Importance of bacterial growth phase in determining minimal bactericidal concentrations of penicillin and methicillin

Abstract

The minimal inhibitory concentrations of penicillin against 96 strains of group B streptococci and of methicillin against 10 strains of Staphylococcus aureus were unrelated to the growth phase of test bacteria. However, the minimal bactericidal concentrations were significantly higher in the stationary phase than the logarithmic phase for both organisms (P < 0.001 and < 0.05, respectively).The in vitro determination of bacterial susceptibility to antimicrobial agents is commonly done by disk diffusion. More quantitative determination of growth-inhibiting activity (minimal inhibitory concentration; MIC) is accomplished by agar or broth dilution, and determination of bacterial-killing activity (miniimal bactericidal concentration; MBC) is accomplished by a modification of the latter. Although there have been attempts at standardization of these procedures, various methods have been recommended for the preparation of the bacterial inoculum. These include logarithmic, early-stationary, and overnight cultures to be appropriately diluted as the inoculum for the dilution tests (2, 5, 7). In studies of antibiotic tolerance of group B streptococci, we observed that normal, nontolerant organisms were killed more rapidly when exposed to penicillin in the logarithmic phase of growth than when tested after overnight incubation (K. S. Kim and B. F. Anthony, Pediatr. Res. 14:560, abstr. no. 808, 1980). The present study extends these observations to examine the effect of the bacterial growth phase on the MICs and MBCs of penicillin against group B streptococci and of methicillin against Staphylococcus aureus.Ninety-six strains of group B streptococci were shown to be nontolerant for penicillin. Briefly, the criteria for tolerance were (i) delayed killing by penicillin concentrations of 16 times the MIC during the logarithmic phase of growth, (ii) additive rather than synergistic response to penicillin-gentamicin combinations, and (iii) relatively deficient autolytic enzyme activity (Kim and Anthony, Pediatr. Res. 14:560, abstr. no. 808, 1980). The organisms were isolated from either blood or cerebrospinal fluid of septic infants at the Harbor-UCLA Medical Center, the University of Minnesota Hospitals, the University of Alabama Medical Center, and the Charity Hospital of Louisiana. They were grouped and typed by the methods of Lancefield (4) and Wilkinson et al. (8) with sera provided by the Centers for Disease Control, Atlanta, Ga. Ten strains of penicillin-resistant S. aureus were isolated from blood cultures at Harbor-UCLA Medical Center and identified on the basis of colonial morphology, Gram strain, and coagulase reaction. The presence or absence of tolerance was not determined.Potassium penicillin G in a potency of 1,595 U/mg and sodium methicillin in a potency of 890 ug/ml (Bristol Laboratories, Syracuse, N.Y.) were dissolved at a concentration of 1 and 10 mg/ml, respectively, in sterile distilled water, passed through a 0.45-ytm pore filter (Gelman Instrument Co., Ann Arbor, Mich.), and stored in aliquots at -70°C fo...

Variables in demonstrating methicillin tolerance in Staphylococcus aureus strains

“…We have failed to demonstrate tolerance with a logarithmic-phase inoculum, confirming the observations of Mayhall and Apollo (8) and supporting the observations of Kim and Anthony (5), who demonstrated that the MBCs were higher with stationary-phase bacteria than with logarithmic-phase bacteria, resulting in a higher MBC/MIC ratio. These observations are in conffict with those of Sabath et al (15) and Raynor et al (14).…”

“…It is clear from our studies and from those of others (1,3,5,6,8,12) that because there is a multiplicity of methodological determinants which affect the status of a given strain, a standardized reference procedure for the determination of tolerance needs to be established.…”

Comparative in vitro bactericidal activity of cefonicid, ceftizoxime, and penicillin against group B streptococci

“…There were no duplicate isolates from the same patient. Aliquots of logarithmic-phase cultures of each strain in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) were stored at -70°C until susceptibility testing was performed.A late-logarithmic-phase culture of each GBS isolate was prepared in Mueller-Hinton broth as previously described (7,8) at -2 x 105 colonyforming units per ml. Potassium penicillin G (Bristol Laboratories, Syracuse, N.Y.), and cefonicid and ceftizoxime (Smith, Kline & French Laboratories, Philadelphia, Pa.) were reconstituted to concentrations of 10,000 ,ug/ml and stored in aliquots at -70°C until the susceptibility testing was performed (within 4 weeks of freezing).…”

“…However, 18% of GBS strains required penicillin MBCs between 0.1 and 1 ,ug/ml, the range of relative penicillin resistance for cerebrospinal fluid streptococcal isolates (6). Four of the 108 strains were "tolerant" to the killing action of penicillin, with MBC/MIC ratios of -16:1 (7,8).…”

The Poisoned Well: Enhancing the Predictive Value of Antimicrobial Susceptibility Testing in the Era of Multidrug Resistance

“…For example, biological variability may be introduced through use of different growth phases (13), inoculum densities, incubation conditions (e.g., duration, temperature, humidity, and oxygen and carbon dioxide concentrations), or media (14). However, some proportion of biological variability is uncontrollable, as individual organisms within clonal populations display phenotypic heterogeneity (15), likely related to stochastic epigenetic effects.…”