A new method to determine postantibiotic effect and effects of subinhibitory antibiotic concentrations

Abstract

It has been shown that bacteria in a postantibiotic (PA) phase exposed to subinhibitory concentrations (sub-MICs) of antibiotics show a long delay before regrowth. This effect has been named the PA sub-MIC effect (PA SME). In the present study, we have used a new method to demonstrate this phenomenon. A computerized incubator for bacteria, Bioscreen C (Lab Systems, Helsinki, Finland), which incubates the bacteria, measures growth continuously by vertical photometry, processes the data, and provides a printout of the results was used. With this method, one may easily test several antibiotics against different bacteria for PA effects (PAEs), PA SMEs, and SMEs. In this study, the effects of benzylpenicillin against 1-hemolytic streptococci and pneumococci were examined. The bacteria were exposed to 2, 10, or 5Ox MIC for 2 h, washed and diluted, incubated in the Bioscreen C incubator, and then exposed to 0.1 to 0.9x MIC. The regrowth was monitored for 20 h. The PAE was calculated as the difference in the time required for the exposed and unexposed bacteria to grow to a defined point (A50) on the absorbance curve. A50 was defined as 50% of the maximum absorbance for the control cultures. The PA SMEs were calculated as the difference in the time required for the reexposed cultures and the unexposed controls to reach Aso. The PAEs ranged between 0.6 and 3.2 h and varied little with the concentration used for the induction of the PAEs. At 0.2x MIC, the PA SMEs were 2 to 3 h longer than the PAEs. Higher sub-MICs increased this delay before regrowth. Most cultures exposed to sub-MICs alone were only slightly affected compared with the controls.Earlier studies and clinical experience have shown that in the treatment of streptococcal and pneumococcal infections, intermittent doses of penicillin are successful even if concentrations in serum and tissues fall below the MIC for the bacteria for long intervals (11, 24). These results may partly be explained by the so-called postantibiotic (PA) effect (PAE), i.e., the suppression of bacterial growth that persists after limited exposure to an antibiotic (3). When intermittent dosing is applied in clinical practice, however, there is a gradual decrease in the antibiotic concentration in which suprainhibitory concentrations will often be followed by a period of subinhibitory concentrations (sub-MICs). The effects of these sub-MIC levels on bacteria may be an additional explanation for the success of intermittent dosage schedules. We have shown earlier that in certain antibioticbacterium combinations, when a PAE is found, there is a long delay before regrowth when the bacteria are reexposed to sub-MICs during the PA phase (PA sub-MIC effect [PA SME]). The PA SMEs have generally been found to be more pronounced than the direct effect of sub-MICs on bacteria not previously exposed to antibiotics (13)(14)(15). In these experiments, we used experimental and control cultures in broth from which serial subcultures were transferred onto solid media, and the numbers of CFUs were counted. Ho...

Postantibiotic and Sub-MIC Effects of Benzylpenicillin against <i>Streptococcus pneumoniae </i>with Different Susceptibilities for Penicillin

“…The PAE was calculated as the difference in time for the exposed cultures and the corresponding control to grow up to a defined point (A 50 ) on the OD curve, where A 50 is defined as 50% of the maximal OD of the control [21][22][23][24].…”

In Vitro Activities of Novel 2-Fluoro-Naphthyridine-Containing Ketolides

“…Studies were performed according to methods described by Loewdin et al (21). Briefly, colonies of the indicated S. pneumoniae strains were resuspended in CAMHB with HB to 0.5 McFarland standard and cultured in the absence or presence of antibiotic (4ϫ the MIC) for 2 h. Cells were washed twice through centrifugation with an equal volume of medium, resuspended, and diluted 10-fold in CAMHB with 2% HB (one to three serial dilutions).…”

Postantibiotic, Postantibiotic Sub-MIC, and Subinhibitory Effects of PGE-9509924, Ciprofloxacin, and Levofloxacin

“…The plates were then incubated in the BioScreen C. The MIC was defined as the lowest concentration that prevented growth, as determined by measuring optical density. The lowest detectable level of optical density corresponded to approximately 5 ϫ 10 5 CFU/ml (9,19). The PAEs, PA SMEs, and SMEs of PGE-9509924 were determined with the BioScreen C and compared with those of levofloxacin and ciprofloxacin.…”

“…The SME was defined as the difference in lengths of time for the cultures exposed only to the sub-MICs and the controls to reach A 50 (9,15,16,17,19). All experiments were performed twice for each antibiotic-bacterium combination.…”

“…The PAE was calculated as the difference in the lengths of time for the exposed cultures and the corresponding control to grow to a defined point (A 50 ) on the OD curve, defined as 50% of the maximal OD of the control (9,15,16,17,19).…”