Journal of Immunologwal Methods, 58 (1983) 375-381

3'75

Elsewer Biomedical Press

Kinetic Analysis of Bacterial Clearance in Mice Using the ESTRIP c and KINET Microcomputer Programs *
Boonlert Cheewatrakoolpong, Earl K. Steffen, R.Don Brown 1 and

Rodney D. Berg
Department of Mtcrobwlogv and Immunology, I Department of Pharmacology, Loutstana State Umverstty Medtcal Center, School of Medtcme m Shreveport, P 0 Box 33932, Shreveport, LA 71130, U S A

(Received 29 July 1982, accepted 15 October 1982)

Two BASIC rmcrocomputer programs, ESTRIP c and KINET, were used to analyze the kanetlcs of bacterial clearance from the blood and mesentenc lymph nodes of rmce Because of the slrmlantles between the clearance of bacteria and the clearance of drugs from tissue, blood pharmacol~neuc techmques were apphed to the analysis of bacterial clearance data The ESTKIP c program, developed for pharmacolaneuc analysis and modified for the study of bacterial clearance, was employed to fit the experimental data of bacterial surwval versus t~me to a polyexponentml equation wRh 1, 2, or 3 terms The K I N E T program, written specifically for kanet~c analysis of bacterial clearance, uses the b~exponentml equauon constants derived vath ESTRIP c to calculate half-hfe values, rate constants, and other useful k m e u c parameters The combined use of these programs perrmts precise comparisons of the clearance rates of different bactenal species from the blood or t~ssues of experimental ammals Key words microcomputer program - - E S T R I P c _ K I N E T - - bactertal clearance - - kmettc analys,s - ammal model - - bacteremta

Introduction Intravenously adnumstered wable bactena are rapidly ehmlnated from the blood of rmce unui the concentration of bactena m the blood is about 10% of the rejected dose, at wluch ttme there is a decreased bactenal clearance rate (Benacerraf et al, 1959) Therefore, bactenal clearance may be considered a phenomenon that occurs m 2 distract phases Although the clearance rates m these 2 phases are different, both population declines are exponential (Benacerraf et al., 1959) Thus, a blexponentml mathematical expression is necessary to adequately describe the blphaslc bactenal clearance process

* Copies of the modified ESTRIP c program and the K I N E T program m a y be obtmned upon written request 0 0 2 2 - 1 7 5 9 / 8 3 / 0 0 0 0 - 0 0 0 0 / $ 0 3 00 1983 Elsevier Science Pubhshers B V , A m s t e r d a m

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A btphaslc pattern of clearance has also been observed m pharmacokmetlc stu&es of intravenously adrmmstered drugs (Glbal& and Perrler, 1975) The analysxs of such data is usually performed by fitting the data sets to a polyexponentml equation by the least squares method Kinetic parameters describing drug clearance can then be derived from constants m tins equation (Glbal& and Perner, 1975, Wagner. 1975, Cocchetto et al, 1980) The general polyexponentml equation descnbmg drug clearance is C(t) = Al e-B't + A2e -B2t + mne -B"t

where C ~s the concentration of drug at time t, n ,s the number of exponentml terms, and A and B are the hybrid constants to be determined Once the values of A and B are known, the half-hfe and clearance rate of a drug can be easdy computed Recently, Brown and Manno (1978) described a BASIC rmcrocomputer program, ESTRIP c for esttmatlng these hybrid constants Since there are strmlartttes between the patterns of bacterial clearance and drug clearance, tins pharmacokmet~cs data analysis method should also be apphcable to the study of the clearance of bacteria from blood and the lymphoid organs Tins paper reports the adaptaUon of ESTRIP ~ to determme the equations (n = 1 to 3) describing the clearance of vtable bacteria from the blood and mesentenc lymph nodes of mice A second program, K I N E T , was developed winch uses the hybrid constants of the b~exponentml equation (n = 2) derived w~th the ESTRIP c program to calculate kmeUc parameters and to create a permanent file of these data and experimental detads

Methods Analyses of the klnettcs of bacterial clearance using the ESTRIP ~ and K I N E T programs were performed on an Apple II 48K rmcrocomputer (Apple Computer, Cupertino, CA) A detmled description of the ESTRIP c program has been reported previously (Brown and Manno, 1978) Tins program was modified to make it specifically apphcable to bacterial clearance studies Prehrmnary attempts to describe bacterial clearance using equations w~th 4 or more exponenttal terms were unrewardmg, the fit of the b~exponentml model was always as good as those of the Ingher models and, m some instances, ~t was better Therefore, only the exponentml equations wtth n = 1, 2, or 3 are esttmated m the modified ESTRIP c The ongjnal ESTRIP c program also mcludes absorption study options winch were not utlhzed Finally, a headmg section in the modified program enables the user to record experimental detads The K I N E T program was written spectfically for bacterial clearance analys~s and has 2 major opttons The first option allows data (b~exponentlal hybrid constants obtained from ESTRIP c analysts) and experimental mformatton to be entered and stored as a sequentml text file on a &skette The second optton enables the user to retrieve these data and generates the addlUonal kanetlc parameters described m Table I Detailed explanations of these parameters are reviewed elsewhere (Gtbal& and Perrler, 1975)

377 TABLE I DESCRIPTION A N D DERIVATION OF K I N E T I C PARAMETERS G E N E R A T E D U S I N G THE K I N E T PROGRAM Kinetic parameter Clearance rate Description A relatwe estimate of the rate of bacterial clearance from the central compartment Calculation Dose (bacteria mjected per gram average body weight) dlvaded by area under the curve from 10 mm through mfimty a l/clearance rate

Persistency index

A relative estimate of bacterial persistence in the central compartment Half-hfe of bacteria in primary phase of clearance (B 2 = a In pharmacokmetlc terms) Half-hfe of bacteria m secondary phase of clearance (B I ffi ~ in pharmacokanetlc terms) A rate constant for the transfer of vmble bacteria to the central compartment from the peripheral compartment A rate constant for the elmunation of bacteria from the central compartment (Kel is also called Klo ) A rate constant for the transfer of bacteria from the central compartment to the peripheral compartment

Half-hfe (A-phase)

0 693/B2 = Ln(2)B2 b

Half-hfe (B-phase)

0 693/B 1 = Ln(2)/B l b

K2!

(A2BI + A I B 2 ) / ( A 2 + A 1)

Kcl

(B2B l)/K21

Kin2

B2 + B 1 - K2t - Ke~

a The area under the curve dunng the first 10 nun after mjecUon of bacteria cannot be employed since there is no rehable way to obtmn accurate bacterial survwal values in tins time interval, the lmtlal rate constant ~s so large that a small change in ~t could dramatically change the area estimate dunng tins time interval b Ln(2) is used because exponential stripping procedure is performed on the natural logarithms of the concentrations

To dlustrate the performance of the ESTRIP c and K I N E T programs, groups of 5 CD-1 mice were rejected intravenously w~th 0 1 ml each of sahne contaamng 2 25 10 7 washed, log phase cells of Klebszella pneumomae strata SPF9 The numbers of vmble bacteria m the blood ( C F U / m l ) and middle mesentenc lymph nodes (MLN, C F U / M L N ) were deterrmned after 10, 30 nun and 1, 2, 3, 4, and 24 h by standard plate count methods on MacConkey agar (Steffen and Berg, 1982) The total number of intervals and the geometnc means of the numbers of bactena

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survwlng m the blood o r M L N after each interval were entered into the ESTRIP ~ program at hne numbers 9900 and 9901 t o 9910, r e s p e c t w e l y , as d e s c r i b e d m instructions displayed m the mmal section of the program

Results The values of the kinetic parameters, generated by the ESTRIP c from the analysis of the clearance of bacteria from the blood stream are shown m a sample output of the KINET program (Fig 1) The calculation and interpretation of the F and R square parameters and the areas under the curve have been reported prewously (Brown and Manno, 1978) Briefly, the F value is the sum of the squares of the differences between observed and estimated values and ~s used to evaluate the 'goodness of fit' The R square value is the squared correlation coefficient The F and R square values indicate that the blexponentlal equaUon fits well to the experimental data The R square value also shows a htgh degree of assocmtlon between t~me and the reduction of bacterial concentration m the bloodstream A b]exponentml profile of K p n e u m o m a e SPF9 clearance is shown m Fig 2 Slrmlar results were obtained when the data of the survival of bacteria m the M L N were used to generate these equations (results not shown) The general equation descrlb-

F'aCTERiC: ~. P h E b ' i 3 5 i P E '_--,"F3 NO. I H J E C T E D : 2i 2 5 E 7 A N i P A L D_-,DEL: E D - ! t~Eilj~,E C Ot,FPRTHE;~T: E:!_OOD DATE: ~ - -2,U-,z,." J '-"~-' E : ' P E R I h E l i T HA,HE: # 3
E:IE::;' EF, IJA: E, T , = q 2 + E , P , - B 2 T ,+~!-,-E:'P(-B1T,

P2= A 1=

t"90955. 4 3 2 I'-- 2. 7 7 0 8 5 !

B 2 = 12. 0766::::E:6 E:I= 2 4 5 7 a 5 2 2 7 11~ 1 ! 0. 4 5 4 2

R , '-,OUARE ,= . 99999:-:007 F =

T O T A L HO. E A C T E R I A INJECTED= 22500000 CFU AtERRF, E_ HOI_ISE H T . = L,.-'~ 2 3 GRAb'-, g ~ E q I_I~OER THE flt,q, l.l E.= . " ~ - r,: .... U, "- L E F I J - H R - H L :' -~ . . ELEARANEE RATE= 28.70:6699 tiL G~A,h-HF. u,-,, ,~F-l_~..Tcc , A - P H Q E E '= . 05739-Z_"~'797 H R HALF-LIFE ( B - P H A E E ~= 2. 8 1 8 9 9 3 7 3 HA, PERSISTENCY IHDE:',= . 0 3 4 8 3 9 9 6 4 5 F12= 1.17057E02 ,'I'HR, 2 ! = .3727~:B--'66 c I. H R ' i EL= I0.8790596 ' !'HR ' 6RqH-HR "HL

E,LOOD ,

Fig 1 Pnntout of loneUc parameters of the clearance of K pneumomae SPF9 from the blood using the KINET program

379
IF.6-

IE~

tEu,I

~' l E3-

1E2-

1 E1

0
TIHE th~tJ~l Fig 2 Blexponentlal curve of the survival of K pneumomae SPF9 m the CD-I mouse generated with the F.STRIP c program C ( t ) = A2 e-a2t + A l e -al: where C ( t ) ~ 790955432e-]2766886t + 1812 77085e - 245745227t The R square value (see text) was equal to 0 999998007 for ttus blexponenual representation of the data

mg the model for the clearance of viable bacteria from the blood or M L N can be written as C(t) - A2 e-a2i + Ale -a't

INPUT DOSE

Central Compartment
"~

Kel

Peripheral

Compartment

Fig 3 A schemaac drawing of the 2-compartment model

380 Since the ESTRIP program analyzes the data from last to first points by the process of exponential stripping, the terms A2 and B2 are the hybrid constants for the first phase (A-phase) of clearance while A 1 and B 1 are the constants for the second phase (B-phase) of clearance A schematic representation of the biexponentlal or 2-compartment model of bacterial clearance is shown in Fig 3 For purposes of these analyses, the central compartment m the previous examples was assumed to be either the bloodstream or the M L N and the peripheral compartment was the remainder of the animal The definitions of the constants K12, K21, and Kel are given in Table I These 3 rate constants and 4 other lonetic parameters were subsequently calculated from the E S T R I P c parameters using the K I N E T program and are shown in Fig 1 Discussion The survival of intravenously injected bacteria has been represented in previous studies by using 1 of 3 basic methods (a) graphical presentation of bacterial concentration or log of bacterial concentration versus time (Derby and Rogers, 1961, Bakker-Woudenberg et a l , 1982), (b) phagocytic index of bacteria (Benacerraf et al, 1959, Nlchaus et al, 1980), or (c) percent recovery of bacteria compared with the injected dose (Rogers, 1960, Harrow et al, 1975) Combinations of these methods also are used, such as the graplucal presentation of percent of viable bacteria recovered (Brown et a l , 1981) Graphacal representation of bacterial survival versus time is the only one of the 3 above methods in whach the dynarmcs of bacterial clearance can be easily discerned However, the clearance of bacteria is difficult to represent graphically because of the hagh numbers of bacteria initially present in the blood versus the few survlxqng bacteria that persist in the blood dunng the second phase Plotting of log surviving bacteria versus time has been used in attempts to allevmte this problem Nonetheless, conclusions often are based solely on the shape or slope of the curves without benefit of mathemaUcal treatment of the data Thus, the interpretation of graphical representations of bacterial clearance data is somewhat subjective Comparisons of phagocytic indices or percent recoveries between bactenal strains or between experimental groups at a gwen time are commonly used procedures in clearance studies The criteria employed in choosing a certain time interval are generally not stated, the interval is presumably chosen on the basis of a 'large' decrease m the surviving population of one or more experimental groups However, bacterial clearance is a relatively complex polyexponential function of time Conclusions based solely upon differences at one time interval may be erroneous It is possible that apparent differences in clearance rates between experimental groups at one time interval do not exast when comparisons are made using parameters derived from data taken over longer periods of time Treatment of bacterial clearance using a bxexponential expression and kinetic analysis circumvents m a n y of the shortcomings inherent in the 3 above methods We have presented an alternative method for the analysis of bacterial clearance in which pharmacokmetic techmques are used to analyze the data The combined use

381 of the E S T R I P c a n d K I N E T programs perrmts the d e n v a U o n of a b l e x p o n e n U a l e q u a t i o n describing the clearance of bacteria from the b l o o d or l y m p h o i d organs a n d the calculaUon of useful lonetic parameters The values for the clearance rate, persistency index, rate constants, a n d half-lives of bacteria i n different phases derived by our m e t h o d can be used to c o m p a r e bacterial clearance between different bacterial strains or between control a n d experimental groups injected with the same bacterial species T h e i m p r o v e d q u a n t i t a t i v e t r e a t m e n t of the data from bacterial clearance studies utilizing the E S T R I P c a n d K I N E T programs p~rmlts a more rigorous a n d defmlUve i n t e r p r e t a t i o n of results than was previously available

References
Bakker-Woudenberg, I A J M, P De Bos, A L E M Van Gerwen, W B Van Leeuwen and M F Michel, 1982, J lnfe~t 4, 17 Benacerraf, B, M M Sebestyen and S Schlossman, 1959, J Exp Med 110, 27 Brown, R D and J E Manno, 1978, J Pharm Scl 67, 1687 Brown, E J, S W Hosea and M M Frank, 1981, J Rettculoendothel Soc 30, 23 Cocchetto, D M , WA Warfonand JW Crow, 1980, J Pharm B~opharm 8, 539 Derby, B and DE Rogers, 1961, J Exp Med 113, 1053 Glbal&, M and D Pemer, 1975, m Drugs and Pharmaceutical Sciences, Vol 1, ed J Sworbnck (Marcel Dekker, New York) p 45 Harrow, E M, G J Jakab, A R Brody and G M Green, 1975, Amer Rev Resp Dis 112, 7 Nlchaus, G D, P R Schumacker and T M Sabu, 1980, Ann Surg 191,479 Rogers, D E, 1960, Bact Rev 24, 50 Steffen, E K and R D Berg, 1982, Infect lmmun 39, m press Wagner, J G, 1975, Fundamentals of Chmcal Pharmacohnet~cs (Drug Intelhgence Pubhcailon, Hamilton, IL)