial agent was declared lethal (a 0.999 or greater reduction in the final inoculum). (Adapted with permission from R. D. Pearson, R. T. Steigbigel, H. T. Davis, and S. W. Chapman. 1980. Method for reliable determination of minimal lethal antibiotic concentrations. Antimicrob. Agents Chemother. 18:699–708.) A 5% error (pipette error plus full sampling error for determination of inoculum) was used. Error was based on duplicate samples for determination of the final inoculum size.

As determined from colony count plate.

Number of colonies.

Sensitivity and specificity were calculated for each specific final inoculum concentration and rejection value.

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
Generic image for table

MBC testing conditions for various bacteria

Inhibitory portion of test is to be incubated for 24 h. This table applies to broth macrodilution and microdilution.

Specified broth appropriate for growing inoculum to log phase and for time-kill assays. HTM, test medium; LHB, lysed horse blood; THB, Todd-Hewitt broth.

All incubations were at 35°C.

Add 2% NaCl for penicillinase-resistant penicillins (oxacillin, methicillin, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin).

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
Generic image for table
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* within acceptable counting range

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
Generic image for table

* within acceptable counting range

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
Generic image for table

* Plot actual or log10 CFU

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10
Generic image for table

* Plot actual or log CFU

Citation: Garcia L. 2010. Tests to Assess Bactericidal Activity, p 89-123. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.10

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