Journal of Antimicrobial Chemotherapy (2006) 57, 714–719

doi:10.1093/jac/dkl041
Advance Access publication 21 February 2006

Urine bactericidal activity against resistant Escherichia coli in an

in vitro pharmacodynamic model simulating urine concentrations
obtained after 2000/125 mg sustained-release co-amoxiclav and
400 mg norfloxacin administration

Luis Alou, Lorenzo Aguilar, David Sevillano, Marı́a-José Giménez, Fabio Cafini,
Eva Valero, Marı́a-Teresa Relaño and José Prieto*

Microbiology Department, School of Medicine, Universidad Complutense, Avda. Complutense s/n,

28040 Madrid, Spain

Downloaded from jac.oxfordjournals.org by guest on January 28, 2011

Received 29 September 2005; returned 6 December 2005; revised 25 January 2006; accepted 30 January 2006

Objectives: To explore the urine bactericidal activity of co-amoxiclav and norfloxacin against Escherichia
coli in an in vitro pharmacodynamic model simulating the human urinary concentrations observed after
administration of a single oral dose of 2000/125 mg sustained-release co-amoxiclav and 400 mg norfloxacin.
Methods: Six E. coli isolates exhibiting amoxicillin/clavulanic acid MICs of 4/2 (two strains), 8/4, 16/8, 32/16
and 64/32 mg/L and norfloxacin MICs of £0.25 mg/L (three strains), 32, 64 and 256 mg/L were used. Colony
counts were determined over 12 h and differences between the bacterial counts of initial inocula and the
bacterial counts at each sampling time-point were calculated.
Results: With co-amoxiclav, bactericidal activity (>3 log10 reduction) was obtained against the susceptible
(MIC £ 8/4 mg/L) and intermediate (MIC = 16/8 mg/L) strains from 3 to 12 h, and from 3 to 10 h against
the resistant strains (MIC ‡ 32/16 mg/L), which exhibited a 2 log10 reduction at 12 h. With norfloxacin,
bactericidal activity was obtained against the susceptible strains from 4 to 12 h and from 8 to 12 h against
the resistant strain with an MIC of 32 mg/L. Regrowth, with respect to initial inocula, occurred from 8 h
onwards with the strain with MIC = 64 mg/L and from 3 h onwards with the strain with MIC = 256 mg/L.
Conclusions: While regrowth occurs after exposure of high norfloxacin-resistant E. coli to urine physio-
logical concentrations of norfloxacin, this study suggests that clavulanic acid can be given twice daily
(to protect amoxicillin activity) with respect to uncomplicated cystitis due to E. coli exhibiting amoxicillin/
clavulanic acid MICs up to 64/32 mg/L.

Keywords: pharmacodynamic modelling, E. coli, urine bactericidal activity, amoxicillin, clavulanic acid

Introduction A new oral pharmacokinetically enhanced formulation of

co-amoxiclav has been developed that prolongs the duration of
The incidence of amoxicillin resistance in Escherichia coli clini- adequate amoxicillin concentrations after dosing.6 In previous
cal isolates exceeds 50% in many parts of the world,1 and in studies the pharmacodynamic potential of this new formulation
Spain only 42% of E. coli community urinary strains are sus- against amoxicillin non-susceptible Streptococcus pneumoniae
ceptible to ampicillin.2,3 Most of these isolates are resistant due to was shown,6 and ex vivo serum bactericidal activity was deter-
production of b-lactamase, and addition of clavulanic acid mined in healthy volunteers.7 The serum bactericidal activity was
increases the ampicillin/amoxicillin susceptibility rate to 90– determined using kill curves, where fresh inoculum was added to
98%.3,4 Quinolone resistance in E. coli has spread in Spain, the serum sample (containing antibiotic) at each time-point.7
with resistance rates in the community reaching 22.8% in urinary However, in infections, the infecting organism is exposed to
isolates3 and 24% and 26% in faecal isolates from healthy adults changing antibiotic concentrations, and for this reason in vitro
and children, respectively.5 pharmacodynamic simulations mimicking this in vivo situation
.............................................................................................................................................................................................................................................................................................................................................................................................................................

*Corresponding author. Tel: +34-91-3941508; Fax: +34-91-3941511; E-mail: jprieto@med.ucm.es

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714

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 Co-amoxiclav urine bactericidal activity against E. coli
were performed against amoxicillin non-susceptible pneumococ-
cal strains.8
Although it is relatively simple to undertake a pharmacody-
namic analysis for systemic infections, the situation is more
complex when considering urinary tract infections, and particu-
larly when b-lactamase-producing pathogens are being tested
against co-amoxiclav. In a previous Phase I study,9 significant
antibacterial activity was obtained up to 12 h for the susceptible
(8/4 mg/L) and intermediate (16/8 mg/L) strains and up to 8 h
with the resistant (32/16 and 64/32 mg/L) strains with the new
co-amoxiclav formulation. However, bactericidal activity
(>3 log10 reduction; >99.9% killing) was obtained only up to
8 h for the susceptible and intermediate strains and at the 2–
4 h interval for the resistant strains.9 Results of the study may
have been influenced by the fact that a fresh inoculum was
exposed to each urine sample. This poorly reflects the in vivo
situation, where bacteria in the urine are contained in the bladder
and exposed to changing antibiotic concentrations over time. In
this situation, the reduction in bacterial density would result in a
reduction in the b-lactamase level, and in turn prolongation of the
in vivo bactericidal activity beyond that obtained ex vivo. This has
been suggested in experiments using animal models to measure
serum bactericidal activity against b-lactamase-producing E. coli
after co-amoxiclav treatment.10
This study evaluates the urine bactericidal activity of co-
amoxiclav against E. coli in an in vitro model simulating
urine concentrations obtained in humans up to 12 h after admin-
istration of a single dose in comparison with norfloxacin.
Materials and methods
Bacterial strains
Six E. coli clinical isolates from community-acquired cystitis
were studied in this in vitro pharmacodynamic model. Four of
them (FJ8, FJ16, FJ32 and FJ64) were those previously used in
ex vivo determinations in a Phase I study.9
Antibiotics
Amoxicillin trihydrate and lithium clavulanate laboratory reference
standards were supplied by GlaxoSmithKline (Worthing, UK). The
norfloxacin laboratory reference standard was purchased from
Sigma Chemical Co. (St Louis, MO, USA).
MIC determination and b-lactamase gene sequencing
MICs were determined by a microdilution method following CLSI
methodology11 prior to and after the simulation process. All deter-
minations were performed five times and modal values are presented.
Amplification of TEM, SHV and CTX-M b-lactamase genes
was performed by PCR as described previously.12 The PCR products
were purified using a QIAquick PCR purification kit (Qiagen,
Valencia, CA, USA) and then sequenced using a 3730 DNA
Analyser (Perkin Elmer Applied Biosystems, Foster City, CA,
USA). Sequences obtained were compared with the NCBI BLAST
database.
In vitro kinetic model
The changing antibiotic urine concentrations were simulated in a
model based on a two-compartment model described previously.8
The extra-capillary space and the intra-dialyser circulating
715
tubing of the peripheral unit (FX50, Fresenius Medical
Care S.A., Barcelona, Spain) represented the infection site. The
exponential decay of concentrations was obtained by a continuous
dilution–elimination process using computerized peristaltic pumps
(Masterflex, Cole-Parmer Instrument Co., Chicago, IL, USA) at
rates of 3.5 mL/min (period 0–3 h) and 1 mL/min (period 3–12 h)
to simulate the clearance of norfloxacin and at a rate of 4.7 mL/min
for the co-amoxiclav combination. Additional pumps circulated the
antibiotic-medium mixture at a 50 mL/min rate between the central
and peripheral compartment and at a rate of 20 mL/min within the
extra-capillary space through external tubing. A computer-controlled
syringe pump (402 Dilutor Dispenser; Gilson S.A, Villiers-
le-Bel, France) allowed the simulation of concentrations in urine,
by infusion of the antibiotic into the central compartment until the
Cmax was reached.
Kinetic simulations
Pharmacokinetic urine profiles in healthy volunteers of co-amoxiclav
corresponding to the new co-amoxiclav 2000/125 mg SR formula-
tion6 and of 400 mg norfloxacin13 were simulated over 12 h. Linear
Downloaded from jac.oxfordjournals.org by guest on January 28, 2011
decay of both antimicrobials in urine was estimated using the mean
concentration of each documented interval in Phase I studies.9,13 This
approximation resulted in a biphasic clearance of norfloxacin from
urine and was achieved by synchronization of the peristaltic pumps
using the Win Lin software (Cole-Parmer Instrument Co., Chicago,
IL, USA). The co-amoxiclav profile was simulated by the clearance
of clavulanic acid from urine and supplementation of the amoxicillin
concentration from time 3 to 10 h to mimic the amoxicillin con-
centrations obtained in urine with the new formulation. The target
concentrations in urine were: 167, 85 and 30 mg/L at 1, 3 and 12 h,
respectively, for norfloxacin; 814.7, 141 and 21 mg/L at 3, 10 and
14 h, respectively, for amoxicillin; and 41 and 1.5 mg/L at 3 and
10 h, respectively, for clavulanic acid. Figure 1 shows target
and experimental concentrations of norfloxacin, amoxicillin and
clavulanic acid.
Killing curves and the pharmacokinetic analysis
Bacterial suspensions in Mueller–Hinton broth supplemented with
Ca2+ (25 mg/L) and Mg2+ (50 mg/L) were allowed to grow to a
density of 5 · 107 cfu/mL, as measured by a UV-spectophotometer
(Hitachi U-1100). An aliquot of 60 mL of this inoculum was intro-
duced into the peripheral compartment. Samples (0.5 mL) from the
peripheral compartment were collected at 0, 1, 3, 4, 8, 10 and 12 h,
and, if necessary, serially diluted in 0.9% sodium chloride. At least
four dilutions of each sample were spread onto Mueller–Hinton
agar plates supplemented with 5% sheep blood and incubated at
37 C, and colonies were counted after 24 h. The limit of detection
was 5 · 10 cfu/mL, and each experiment was performed in triplicate.
In addition, aliquots were taken from the central compartment at
0, 1, 3, 4, 6, 8, 10 and 12 h, and stored at –50 C for the measurement
of simulated urine concentrations. Samples were assayed by bioassay
using Micrococcus luteus ATCC 9341 and Klebsiella pneumoniae
NCTC 11228 as indicator organisms for amoxicillin and clavulanic
acid, respectively. E. coli NCTC 10418 was used as the indicator
organism for norfloxacin.14 Standards and samples were prepared
and diluted in Mueller–Hinton broth as required. The limit of detec-
tion was 0.015, 0.125 and 0.25 mg/L for norfloxacin, amoxicillin and
clavulanic acid, respectively. The intra-day and inter-day coefficients
of variation between assays were 3.8% and 2.25% for norfloxacin,
2.1% and 4.3% for amoxicillin, and 7.3% and 4.3% for clavulanic
acid, with internal control concentrations of 0.75 mg/L for nor-
floxacin and clavulanic acid and of 0.3 mg/L for amoxicillin. The
 Alou et al.

(a) Table 1. b-Lactamases and MICs (mg/L) for the six E. coli strains
1050 used in this study
Concentration (mg/L)

800
Amoxicillin/ Norfloxacin
550 Strain b-Lactamase clavulanic acid MIC MIC
300
MR110 TEM-1 4/2 256
50
MR61 TEM-116 4/2 64
FJ8 TEM-1 8/4 32
40 FJ16 TEM-1 16/8 £0.25
20 FJ32 TEM-1 32/16 £0.25
FJ64 TEM-1 64/32 £0.25
0
0 2 4 6 8 10 12
(b) 100 Time (h)
Concentration (mg/L)

80 strains were susceptible to amoxicillin/clavulanic acid (MICs of

4/2, 4/2 and 8/4 mg/L), one was intermediate (16/8 mg/L) and
60 two were resistant (MICs of 32/16 and 64/32 mg/L), according to
CLSI breakpoints.15 The three amoxicillin/clavulanic acid

Downloaded from jac.oxfordjournals.org by guest on January 28, 2011

40 susceptible strains were resistant to norfloxacin (MICs of 256,
64 and 32 mg/L), while the three amoxicillin/clavulanic acid
20
non-susceptible strains (MICs ‡ 16/8 mg/L) were susceptible
to norfloxacin (MICs £ 0.25 mg/L) according to CLSI
0
0 2 4 6 8 10 12 breakpoints.15
Time (h)
Experimental Cmax (mg/L), Tmax (h) and AUC0–24 (mg·h/L)
(c) 300 values were 821.8 – 19.2, 3 – 0 and 4647.7 – 27.7 for amoxicillin,
42.2 – 3.1, 3 – 0 and 155.6 – 11.1 for clavulanic acid, and
152.5 – 13.5, 1 – 0 and 1038.0 – 85.5 for norfloxacin,respectively.
Concentration (mg/L)

200 In antibiotic-free simulations, the bacterial load increased

100
0
0 2 4 6 8 10 12
Time (h)
Figure 1. Experimental (black continuous line) and target (black broken line)
concentrations of amoxicillin (a), clavulanic acid (b) and norfloxacin (c).
Staircase maximum and minimum concentrations are shown as grey dotted
lines.
pharmacokinetic analysis was based on a non-compartmental
approach (WinNonlin, Pharsight, Mountain View, CA, USA).
Statistical analysis
Differences in log10 colony counts at each sampling time with
respect to initial inocula were calculated. Inter-strain differences
at each time-point were determined using Tukey’s multiple compari-
son test. P < 0.001 was considered statistically significant. The
P value was adjusted by the Bonferroni correction method, taking
into consideration the multiple comparisons performed in the
analysis.
Results
Table 1 shows the b-lactamase profile and MICs of co-amoxiclav
and norfloxacin for the E. coli strains used in this study. Three
from initial inocula (log10 cfu/mL) to bacterial counts at 12 h
(log10 cfu/mL) as follows: from 7.66 – 0.04 to 10.81 – 0.01 for
the strain FJ8; from 7.69 – 0.09 to 10.83 – 0.05 for the strain
FJ16; from 7.75 – 0.17 to 10.92 – 0.06 for the strain FJ32; from
7.66 – 0.08 to 10.98 – 0.12 for the strain FJ64; from 7.62 – 0.21
to 11.07 – 0.03 for the strain MR61; and from 7.64 – 0.17 to
12.04 – 0.07 for the strain MR110.
Table 2 shows the initial inocula decrease over 12 h with the
simulated urinary co-amoxiclav concentrations. These concentra-
tions gave mean values for t > MIC of 100% for all strains except
for the most resistant strain (MIC of 64/32 mg/L), where t > MIC
was 92%. At least a 2 log10 reduction with respect to initial
inocula (log10 cfu/mL initial – log10 cfu/mL at the corresponding
sampling time) was obtained for all strains from the 3 h time-
point onwards, with no significant differences between the
strains. However, a ‡1 log10 increase in bacterial counts with
respect to the previous sampling time-point occurred at 10 h
(versus 8 h) and at 12 h (versus 10 h) with the resistant strains
when the amoxicillin concentrations were lower than 3 · MIC
(amoxicillin/clavulanic acid) and clavulanic acid concentrations
were £1.5 mg/L. Antibacterial activity could be considered bac-
tericidal (>3 log10 reduction with respect to initial inocula) from 3
to 12 h against the susceptible and intermediate strains and from
3 to 10 h against the two resistant strains.
Table 3 shows the initial inocula decrease over 12 h with the
simulated urinary norfloxacin concentrations. These produced
mean values for t > MIC of 100% for all susceptible strains
(MICs £ 0.25 mg/L) and of 95.8%, 42.7% and 0% for the resis-
tant strains (MICs of 32, 64 and 256 mg/L). Significant inter-
strain differences were observed, with bacterial regrowth, with
respect to the initial inocula, occuring when norfloxacin

716
 Co-amoxiclav urine bactericidal activity against E. coli

Table 2. Simulated co-amoxiclav urine concentrations (mean – SD) after a single 2000/125 mg dose, E. coli initial inocula reduction over time
(log10 cfu/mL initial inocula – log10 cfu/mL at the corresponding sampling time) and mean urine t > MIC

Mean t >
Strain MIC (mg/L)a 0h 1h 3h 4h 8h 10 h 12 h MICb (%)

MR110 4/2 0.0 – 0.0 0.5 – 0.2 2.6 – 0.2 3.1 – 0.3 4.7 – 0.2 4.4 – 0.5 5.9 – 0.2 100
MR61 4/2 0.0 – 0.0 2.8 – 0.7 3.6 – 0.2 3.8 – 0.4 5.7 – 0.9 5.2 – 0.4 5.5 – 0.4 100
FJ8 8/4 0.0 – 0.0 2.4 – 1.3 4.3 – 0.8 5.3 – 0.7 5.9 – 0.6 6.0 – 1.1 5.5 – 1.9 100
FJ16 16/8 0.0 – 0.0 2.3 – 0.4 4.3 – 0.4 4.7 – 0.3 5.5 – 0.7 5.1 – 1.4 4.6 – 1.8 100
FJ32 32/16 0.0 – 0.0 1.0 – 0.9 3.4 – 0.4 4.1 – 0.3 4.9 – 0.3 4.1 – 0.1 2.0 – 0.3 100
FJ64 64/32 0.0 – 0.0 0.9 – 0.4 3.9 – 0.8 3.4 – 1.3 5.0 – 0.2 4.1 – 0.5 2.0 – 0.1 92
Amoxicillin concentration (mg/L) – 462.4 – 7.0 821.8 – 19.2 583.1 – 17.1 252.3 – 2.6 124.5 – 3.5 40.8 – 6.2
Clavulanate concentration (mg/L) – ND 42.2 – 3.1 26.4 – 1.9 4.0 – 0.3 1.5 – 0.1 0.6 – 0.1

ND, not determined.

a
Amoxicillin/clavulanic acid MIC.
b
t > MIC = % dosing interval (12 h) where amoxicillin concentrations in urine exceed amoxicillin/clavulanic acid MIC.

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Table 3. Simulated norfloxacin urine concentrations (mean – SD) after a single 400 mg dose, E. coli initial inocula reduction over time
(log10 cfu/mL initial inocula – log10 cfu/mL at the corresponding sampling time) and mean urine AUC0–12/MIC

Mean
Strain MIC (mg/L) 0h 1h 3h 4h 8h 10 h 12 h AUC0–12/MIC

FJ64 £0.25 0.0 – 0.0 1.7 – 0.3 3.3 – 0.3 3.9 – 0.4 6.1 – 0.5 6.3 – 0.4 6.0 – 0.3 ‡3092.43
FJ16 £0.25 0.0 – 0.0 1.3 – 0.6 2.9 – 0.6 3.5 – 0.5 5.8 – 0.6 5.7 – 0.3 6.0 – 0.4 ‡3092.43
FJ32 £0.25 0.0 – 0.0 0.8 – 0.7 1.8 – 1.3 2.1 – 1.1 3.1 – 1.3 3.5 – 1.5 3.8 – 1.3 ‡3092.43
FJ8 32 0.0 – 0.0 0.1 – 0.1 1.1 – 0.8 2.1 – 0.7 3.9 – 0.4 4.0 – 0.3 3.5 – 0.4 24.16
MR61 64 0.0 – 0.0 0.1 – 0.3 0.3 – 0.2 0.5 – 0.3a –0.1 – 0.1c –0.2 – 0.2c –0.8 – 0.3c 12.08
MR110 256 0.0 – 0.0 0.5 – 0.8 –1.6 – 0.3a –2.6 – 0.3b –2.8 – 0.4c –2.9 – 0.3c –3.0 – 0.1c 3.02
AUC0–12 (mg·h/L)
Concentration (mg/L) – 152.5 – 13.5 79.3 – 11.1 71.6 – 13.5 47.3 – 7.2 39.7 – 6.7 29.3 – 2.7 773.10 – 68.60

Negative values (given in bold) correspond to regrowth with respect to initial inocula.
a
P < 0.001 versus FJ64 and FJ16.
b
P < 0.001 versus all other strains.
c
P < 0.001 versus FJ64, FJ32, FJ16 and FJ8.

concentrations fell below the MIC for the isolate. This occurred
at 3 h in the case of the strain with an MIC of 256 mg/L and at 8
h in the case of the strain with an MIC of 64 mg/L. The AUC0–12/
MIC ratio was <24 in those strains that showed regrowth (nor-
floxacin MICs of 64 and 256 mg/L). Bactericidal activity (>3
log10 reduction) was generally observed from 4 to 12 h for the
susceptible strains (norfloxacin MICs £ 0.25 mg/L) and from 8 to
12 h for the strain with a norfloxacin MIC of 32 mg/L, but was
never observed for the strains with norfloxacin MICs of 64 and
256 mg/L.
Bactericidal activity (against all strains with co-amoxiclav and
against four strains with norfloxacin) was observed at earlier
time-points with co-amoxiclav than with norfloxacin (3 h versus
4–8 h).
Isolates recovered at the end of the experiments exhibited
amoxicillin/clavulanic acid MIC values equal to those prior to
co-amoxiclav exposure. However, after norfloxacin exposure, the
strain FJ8 exhibited an MIC of 256 mg/L (three dilutions higher
than the one pre-exposure: 32 mg/L) while the remaining strains
exhibited the same MIC values.
Discussion
Owing to the high prevalence of resistance to amoxicillin or
ampicillin in E. coli,16,17 their use in cystitis is not advocated
without a b-lactamase inhibitor.18 Susceptibility rates of E. coli
to amoxicillin/clavulanic acid are 90% in Spain3 and have
remained at this level over time because hyperproduction of
TEM-1 b-lactamase (or its derivative IRT) has a low frequency
in Spain.3 Extended-spectrum b-lactamases are not often found in
E. coli from outpatient urine culture in Spain (1.4%), and those
that are found are not usually TEM derivates.12 It has been
suggested that fluoroquinolones are a logical choice for empirical
therapy of uncomplicated cystitis,19 and in early studies nor-
floxacin was associated with good efficacy in the treatment of
uncomplicated cystitis.19,20 However, this was at a time when
resistance rates were not higher than 15% in Spain, and since then
an 1% per annum increase in the resistance rate for E. coli to
fluoroquinolones has occurred.17 As a consequence, resistance
rates in E. coli to norfloxacin now exceed the 20% level in
many parts of Spain.

717
 Alou et al.
In the assessment of an antibiotic for uncomplicated cystitis,
bactericidal activity in urine is a relevant pharmacodynamic
parameter because the gradual decrease in antibiotic concentra-
tion (which is not possible to mimic in standard in vitro testing)
could lead to concentrations below the MIC, permitting bacterial
regrowth that may result in therapeutic failure.21 One of the
earlier attempts to use in vitro dynamic models to simulate
bladder infections measured the effects of antibiotics by photo-
metrically studying bacterial growth in artificial media and simu-
lating micturation by alterations of the volume and flow rate of
the medium.22 Despite criticisms that such spectrophotometric
measurements included both non-viable and viable bacteria
and that they were not reliable for dense bacterial populations
(106–109 cfu/mL), this bladder model correlated well with mouse
infection protection models and provided realistic simulations of
the response seen in patients in clinical trials.23–25
In the present pharmacodynamic simulation, the system
used was closed (bacteria were not eliminated with alterations
in the flow rate), and the changes in bacterial counts resulted
solely from the action of the antimicrobial agent. Although
generally bacteria grow more quickly in Mueller–Hinton broth
than in urine, fluoroquinolone activity can be decreased in urine
due to magnesium concentrations present. In this study the
Mueller–Hinton broth used was supplemented with Ca2+ and
Mg2+ to better simulate the urinary situation.
In this study bactericidal activity with norfloxacin was
obtained from 4 to 12 h against strains with MIC values of
£32 mg/L, but regrowth (with respect to the initial inoculum)
occurred when concentrations were below the MIC for resistant
strains with MICs ‡ 64 mg/L. Moreover, with the strain with an
MIC of 32 mg/L the survival bacterial population at the end
of the simulation exhibited an increased MIC (256 mg/L).
This suggests that despite the original bactericidal activity
obtained against this strain with norfloxacin, categorization of
this strain as resistant is correct taking into account the highly
resistant subpopulation selected, as may occur in vivo.26
The results obtained in this study with co-amoxiclav contrast
with those obtained previously in a Phase I study in healthy
volunteers, where the bactericidal activity against four of
the six strains used in this study (those strains with MICs of
8/4, 16/8, 32/16 and 64/32 mg/L) was assessed.9 In the earlier
study, bactericidal activity (>3 log10 reduction) was obtained for
up to 8 h against the susceptible and intermediate strains, and
only at the 2–4 h post-dosing interval against the two resistant
strains. In the present in vitro simulation, bactericidal activity was
obtained up to 12 h (the whole dosing interval) against the
susceptible and intermediate strains, and up to 10 h against
the resistant strains. We believe that the difference between
the two studies is due to the fact that in the Phase I study a
fresh inoculum was exposed to the urine sample (containing the
antibiotic) at each time-point, while in this simulation the same
inoculum is exposed to changing concentrations over time, which
better mimics the in vivo situation.
In previous in vitro27 and ex vivo studies in animal models10
and in humans,9 where fresh E. coli inocula were exposed to
fixed co-amoxiclav concentrations at different time-points (static
experiments), a clavulanic acid threshold for the activity of
amoxicillin was observed. In contrast, in this study, where expo-
sure of the same inocula to changing co-amoxiclav concentrations
was used, this threshold was not observed. From this perspective
the results of this study suggest that clavulanic acid can be given
718
twice daily (to protect amoxicillin activity) in the treatment
of uncomplicated cystitis due to E. coli exhibiting amoxicillin/
clavulanic acid MICs up to 64/32 mg/L. This is a higher value
than those found for strains with b-lactamase hyperproduc-
tion.28,29 In contrast, quinolone treatment should be used with
caution in countries such as Spain, where resistance rates are
higher than 15%.
Acknowledgements
We thank F. Soriano and M. C. Ponte (Fundación Jiménez Dı́az)
for the supply of the FJ E. coli strains and O. Echeverrı́a for her
technical assistance in this study. This study was supported by an
unrestricted grant from GlaxoSmithKline S.A., Madrid, Spain.
Transparency declarations
None to declare.
Downloaded from jac.oxfordjournals.org by guest on January 28, 2011
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