The bactericidal effect of gentarrucin on Pseudomonas aeruginosa ATCC 27853 was investigated in a computer controlled dynamic in-vitro model, which allows the simultaneous simulation of three different dosing regimens for several days. The same total dose reduced cfu-counts of Pseudomonas aeruginosa most effectively, when administered with peak concentrations of 32 mg/l every 32 h, whereas the other dosing regimens with peak concentrations of 16 mg/l every 16 h and 8 mg/l every 8 h were distinctly less effective following the second and subsequent doses. It was shown that the use of a microcomputer facilitates the in-vitro investigation of multiple dosing regimens but counting of cfu cannot be substituted by automatic measurements of turbidity when rapid bactericidal effects occu

Blaser, J.

Ledergerber, B.

Lüthy, R.

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Journal of Antimicrobial Chemotherapy (1985) 15, Suppl. A, 169-173Computer-controlled in-vitro simulation of multiple dosing regimensB. Ledergerber, J. Blaser and R. LutfayDivision of Infections Diseases, Department of Medicine,University Hospital Zurich, CH-8091 Zurich, SwitzerlandThe bactericidal effect of gentarrucin on Pseudomonas aerugwosa ATCC 27853 wasinvestigated in a computer controlled dynamic in-vitro model, which allows thesimultaneous simulation of three different dosing regimens for several days. Thesame total dose reduced cfu-counts of Pseudomonas aerugmosa most effectively,when administered with peak concentrations of 32 mg/1 every 32 h, whereas theother dosing regimens with peak concentrations of 16 mg/1 every 16 h and 8 mg/1every 8 h were distinctly less effective following the second and subsequent doses. Itwas shown that the use of a microcomputer facilitates the in-vitro investigation ofmultiple dosing regimens but counting of cfu cannot be substituted by automaticmeasurements of turbidity when rapid bactericidal effects occur.IntroductionWith few exceptions, such as gonorrhoea or uncomplicated urinary tract infections, thetreatment of infectious diseases requires administration of antibiotics in multiple dosesfor several days. It therefore seems appropriate to perform m-vitro investigations ofmultiple dosing regimens which can yield important information concerning thebactericidal or bactenostatic action of the second or subsequent doses and thepossibility of emerging resistance, phenomena which escape the m-vitro studies ofsingle doses.The use of a microprocessor with the capacity to control simultaneously multipleindependent sets facilitates the performance of long-term experiments and improvestheir reproducibility. The feedback of certain data to the computer offers additionaladvantages such as (i) information concerning the actual state of the experiment bydisplaying and recording turbidity data which can be used as a guideline for dilutingthe samples for cfu-determinations and (li) maintenance of constant pharmacokineticconditions by verification of liquid levels.Turbidity data can further be used to initiate automatically active intervention in anexperiment, e.g. when lack of action of a drug is detectedThe purpose of the present study was to prove the feasibility of the computer controlledin-vitro model to determine the effect of three different multiple dosing regimens ofgentamicin on the viable counts of Pseudomonas aeruginosa.Materials and methodsMediaMueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA) were obtained fromDifco (Brunschwig AG, Basel, Switzerland). MHB was supplemented with Mg + + andCa + + (MHB-S) according to NCCLS standards (1983).1690305-7453/85/15A169 + 05 $02 00/0 © 1985 The British Society for Antimicrobial Chemotherapy170AntibioticsB. Ledergerber et aJ.Gentamicin laboratory reference powder was obtained from Schering (Essex Chemie,Luzern, Switzerland).Bacterial strainLog-phase cultures of Ps. aeruginosa ATCC 27853 were photometrically adjusted bydiluting them with MHB-S to yield a final inoculum of 105 cfu/ml. Glass flasks(volume 125 ml) were filled with 100 ml of the bacterial suspension. The minimalinhibitory concentration (MIC) of gentamicin in MHB-S was 20 mg/1.Viable counts of bacteriaTo detect > 100 cfu/ml, three drops of 20 fi\, each from appropriate tubes of serial ten-fold dilutions, were plated on MHA. Less than 100 cfu/ml were quantified bymembrane filtration of 1 ml and 10 ml of undiluted samples, yielding a sensitivity of0-5 cfu/ml. Filters were washed with 100 ml of 0-9% NaCl in water to eliminateresidual drug and placed on MHA. Viable counts were not adjusted for the loss whichoccurred through the dilution with drug free broth.Simulation of multiple dosing regimensThe in-vitro model (designated apparatus model I) described by Grasso et al. (1978)was modified by adding a second peristaltic pump which drained the culture flask andthereby maintained a constant fluid level (Figure 1, pump 3). In addition, this pumpbrothreservoirI pumpP 1 (c dramFigure 1. Single set of the in-ntro model. Pump speed of pump PI was adjusted to produce the desiredelimination half-life, whereas pump P3 (drain) was suctioning at a higher flow rate to maintain the constantvolume and to provide the ventilation of the head space with filtered air (pore size: 0-2 /an). Pump P2 wasoperated according to the dosage regimens.Computer-controlled in-vitro model 171irtJtor prtattr (tiaJitIrad-llidockTmicrocomputerIZcprocess-interface220VlwiKwJ 3.1 Ml tapmiiiii r rONInWl i / D -|olog wpwi-TTT*ok*m-co<tfrotomognttfc ttlrrtr;Ycontrol of three m-vitro setsFigure 2. Diagram of the computer system The process interface was designed to control simultaneouslythree m-vilro sets as shown in Figure 1provided continuous ventilation of the head space of the culture flask through a portfor filtered air which allowed aerobic growth conditions. Three sets of the in-vitro modelwere simultaneously operated in an incubator at a temperature of 370°C, simulatingthree multiple dosing regimens (A, B and C) with identical total doses, which producedpeak gentamicin concentrations of 8, 16 and 32 mg/1 at dosage intervals of 8, 16 and32 h, respectively. The infusion periods were 30 min each. Human one-compartmentpharmacokinetics with an elimination half-life of 2-2 h were simulated by continuousdilution with drug free broth.TurbidimetryThree photometers were built with extra-bright light-emitting diodes, wave-length660 nm (Stanley ESBR 5501, Dewald AG, Zurich, Switzerland), matched pairs ofphotodiodes (United Detector Technology PIN-3DP, Traco AG, Zurich, Switzerland)and a log ratio amplifier (LOG 100, Burr-Brown AG, Ruschlikon, Switzerland)providing a compensated operation with a threshold for the detection of> 200,000 cfu/ml. Turbidity was measured in each culture flask over a path length of4-6 cm and recorded on the disc of the computer at 5 min intervals.Computer controlAn Apple II® plus computer (Apple Computer Inc., Cupertino, California), suppliedwith a specially designed interface was used to control simultaneously three sets of thein-vitro model as shown in Figure 2.ResultsMultiple dosing regimensThe gentamicin concentration versus time curves for the three different dosingregimens are displayed in Figure 3. The resulting viable counts and optical density172 B. Ledergerber et al.320-16-0-80-40-20-1-0-0-5-0-25-0-125-00625-0031200156-8 mg/ l every 8h16 mg/ I every 16 h32 mg/ l every 32 hI16 —t24HouriFigure 3. Concentration versus time curves of the three gentamian dosing regimens A, B and C with anidentical total dose yielding peak concentrations of 8, 16 and 32 mg/l at intervals of 8, 16 and 32 h,respectively8 mg/l every 8 h* 16 mg/l every 16 ho32 mg/l every32 h48Hours00100001Figure 4. Cfu and turbidity versus time curves of Ps aeruginosa ATCC 27853, exposed to the three dosingregimens of gentamian which are displayed in Figure 3 The photometers, operated at 660 nm, yielded athreshold of the detection of > 200,000 cfu/mlComputer-controlled in-vitro model 173versus time curves (Figure 4) show a rapid and dose dependent decline after the firstdose, followed by regrowth within 4 to 8 h. Apart from a slight reduction of cfu at24 h, the subsequent doses of regimen A (8 mg/1 peak concentrations every 8 h) showedno apparent bactericidal activity. The second and third dose of regimen B, yielding apeak concentration of 16 mg/1 at 16 and 32 h were marginally effective, and at 48 h, theeffect of the fourth dose was completely lacking. This is in contrast to the obviousbactericidal effect of the second dose of regimen C (32 mg/1 peak concentration) at 32 hwhich resulted in a decrease of approximately 4 log cfu, comparable to the effect of thefirst dose.Turbidity versus time curves showed a good correlation with the cfu-counts whengrowth occurred. In periods of rapid bactericidal action (e.g. regimen C at 32 h),turbidity was determined by the washout of dead bacteria through dilution with drugfree broth and deviated markedly from the determinations of viable counts.DiscussionThe effect of multiple doses of gentamicin on Ps. aerugmosa ATCC 27853 was studiedin a computer controlled in-vitro model. It was demonstrated that identical total dosesof gentamicin were most effective, when administered at intervals of 32 h. Although theconcentration of gentamicin for that regimen (C) was below the MIC forapproximately 3/4 of the dosage interval, cfu's did not exceed the counts observed withregimen A within the first 24 h. Indeed, the long period of subinhibitory concentrationsmay be the reason for the retained bactericidal action of the second dose.The on-line measurement of turbidity showed a good correlation with> 200,000 cfu/ml as long as growth occurred and was very useful as a guideline fordiluting the samples for cfu-determinations. Due to insufficient sensitivity and theinability to quantify rapid bactericidal effects, as observed with aminoglycosides, thesubstitution of cfu-counts by turbidity measurements is not advisable.The use of a microcomputer was a time-saving and reliable method to simulatemultiple dosing regimens for several days. Limitations in simultaneously performingmultiple dosing regimens were found to be the processing of samples, increasingnumber of pumps and space in the incubator.Further development of the model includes the analogue control of pump speeds tosimulate combination therapies of drugs with different half-lives and the automatic useof turbidity data to evaluate optimal timing of synergistic combinations.ReferencesGrasso, S, Meinardi, G., Carnen, I. & Tamassia, V. (1978). New in vitro model to study theeffect of antibiotic concentration and rate of elimination on antibacterial activityAntimicrobial Agents and Chemotherapy 13, 570-6Tentative Standard. (1983). Methods for dilution antimicrobial susceptibility tests for bacteriathat grow aerobically National Committee for Clinical Laboratory Standards, Villanova,Pa

Computer-controlled in-vitro simulation of multiple dosing regimens

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