ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1989, P. 1046-1051 Vol. 33, No.

7
0066-4804/89/071046-06$02.00/0
Copyright C 1989, American Society for Microbiology

In Vitro Evaluation of the Determinants of Bactericidal Activity of

Ampicillin Dosing Regimens against Escherichia coli
CATHERINE A. WHITE,1 ROGER D. TOOTHAKER,2 ARNOLD L. SMITH,3 AND JOHN T. SLATTERYl*
Department of Pharmaceutics, University of Washington, Seattle, Washington 981951; Department of Pharmacokinetics
and Drug Metabolism, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Michigan
481052; and Department of Pediatrics, University of Washington, and Division of Infectious Diseases,
Children's Hospital and Medical Center, Seattle, Washington 981053
Received 2 October 1987/Accepted 5 April 1989

An in vitro flow model was used to examine the influence of peak concentration (Cmax), the area under the

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antibiotic concentration-time curve (AUC), the magnitude of AUC above the MIC, and the aggregate time the
antibiotic concentration exceeds the MIC (TMIC) on the bactericidal effect of ampicillin against Escherichia coli
ATCC 12407. Bacteria in the log phase were exposed to therapeutically realistic drug regimens. Ampicillin
concentration and bacterial density (CFU per milliliter) were measured over time. Four parameters reflecting
bactericidal activity were quantitated: difference between initial and minimum and initial and final bacterial
densities, area under the bacterial density-time curve, and a fourth parameter, which is a function of these
Z,

three. Multiple regression analysis confirmed AUC as the major factor in predicting bactericidal activity. An
AUC of >70 ug- h/ml correlated with the lack of emergence of resistance.

Since penicillin G was introduced in 1941, the dependence
of therapeutic -effect on antibiotic dose and administration
schedule has been the focus of considerable investigative
effort. The importance of this issue was demonstrated by
Eagle et al. (5) and Miller et al. (20) who observed that the
median effective total amount of penicillin G administered
varied 100-fold depending on the size and frequency of
individual doses. It is clear from such work that the time
course of antibiotic concentration in serum is an important
determinant of efficacy, but it has been difficult to identify a
descriptor(s) of the P-lactam concentration-time curve with
which antibacterial activity will be correlated. Among those
considered have been the area under the antibiotic concen-
MATERIALS AND METHODS
Kinetic model. A modified Grasso in vitro kinetic dilution
model was used to expose E. coli to various ampicillin
concentration-time profiles (Fig. 1) (11). The flow of drug-
and bacteria-free medium into the bacterial flask in this
model allows a range of drug half-lives to be simulated.
However, flow also results in the dilution of bacteria, which,
if not corrected for, could not be distinguished from the
bactericidal activity of the antibiotic. The correction for flow
is made with the following equation:
Nt'' = (Nmax -(NmaxNt)
I

tration-time curve (AUC), the magnitude of the AUC above
the MIC (AUC > MIC), the total time that antibiotic
concentration exceeds the MIC (TMIC), and the maximum
antibiotic concentration attained during a dosing interval
(Cmax) (3, 6-9, 11, 12). These descriptors of dosing regimen
are a function of dose and frequency of administration as
well as clearance and volume of distribution.
Ampicillin is most commonly administered as a series of
intermittent bolus doses. The regimen is designed to main-
tain antibiotic concentrations in serum above the MIC for
some portion of the dosing interval, but it is not clear that
time above the MIC should be considered a more important
feature of the dosage regimen than other characteristics,
e.g., Cmax or AUC (7, 8, 14, 19). Further, it is not clear
whether there is an independent effect of half-life (which is a
function of clearance and volume of distribution) or dosing
interval or whether these factors are important only insofar
as they affect AUC, AUC > MIC, Cmax, and TMIC.
We examined the dependence of the bactericidal activity
of ampicillin on AUC, AUC > MIC, TMIC, and Cmax in in
vitro studies with Escherichia coli ATCC 12407 in which
ampicillin half-life and dosing interval were varied.
*
Corresponding author.
1046
N,) e - ket + N,
where N, is the measured bacterial density, Nmax is the
maximum attainable bacterial density had there been no
flow, ke is the elimination rate constant, and N,' is the
bacterial density which would have been observed had there
been no flow (27). In each study, bacteria were exposed to
the drug for 12 h. Doses of ampicillin were repeated as
required by the dosing interval to maintain a 12-h total
exposure. Dosing interval was varied to yield a range of
AUC, Cmax, and TMIC.
Bacteria. E. coli ATCC 12407 was used in these experi-
ments. The general experimental procedure was as follows.
An overnight culture of E. coli in Mueller-Hinton broth
(Difco Laboratories, Detroit, Mich.) was diluted to 102
CFU/ml and allowed to grow to 10i CFU/ml in the same
medium. A 1-ml sample of the 107-CFU/ml culture was
added to the test flask, resulting in a density of 105 CFU/ml.
When the bacteria reached a density of 107 CFU/ml (in the
logarithmic growth phase), the dose of ampicillin required to
obtain the desired initial drug concentration was added to the
test flask and the peristaltic pump was started. Samples for
determination of bacterial density were obtained at 0.5-h
intervals. Bacterial density prior to the addition of drug was
determined by measurement of A6.. After the addition of
ampicillin, bacterial density was determined by serially
diluting samples in sterile phosphate-buffered saline contain-
VOL. 33, 1989
D -Es_
I I
FIG. 1. Schematic representation of in vitro dilution model.
Medium is transferred from the reservoir flask (A) to the test flask
(B) by the pump (C). Flask B maintains a constant volume; bacteria,
drug, and medium are sent to waste (D).
ing 0.1% gelatin. Triplicate 10-pI aliquots of each dilution
and 100 ,ul of undiluted sample were streaked onto nutrient
agar plates (Difco). The plates were incubated at 37°C for a
minimum of 12 h. Preliminary studies determined that the
small amount of ampicillin in the sample had no effect on the
estimation of bacterial density (data not shown), most prob-
ably because of dilution of the drug during preparation of the
sample prior to streaking on the plates. The MIC and MBC
of ampicillin were determined by broth dilution by standard
methods (25, 26). The MIC is defined as that ampicillin
concentration which inhibited visibly detectable growth in
broth containing 5 x 105 CFU. The MBC was the ampicillin
concentration which resulted in killing of .99.9% of the
same inoculum. The MBC was the same as the MIC when
tested as described above and at an inoculum of 103 CFU.
Rejection values of 3 and 11 were used for the inocula of 5 x
105 and 1 x 103 CFU, respectively (22).
Determination of bactericidal parameters. These studies
required quantitative measures of bactericidal activity. The
four parameters used were ANmax, ANtot, BAUC, and X,
which (except for E) are illustrated in Fig. 2. The values
reported for each parameter were determined over the entire
12-h period of exposure of bacteria to antibiotic. ANmax is
the difference between the initial and minimum bacterial
I-
----T
z inoculum size. BAUC is a time-integrated parameter and is
uJ
a
/< __ __ANtot
-J
Ci -
-------
- --7-- -- --------
uJ
cc O AN 1max
m
ot
'-BAUC- ---
0 T1 2T'
TIME
FIG. 2. Illustration of the bactericidal effect parameters: ANmax,
ANto,, and BAUC. X is calculated from the three parameters
depicted here as described in the text. BAUC is the shaded area
under the solid curve of bacterial density. The stippled lines identify
the bacterial density-time points used to calculate the differences
ANmax and ANtot. t is the interval between doses of antibiotic.
DOSING REGIMEN AND AMPICILLIN ACTIVITY 1047
E
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121
E 10
0
8
0
o 6
-o
C:
~0 4
a)
0
2
co
I
0 2 4 6 8 10 12
Time, hours
FIG. 3. Time course of bacterial density and ampicillin concen-
tration in a representative study with E. coli ATCC 12407. Ampicil-
lin was dosed at 0 and 6 h and removed by flow with a 1.0-h half-life.
Drug concentration exceeded the MIC for 6 h of the 12-h study.
Arrows identify times at which ampicillin was added.
densities and represents the initial decrease in viable cell
density associated with antibiotic addition. ANtot is the
difference between the bacterial density at the beginning and
end of the 12-h study and represents the regrowth phase (in
which the emergence of resistance becomes apparent) over
two or more dosing intervals. BAUC is the area under the
bacterial concentration-time curve divided by the initial
affected by cell death, stationary, and regrowth phases. The
parameter L is introduced in an attempt to obtain a single
parameter which is a function of the three fundamental
parameters. l is calculated by assigning a rank of 10 to the
lowest value of each respective fundamental parameter
(ANmax, ANtot) and BAUC) observed in all studies con-
ducted with a given strain of bacteria. A score of 1 is
assigned to the maximum value of each parameter. Scores
are assigned to other values of a given parameter by linear
interpolation between these extremes. l is the sum of the
ranks thus assigned to the values of the respective parame-
ters. A value of E is calculated for each exposure condition.
Ampicillin assay. Ampicillin concentration was determined
by a standard disk diffusion microbiological method (24).
Sarcina lutea ATCC 9341 or Bacillus subtilis (Difco) was
used as the assay organism (2, 15). Ampicillin standards
(Sigma Chemical Co., St. Louis, Mo.) were prepared in
1048 WHITE ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Reproducibility of results with E. coli 12407

(pigAUC
Cmax
(logANmax
Study Dosing(h)
interval t1/2
(h)" (,ug/ml) TMIC
(h) h/ml)
AUC
(pLg. >h/ml)
MIC CFU/mi) (log ANtot
CFU/ml) BAUC* h/mi)
(log CFU I
1 6 0.87 10.1 5.8 25.0 17.0 -3.6 +1.9 106 10.7
2 6 0.88 9.1 5.6 23.0 15.0 -3.3 +1.9 113 9.5
3 6 0.9 9.0 5.6 22.9 15.0 -3.3 +1.9 113 9.5
4 6 1.1 7.2 6.0 21.2 12.7 -2.2 +2.4 125 6.6
a t12, Half-life.

Mueller-Hinton broth. The lower limit of detection was 0.5 the MIC. Bacterial density and ampicillin concentration-time
,ug/ml, and the coefficient of variation of the slope of the curves from a representative study are shown in Fig. 3;
standard curve was 6.6% over a 2-year period. parameter values calculated from these raw data are sum-
Determination of pharmacokinetic parameters. The in vitro marized in row 3 of Table 1. The dosing interval was 6 h and

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system is designed such that antibiotic concentration will
decline monoexponentially. The actual elimination rate con-
stant, ke and maximum concentration, Cmax, were deter-
mined by linear regression. AUC and AUC > MIC were
calculated by the trapezoidal rule over the 12-h period of
each study (10). The aggregate time during which concentra-
tions were above the MIC for the initial inoculum was
calculated by TMIC = kI-1 ln(Cmax/MIC).
Statistical analysis. Multiple regression analysis was per-
formed to determine the importance of each of the descrip-
tors of the antibiotic concentration-time profile as a deter-
minant of ANmax, A&N,01, BAUC, and Multiple regression
Z.
was performed by the stepwise linear regression procedure
with a significance level of 0.05 (17). Stepwise regression is
an improved version of forward regression which permits
reexamination at every step of the variables incorporated in
the model in previous steps. At each step, a partial F test for
each variable presently in the model is made, treating it as
though it were the most recent variable entered. This pro-
cedure allows removal of variables that were entered at an
early stage which have become superfluous because of their
relationship with other variables subsequently evaluated.
RESULTS
The goal of this investigation required the simulation of a
large number of dosing regimens. The general strategy was
to evaluate ranges of dosing interval and half-life which are
encountered clinically after intravenous ampicillin adminis-
tration. Cmax was varied from approximately 1 to 100 times
the half-life of ampicillin was approximately 1 h; this allowed
drug concentration to exceed the MIC for 6 h of the 12-h
study. Ampicillin was effective in killing bacteria (Cmax was
nine times the MIC and ANmax was -3.3 log CFU/ml, i.e.,
10-3-3 CFU/ml). However, there was a net growth of bacte-
ria over the period of exposure; AN,., was 1.9 log CFU/ml.
Net growth began 2 h after the first dose and 1 h after the
second dose. Table 1 shows the reproducibility of these
results in a series of four replicate studies. The replicates
shown represent data collected over 1 year.
The results obtained from the various exposure conditions
are summarized in Table 2. Data from individual studies
were analyzed by multiple linear regression to determine the
descriptor(s) of the antibiotic concentration-time profile
most strongly associated with bactericidal activity. Log
transformations of Cmax and AUC, referred to as log Cmax
and log AUC, were used in the analysis because the log
transformations improved the correlation between these
parameters and bactericidal efficacy. Log transformation did
not improve the correlation between measures of efficacy
and any other descriptors of dosage regimen (e.g., TMIC).
The results of the regression analysis are shown in Table
3. A value of 0 for a coefficient means that inclusion of the
parameter with which it is associated does not improve the
overall correlation. The absolute value of the regression
coefficient is a reflection of the relative importance of a
parameter as a determinant of the bactericidal activity of a
regimen. The relationship between the various bactericidal
parameters and log AUC for E. coli is shown in Fig. 4.
The apparent emergence of resistance was encountered in

TABLE 2. Influence of ampicillin concentration-time profile on bactericidal activity against E. coli ATCC 12407
Dosing t112 Cmax TMIc AUC AUC > MIC ANmax ANt., BAUC
interval (h) (h) (pig/mi) (h) (pLg. h/ml) (pig. h/ml) (log CFU/mi) (log CFU/mi) (log CFU h/ml)
2a 1.0 2.5 7.5 15.9 5.2 -1.5 +1.7 124 6.5
2a 1.1 25.5 12.0 169 157 -7.9 -5.0 60 26.5
2 1.1 62.0 12.0 413 201 -7.9 -7.9 53 30.0
2 4.0 1.3 8.2 13.2 1.7 -2.6 +0.6 118 9.3
30 0.9 5.2 8.5 24.0 13.2 -1.9 +2.1 118 7.3
3a 1.0 8.8 11.2 42.0 30.0 -3.7 +1.8 104 11.2
3 10.0 8.4 12.0 91.0 99.0 -5.5 -5.2 69 23.0
6 1.1 4.8 4.7 14.0 9.6 -3.6 +1.1 106 11.4
6a 0.9 8.8 5.8 23.0 14.9 -3.1 +2.0 114 9.1
6 1.1 80.4 12.0 253 241 -7.9 -3.1 64 24.6
6 4.1 2.1 8.3 15.3 3.9 -2.5 +0.3 118 9.5
6 5.8 8.4 12.0 72.0 60.0 -5.1 -5.1 74 21.9
12 1.0 10.2 3.3 14.4 11.1 -5.0 +1.3 101 13.3
120 1.0 80.4 6.1 111 105 -7.5 -1.2 71 21.7
Data represent the mean of two to four individual incubations when dosing interval and half-life U1,2) were constant and peak concentration varied less than
10%. n = 1 if no superscript.
VOL. 33, 1989 DOSING REGIMEN AND AMPIC1LL1N ACTIVITY 1049

TABLE 3. Multiple regression summary for E. coli ATCC 12407 TABLE 4. Relationship between emergence of resistance
and ampicillin AUC
Regression coefficient"
Parameter Presence of resistant strains of
bh C d , Ampicillin AUC E. coli ATCC 12407
(p.g h/ml)"
ANnlrx 2.26 -5.78 0 0.30 0.83 No. positive No. negative
ANto,t 13.0 -18.9 10.2 0.84 0.83
BAUC 176 -48.9 0 0 0.84 >70 0 7
M; -10.1 15.2 0 0 0.81 <70 12 1
"General regression equation: parameter = a + b log AUC + ( log Cnflax + Resistance identified by MIC. Presence of resistance was positive when
dTMIc. Regression coefficient is 0 when inclusion of the respective descriptor bacteria achieved an MIC three times the original.
did not improve the value of r2. Parameters not listed, i.e., half-life, dosing
interval, and AUC > MIC, always had coefficients of 0.
Grasso model, regrowth of bacteria has been shown under
certain conditions by Haag et al. (13) to be a consequence of
more than half of the studies conducted. The frequency of adherence of bacteria to the wall of the incubation flask,

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mutation for resistance to 10 p.g of ampicillin per ml was 6.18
X 10-7 for E. coli ATCC 12407, as determined by the
modified Luria-Delbruck fluctuation test (19). The frequency
of mutation for resistance to ampicillin at 20 jig/ml was 1.1 x
10'. Thus, resistance to ampicillin at 10 jig/ml apparently
developed as a consequence of the emergence of a resistant
clone present in the original inoculum of E. coli ATCC 12407
(23). The MIC was regularly determined over the study
period. The initial population of E. coli ATCC 12407 (107
CFU/ml) was tested to determine what fraction of the
population was resistant to ampicillin concentrations equal
to the MIC, twice the MIC, four times the MIC, and eight
times the MIC. Of the initial bacterial population, 0% was
resistant at these ampicillin concentrations.
Table 4 shows that the emergence of resistance (defined by
a minimum of a threefold increase in MIC) was associated
with an ampicillin AUC of <70 [Lg h/mi. In variants of the
forming a thin film. The film sheds bacteria into the medium,
resulting in apparent regrowth. We evaluated the potential
role of this artifact in our system by conducting (in duplicate)
incubations without drugs and with a medium flow rate such
that the drug half-life would have been 15 min. Results (mean
values of the duplicate studies) are shown in Fig. 5. Bacterial
density declined log linearly for the first 4 h of the incubation
and then became constant at approximately 3.5 log CFU/ml.
The results suggest that some bacteria adhere to the model
(in that bacterial density should have been reduced to 1 log
CFU/ml at this flow) but that adherence per se cannot
account for the regrowth observed in our studies with
ampicillin. In addition, growth rate constants at ampicillin
concentrations below the MIC were dependent on medium
flow rate. The growth rate constant at a medium flow rate of
1.2 ml/min was 0.58 ± 0.13 h-1 (range, 0.3 to 0.74 h-<), and
at a medium flow rate of 0.25 ml/min, it was 1.0 ± 0.16 h-'

0
-1
3-1 0
0
000
1. .2C.) -3I
0 0 0
' -4
.0%
o0 0
c) -3' 0 0
I
0
0 -5 0
0
E -6
z Z
-7 0
.7-
-81 OD 0 0
0 0

0 100 100oo -10 1o000

30T 00
0
o O
25t
8
0°
C.R oo 20c
0
I 0 0 0
0) 0
0
15
0
-m 8
10 (;
0 0 o 0
0 0
m 5o
0 0
0 0

1 oon 1-10( 100 100oo

AUC, gg hr/ml -

FIG. 4. Scatter plots illustrating the correlation between the various measures of bactericidal efficacy and log AUC.
1050 WHITE ET AL.
E 8-
CD)
0)6-
C)
w
0
< C° I signs of the coefficients of AUC and Cmax (Table 3) are
m 0 2 4 6 8 10 12
TIME, hours
FIG. 5. Time course of bacterial density in an incubation without
ampicillin and a flow rate such that drug half-life (had drug been
incorporated) would be 15 min. Data shown are mean values of
duplicate incubations.
(range, 0.8 to 1.2 h-1). When the film accounted for the
apparent resistance in the studies by Haag et al. (13), the
growth rate constant was independent of medium flow rate.
DISCUSSION
The analysis of data obtained in dynamic in vitro studies of
antibiotic effect has been complicated by the lack of quanti-
tative measures with which to characterize bactericidal
activity. We defined four parameters with which this objec-
tive can be accomplished. ANmax, AN,,, and BAUC (de-
fined in Materials and Methods) reflect different facets of the
bactericidal activity of a dosing regimen. The term X was
introduced to provide a method to sum all these measures in
a single term and to serve as an overall index of bactericidal
activity. However, a high value of E may not translate into
the most effective regimen in vivo, as the characteristics of
the bactericidal activity of a regimen reflected in ANmax,
ANtot, and BAUC are likely not to be of equal importance
when other factors such as host defense contribute to the
removal of bacteria.
In our studies with ampicillin, AUC was determined to be
the most consistent index of bactericidal activity (Table 3,
Fig. 3) in agreement with the in vitro results of others (7, 9,
16). Studies in mice have also shown a correlation between
AUC and bactericidal activity (3). However, TMIC has also
been suggested to be strongly associated with bactericidal
activity in vitro and in vivo (5, 6, 19). In our studies, TMIC
did not covary strongly with AUC for E. coli (r2 = 0.36).
Thus, a contribution of TMIC to bactericidal activity was not
masked by the correlation obtained with AUC. It is possible
that TMIC was not identified as an important characteristic of
the dosage regimen in our studies owing to the high fre-
quency (>50%) with which resistant strains emerged. When
MIC increases during a study, the period that the antibiotic
concentration exceeds the MIC for the original inoculum
becomes irrelevant. In support of this view, Eagle et al. (5)
noted that a longer TMIC was required to cure mice infected
with group A Streptococcus species when regrowth was
observed between doses. However, their in vivo studies
examined the relationship between concentration of antibi-
otic in plasma and bactericidal effect in tissue, and the
relationship between concentration in tissue and concentra-
tion in plasma was not known.
ANTIMICROB. AGENTS CHEMOTHER.
In in vitro studies with several strains of bacteria and
antibiotics, Blaser et al. (4) observed that dosage regimens
which produced a peak antibiotic concentration eightfold
greater than the MIC effected substantial cell killing and
inhibited the emergence of resistance. In our studies, Cmax
was identified as an important determinant (although second-
ary to AUC) of AN,0, the bactericidal parameter which is
most directly affected by the emergence of resistance. Cmax,
of course, is simply the ampicillin concentration at a single
point in time and provides virtually no information concern-
ing concentrations after that time. A Cmax of >10 times the
MIC can result in a net gain in bacterial concentration
(positive AN,0t,) if the half-life is short, whereas the same
Cmax coupled with a longer elimination half-life can result in
a net loss of bacteria (negative ANt,d) (Table 2). The different
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apparently due to the frequent association of a high Cmax
with a short half-life. A contribution of Cmax to the other
bactericidal parameters may have been masked by the
correlation between Cmax and AUC, r- = 0.56.
AUC was also found to be an important determinant of the
emergence of resistance, defined by a threefold increase in
MIC or by a positive value of ANt,,, (Table 2). There was no
such result with Cmax. This observation is consistent with
that reported by Grasso et al. (11).
As mentioned above, Haag et al. (13) showed that the
emergence of resistance could be an artifact of in vitro
systems owing to the adherence of bacteria to the glass walls
of incubation flasks. A similar experiment conducted with E.
coli ATCC 12407 indicated that the regrowth observed in
these experiments was not an artifact of the in vitro system
(Fig. 5). In addition, resistance was documented by changes
in MIC, and the growth rate constants at an ampicillin
concentration of <1 p.g/ml (the MIC for the original inocu-
lum) in our studies were a function of medium flow rate (i.e.,
ampicillin half-life), whereas Haag et al. (13) pointed out that
a constant growth rate as a function of medium flow rate is a
hallmark of apparent resistance owing to adherence of
bacteria to glass.
Although the emergence of resistance is a problem en-
countered more commonly in vitro than in vivo, suboptimum
antibiotic therapy has been shown to allow the emergence of
resistant strains in animal studies and in certain patients (1,
18, 21). The emergence of resistance is of particular concern
when the host is immunocompromised (9). In immunocom-
promised patients, the results of kinetic in vitro studies may
be directly applicable. The observation that resistance is
least likely to emerge when AUC is high means that resis-
tance will be least likely when relatively large doses are
given frequently. A large dose, frequent administration, or a
long half-life alone may be insufficient.
Maintenance of antibiotic concentration above the MIC
for the initial inoculum also appears to be insufficient to
prevent the emergence of resistance of E. coli to ampicillin.
Indeed, a constant ampicillin concentration just above the
MIC is likely to allow the emergence of resistant E. coli,
whether present initially or arising from a mutation during
exposure to the antibiotic. The results of this and other
studies (4) suggest that the emergence of resistance will be
discouraged if a dosage regimen is adopted which is designed
to eradicate the least susceptible organism present at the
beginning of therapy.
ACKNOWLEDGMENT
C.A.W. was supported by training grant GM07750 from the
National Institutes of Health.
VOL. 33, 1989
LITERATURE CITED
1. Bayer, A. S., D. Norman, and K. S. Kim. 1985. Efficacy of
amikacin and ceftazidime in experimental aortic valve en-
docarditis due to Pseudomonas aeruginosa. Antimicrob.
Agents Chemother. 8:781-785.
2. Bennett, J. V., J. L. Brodie, E. J. Benner, and W. M. M. Kirby.
1966. Simplified accurate method for antibiotic assay of clinical
specimens. Appl. Microbiol. 14:170-177.
3. Bergeron, M. G., B. M. Nguyen, and L. Gauvreau. 1978.
Influence of constant infusion versus bolus injections of antibi-
otics on in vivo synergy. Infection 6(Suppl.):38-46.
4. Blaser, J., B. B. Stone, M. G. Groner, and S. H. Zinner. 1987.
Comparative study with enoxacin and netilmicin in a pharma-
codynamic model to determine importance of ratio of antibiotic
peak concentration to MIC for bactericidal activity and emer-
gence of resistance. Antimicrob. Agents Chemother. 31:1054-
1060.
5. Eagle, H., R. Fleishman, and M. Levy. 1953. Continuous vs.
discontinuous therapy with penicillin. N. Engl. J. Med. 248:
481-488.
6. Eagle, H., R. Fleishman, and A. Musselman. 1950. Effect of
schedule of administration on the therapeutic efficacy of peni-
cillin. Am. J. Med. 9:280-299.
7. Gerber, A. U., H. Brugger, C. Feller, T. Stritzko, and B. Stalder.
1986. Antibiotic therapy of infections due to Pseudomonas
aeruginosa in normal and granulocytopenic mice: comparison
of murine and human pharmacokinetics. J. Infect. Dis. 153:
90-97.
8. Gerber, A. U., W. A. Craig, H. Brugger, C. Feller, A. P. Vastola,
and J. Brandel. 1983. Impact of dosing intervals on activity of
gentamicin and ticarcillin against Pseudomonas aeruginosa in
granulocytopenic mice. J. Infect. Dis. 147:910-917.
9. Gerber, A. U., P. Wiprachtiger, U. Stettler-Spichiger, and G.
Lebek. 1982. Constant infusions vs. intermittent doses of gen-
tamicin against Pseudomonas aeruginosa in vitro. J. Infect. Dis.
145:554-560.
10. Gibaldi, M., and D. Perrier. 1982. Pharmacokinetics, 2nd ed., p.
445-449. Marcel Dekker, Inc., New York.
11. Grasso, S., G. Menardi, I. deCarneri, and V. Tamassia. 1978.
New in vitro model to study the effect of antibiotic concentra-
tion and rate of elimination on antibacterial activity. Antimi-
crob. Agents Chemother. 13:570-576.
12. Guggenbichler, J. P., E. Semenitz, and P. Konig. 1985. Kill
kinetics and regrowth pattern of bacteria exposed to antibiotic
concentrations simulating those observed in vivo. J. Antimi-
crob. Chemother. 15(Suppl. A):139-146.
13. Haag, R., P. Lexa, and I. Werkhauser. 1986. Artifacts in dilution
pharmacokinetic models caused by adherent bacteria. Antimi-
DOSING REGIMEN AND AMPICILLIN ACTIVITY 1051
crob. Agents Chemother. 29:765-768.
14. Hunter, P. A., G. N. Rolinson, and D. A. Witting. 1973.
Comparative activity of amoxycillin and ampicillin in an exper-
imental bacterial infection in mice. Antimicrob. Agents Chemo-
ther. 4:285-293.
15. Jallings, B., A. S. Malmberg, A. Lindman, and L. 0. Boreus.
1972. Evaluation of a micromethod for determination of antibi-
otic concentration in plasma. Eur. J. Clin. Pharmacol. 4:150-
157.
16. Klaus, U., W. Henninger, P. Jacobi, and B. Wiedemann. 1981.
Bacterial elimination and therapeutic effectiveness under dif-
ferent schedules of amoxicillin administration. Chemotherapy
27:200-208.
17. Kleinbaum, D., and L. Krupper. 1978. Applied regression
analysis and other multivariable methods. Duxbury Press,
North Scituate, Mass.
18. Maugh, T. H., II. 1981. A new wave of antibiotics builds.
Downloaded from http://aac.asm.org/ on March 6, 2020 by guest
Science 214:1225-1228.
19. Merrikin, D., and G. N. Rollinson. 1979. Antibiotic levels in
experimentally infected mice in relation to therapeutic effect
and antibacterial activity in vitro. J. Antimicrob. Chemother.
5:423-429.
20. Miller, A. K., D. L. Wilmer, and W. F. Verwey. 1950. Effect of
penicillin dosage schedule on treatment of experimental thyroid
infections in mice. Proc. Soc. Exp. Biol. Med. 74:62-64.
21. Olson, B., R. A. Weinstein, C. Nathan, W. Chamberlin, and
S. A. Kabins. 1985. Occult aminoglycoside resistance in Pseu-
domonas aeruginosa: epidemiology and implication for therapy
and control. J. Infect. Dis. 152:769-774.
22. Pearson, R. D., R. T. Steigbigel, H. T. Davis, and S. W.
Chapman. 1980. Method for reliable determination of minimal
lethal antibiotic concentrations. Antimicrob. Agents Chemo-
ther. 18:699-708.
23. Sherris, J. C., and B. H. Minshew. 1980. Mutational antibiotic
resistance, p. 418-432. In V. Lorian (ed.), Antibiotics in labo-
ratory medicine. The Williams & Wilkins Co., Baltimore, Md.
24. Simon, H. J., and E. J. Yin. 1970. Microbioassay of antimicro-
bial agents. Appl. Microbiol. 19:573-579.
25. Thrupp, L. D. 1980. Susceptibility testing of antibiotics in liquid
media, p. 73-113. In V. Lorian (ed.), Antibiotics in laboratory
medicine. The Williams & Wilkins Co., Baltimore, Md.
26. Waterworth, P. M. 1978. Quantitative methods for bacterial
sensitivity testing, p. 34-38. In D. S. Reeves, I. Phillips, J. D.
Williams, and R. Wise (ed.), Laboratory methods in antimicro-
bial chemotherapy. Churchill Livingstone, Inc., New York.
27. White, C. A., R. D. Toothaker, A. L. Smith, and J. T. Slattery.
1987. Correction for bacterial loss in in vitro dilution models.
Antimicrob. Agents Chemother. 31:1859-1860.