Comparison of two methods for determining in vitro postantibiotic effects of three antibiotics on Escherichia coli

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A turbidimetric assay was developed and validated against Escherichia coli for the quantitation of viable bacterial densities. The Abbott MS-2 research system was employed for continuous 5-min measurements of optical density. A linear standard curve was obtained by regressing the initial bacterial density (log CFU per milliliter) against the time required for bacterial growth causing a 5% decrease in optical transmittance. Slope and intercept values obtained from eight standard curves showed excellent assay reproducibility. Results obtained by the turbidimetric assay compared favorably to those obtained by the conventional pour plate assay.Prior to the application of the new assay, possible interferences of postantibiotic effect induced by the test antibiotics were excluded. The turbidimetric assay, which is presumably more efficient and less expensive, was implemented for the time-kill studies of three different 1-lactams against E. coli.The MIC is the most commonly used measurement to quantitate the antimicrobial effect of antibiotics. However, this endpoint provides little information on the time-dependent changes of a bacterial population during incubation with the antibiotic. Time-kill studies, which rely solely on the pour plate and colony-counting assay, remain the only means to examine these time-related changes. Automated systems such as the spiral plater (6) MHB-S. A volume of 2.5 ml of the adjusted bacterial culture was introduced into the first chamber of the MS-2 cartridge. The initial inoculum size of the adjusted culture was determined by the pour plate assay. Chambers 2 through 11 were each filled with 2.0 ml of sterile MHB-S. To achieve a series of 1 to 5 dilutions for chambers 2 through 10, 0.5 ml of the bacterial culture from the preceding chamber was transferred and mixed with the MHB-S in the next chamber. When this procedure was completed, the final volume in each chamber was 2.0 ml. Chamber 11 served as a negative growth control. When the fully loaded cartridge was inserted into the MS-2, incubation at 35°C and 5-min OD measurements were started. The time required for a 5% decrease in transmittance (T.%) was recorded for each chamber. To obtain a linear standard curve, the initial logl0 CFU per milliliter values for each chamber determined from the initial inoculum of the adjusted culture and the dilution factor were plotted against the corresponding T5% values (see Appendix). Orthogonal linear least-squares regression was used to determine the best fit line through the points. Bacterial density of any test sample was obtained by interpolating its T5% value from the regression equation. From the regression equation, the instantaneous growth rate constant (K) of the test organism was calculated as the absolute value of the regression slope multiplied by 2.303. The x-intercept theoretically represents the time necessary for a single organism to multiply and produce an inoculum causing a 5% decrease in transmittance. The y-intercept approximates the assay threshold, i.e., the bacterial densi...

supporting

confidence: 80%

mentioning

confidence: 99%

New turbidimetric assay for quantitation of viable bacterial densities

Nix

Schentag

1993

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“…Other methods have been used to quantitate the PAE, such as optical density (1,2,12,13) and measurements of intracellular ATP (6-8, 11, 17), electrical impedance (9), and conductance (5) in bacterial cultures. The correlation between these methods and the rather time-consuming standard method has been variable, and some of the alternative methods require equipment that is usually not part of the standard armamentarium found in general clinical microbiology laboratories.…”

mentioning

confidence: 99%

Quantitation of postantibiotic effect by measuring CO2 generation of bacteria with the BACTEC blood culture system

Gottfreðsson¹,

Erlendsdóttir²,

Guðmundsson³

1991

The duration of the postantibiotic effect (PAE) determined by bacterial CO2 production measured by using the BACTEC NR 730 blood culture system was compared with PAEs determined by standard viability counting. PAEs for Staphylococcus aureus after exposure to dicloxacillin, vancomycin, rifampin, gentamicin, and ciprofloxacin and for Escherichia coli after exposure to ampicillin, gentamicin, and ciprofloxacin were quantitated by the two methods, and an excellent correlation (r = 0.93) was demonstrated. The difference in the PAE durations determined by the two methods was 0.1 ± 0.4 (mean ± standard deviation) h. Thus, the BACTEC CO2 generation method provides a simple, alternate way of determining the PAE in vitro.The clinical significance of the postantibiotic effect (PAE) pertains primarily to the impact it may have on the design of antimicrobial dosing regimens in clinical practice (3), where antimicrobial agents inducing long PAEs against the offending organism may be administered with longer dosing intervals than before without loss of efficacy and even with a lower frequency of adverse reactions (15, 16).The most widely used method for determining the PAE in vitro is by exposing the organism to an antimicrobial agent for a limited time and, after removal of drug, monitoring regrowth by serially measuring viable counts (3). Other methods have been used to quantitate the PAE, such as optical density (1,2,12,13) and measurements of intracellular ATP (6-8, 11, 17), electrical impedance (9), and conductance (5) in bacterial cultures. The correlation between these methods and the rather time-consuming standard method has been variable, and some of the alternative methods require equipment that is usually not part of the standard armamentarium found in general clinical microbiology laboratories. Therefore, a simple alternative is needed. If the PAE is to be applied to specific clinical situations, determination of the PAE needs to be simple, require as little technician time as possible, and utilize technology already present in most laboratories.In this regard, we examined CO2 generation by bacteria as measured by the BACTEC NR 730 blood culture system (Becton Dickinson and Co., Sparks, Md.) for determination of the PAE and compared it directly with viable counting.The BACTEC blood culture system detects bacterial CO2 by infrared spectroscopy of gas aspirated from the headspace of a blood culture vial. CO2 absorption measurement by spectrophotometer is expressed in terms of a growth value (GV), which is a unitless measurement derived from a comparison made by the system of the amount of CO2 present in the vial headspace with the amount of CO2 in aerobic (2.5% C02) or anaerobic (5.0% C02) reference culture gas. The manufacturer recommends that a GV of 30 be used as a cutoff for subculturing a blood culture vial. The bacterial viable count corresponding to this number is variable, ranging from 106 to 108 CFU/ml, and depends on the * Corresponding author. bacterial species and the pH, oxygenation, and temperature of t...

“…A lag phase before the regrowth of bacteria previously challenged with antibiotics has been demonstrated and named the postantibiotic effect (PAE) (2,14). This phenomenon is thought to be a possible explanation for the success of intermittent-dosing regimens (2).The PAE has been demonstrated for various antibioticbacterium combinations (1,8,9,22). However, the PAE is a highly artificial parameter, as in vivo, a suprainhibitory concentration of a drug will always be followed by subinhibitory concentrations (sub-MICs).…”

mentioning

confidence: 99%

“…The PAE has been demonstrated for various antibioticbacterium combinations (1,8,9,22). However, the PAE is a highly artificial parameter, as in vivo, a suprainhibitory concentration of a drug will always be followed by subinhibitory concentrations (sub-MICs).…”

mentioning

confidence: 99%

Pharmacodynamic effects of subinhibitory concentrations of imipenem on Pseudomonas aeruginosa in an in vitro dynamic model

Maggiolo

Taras

Frontespezi

et al. 1994

The postantibiotic effect (PAE), sub-MIC effect (SME), and postantibiotic sub-MIC effect (PASME) of imipenem on Pseudomonas aeruginosa were investigated with an in vitro dynamic model reproducing in vivo elimination kinetics of the antibiotic. The PASMEs were constantly longer than the corresponding SMEs, but differences between them were not statistically significant. Both PASMEs and SMEs were initially bactericidal and were significantly longer than PAEs. The mean values of both PASMEs and SMEs were over 12 h. SMEs appear to be more relevant for the bacterial growth kinetics than PAEs.Dosage schedules for antibiotic therapy are generally based on the assumption that drug concentrations must be maintained above the MIC. However, it has been shown that discontinuous therapy is as effective as continuous therapy, even if the concentrations in serum fall below the MIC (24). A lag phase before the regrowth of bacteria previously challenged with antibiotics has been demonstrated and named the postantibiotic effect (PAE) (2,14). This phenomenon is thought to be a possible explanation for the success of intermittent-dosing regimens (2).The PAE has been demonstrated for various antibioticbacterium combinations (1,8,9,22). However, the PAE is a highly artificial parameter, as in vivo, a suprainhibitory concentration of a drug will always be followed by subinhibitory concentrations (sub-MICs).Subinhibitory antibiotic concentrations may have different effects on bacteria previously exposed to suprainhibitory concentrations and on unexposed bacteria (18)(19)(20). In most of the investigated combinations with I8-lactam antibiotics, the postantibiotic sub-MIC effect (PASME) was significantly longer than the sub-MIC effect (SME).The aim of this study was to investigate the occurrence of the PASME and SME of imipenem on The bacteria, in the exponential growth phase, were then exposed to five times the MIC of the antibiotic for 1.5 h at 37°C. The exposed strains were washed three times for 5 min each time at 1,400 x g to eliminate the antibiotic and were then resuspended in fresh medium. Controls, unexposed to the antibiotic, were treated similarly. Exposed cultures and controls were either diluted or left undiluted and inoculated into the dynamic model (see below) to obtain a final concentration of approximately 5 x 105 CFU/ml.Samples were withdrawn at 0 (before and after dilution), 1, 2, 4, 7, 10, 13, 16, and 20 h and, if necessary, diluted with phosphate-buffered saline. Samples were seeded on Iso-Sensitest agar plates and incubated for 20 h, and CFU were counted. CFU were counted only for plates with 10 to 500 colonies. All experiments were performed in triplicate.The PAE was defined according to the previously described formula (2) PAE = T -C, where T is the time required for the viable counts of the antibiotic-exposed cultures to increase by 1 log1o above the counts observed immediately after the seeding of the system and C is the corresponding time for the unexposed cultures.Determination of the effects of sub-MICs. Th...