In vitro activity and in vivo efficacy of clavulanic acid against Acinetobacter baumannii.
ABSTRACT Clavulanic acid (CLA) exhibits low MICs against some Acinetobacter baumannii strains. The present study evaluates the efficacy of CLA in a murine model of A. baumannii pneumonia. For this purpose, two clinical strains, Ab11 and Ab51, were used; CLA MICs for these strains were 2 and 4 mg/liter, respectively, and the imipenem (IPM) MIC was 0.5 mg/liter for both. A pneumonia model in C57BL/6 mice was used. The CLA dosage (13 mg/kg of body weight given intraperitoneally) was chosen to reach a maximum concentration of the drug in serum similar to that in humans and a time during which the serum CLA concentration remained above the MIC equivalent to 40% of the interval between doses. Six groups (n = 15) were inoculated with Ab11 or Ab51 and were allocated to IPM or CLA therapy or to the untreated control group. In time-kill experiments, CLA was bactericidal only against Ab11 whereas IPM was bactericidal against both strains. CLA and IPM both decreased bacterial concentrations in lungs, 1.78 and 2.47 log10 CFU/g (P < or = 0.001), respectively, in the experiments with Ab11 and 2.42 and 2.28 log10 CFU/g (P < or = 0.001), respectively, with Ab51. IPM significantly increased the sterility of blood cultures over that for the controls with both strains (P < or = 0.005); CLA had the same effect with Ab51 (P < 0.005) but not with Ab11 (P = 0.07). For the first time, we suggest that CLA may be used for the treatment of experimental severe A. baumannii infections.
- SourceAvailable from: ncbi.nlm.nih.gov[Show abstract] [Hide abstract]
ABSTRACT: Acinetobacter baumannii is a nosocomial pathogen with a high prevalence of multiple-drug-resistant strains, causing pneumonia and sepsis. The current studies further develop a systemic mouse model of this infection and characterize selected innate immune responses to the organism. Five clinical isolates, with various degrees of antibiotic resistance, were assessed for virulence in two mouse strains, and between male and female mice, using intraperitoneal infection. A nearly 1,000-fold difference in virulence was found between bacterial strains, but no significant differences between sexes or mouse strains were observed. It was found that microbes disseminated rapidly from the peritoneal cavity to the lung and spleen, where they replicated. A persistent septic state was observed. The infection progressed rapidly, with mortality between 36 and 48 h. Depletion of neutrophils with antibody to Ly-6G decreased mean time to death and increased mortality. Interleukin-17 (IL-17) promotes the response of neutrophils by inducing production of the chemokine keratinocyte-derived chemoattractant (KC/CXCL1), the mouse homolog of human IL-8. Acinetobacter infection resulted in biphasic increases in both IL-17 and KC/CXCL1. Depletion of neither IL-17 nor KC/CXCL1, using specific antibodies, resulted in a difference in bacterial burdens in organs of infected mice at 10 h postinfection. Comparison of bacterial burdens between IL-17a(-/-) and wild-type mice confirmed that the absence of this cytokine did not sensitize mice to Acinetobacter infection. These studies definitely demonstrate the importance of neutrophils in resistance to systemic Acinetobacter infection. However, neither IL-17 nor KC/CXCL1 alone is required for effective host defense to systemic infection with this organism.Infection and immunity 05/2011; 79(8):3317-27. · 4.16 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Acinetobacter baumannii is an increasingly problematic pathogen in United States hospitals. Antibiotics that can treat A. baumannii are becoming more limited. Little is known about the contributions of penicillin binding proteins (PBPs), the target of β-lactam antibiotics, to β-lactam-sulbactam susceptibility and β-lactam resistance in A. baumannii. Decreased expression of PBPs as well as loss of binding of β-lactams to PBPs was previously shown to promote β-lactam resistance in A. baumannii. Using an in vitro assay with a reporter β-lactam, Bocillin, we determined that the 50% inhibitory concentrations (IC(50)s) for PBP1a from A. baumannii and PBP3 from Acinetobacter sp. ranged from 1 to 5 μM for a series of β-lactams. In contrast, PBP3 demonstrated a narrower range of IC(50)s against β-lactamase inhibitors than PBP1a (ranges, 4 to 5 versus 8 to 144 μM, respectively). A molecular model with ampicillin and sulbactam positioned in the active site of PBP3 reveals that both compounds interact similarly with residues Thr526, Thr528, and Ser390. Accepting that many interactions with cell wall targets are possible with the ampicillin-sulbactam combination, the low IC(50)s of ampicillin and sulbactam for PBP3 may contribute to understanding why this combination is effective against A. baumannii. Unraveling the contribution of PBPs to β-lactam susceptibility and resistance brings us one step closer to identifying which PBPs are the best targets for novel β-lactams.Antimicrobial Agents and Chemotherapy 08/2012; 56(11):5687-92. · 4.57 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Two mechanisms of resistance to colistin have been described in Acinetobacter baumannii. One involves complete loss of LPS, resulting from mutations in lpxA, lpxC or lpxD, and the second is associated with phosphoethanolamine addition to LPS, mediated through mutations in pmrAB. In order to assess the clinical impact of both resistance mechanisms, A. baumannii ATCC19606 and its isogenic derivatives, AL1851 ΔlpxA, AL1852 ΔlpxD, AL1842 ΔlpxC and ATCC 19606 pmrB, were analyzed for in vitro growth rate, in vitro and in vivo competitive growth, infection of A549 respiratory alveolar epithelial cells, virulence in the Caenorhabditis elegans model and virulence in a systemic mouse infection model. The in vitro growth rate of the lpx mutants was clearly diminished; furthermore, in vitro and in vivo competitive growth experiments revealed a reduction in fitness for both mutant types. Infection of A549 cells with ATCC19606 or the pmrB mutant resulted in higher loss of viability than with lpx mutants. Finally, the lpx mutants were highly attenuated in both the C. elegans and mouse infection models while the pmrB mutant was only attenuated in the C. elegans model. In summary, while colistin resistance in A. baumannii confers a clear selective advantage in presence of colistin treatment, it causes a noticeable cost in terms of overall fitness and virulence, with a more striking reduction associated with LPS loss than with phosphoethanolamine addition. Therefore, we hypothesize that colistin resistance mediated by changes in pmrAB will be more likely to arise in clinical settings in patients treated with colistin.Antimicrobial Agents and Chemotherapy 11/2013; · 4.57 Impact Factor
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2009, p. 4298–4304
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 10
In Vitro Activity and In Vivo Efficacy of Clavulanic Acid against
Alejandro Beceiro,1† Rafael Lo ´pez-Rojas,2*† Juan Domínguez-Herrera,2Fernando Docobo-Pe ´rez,2
Germa ´n Bou,1Jero ´nimo Pacho ´n,2and the Spanish Network for Research in
Infectious Diseases (REIPI)
Servicio de Microbiología-Unidad de Investigacio ´n, Complejo Hospitalario Universitario Juan Canalejo, 15006 La Corun ˜a,1and
Servicio de Enfermedades Infecciosas, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del
Rocío/CSIC/Universidad de Sevilla, 41013 Seville,2Spain
Received 9 March 2009/Returned for modification 11 May 2009/Accepted 15 July 2009
Clavulanic acid (CLA) exhibits low MICs against some Acinetobacter baumannii strains. The present study
evaluates the efficacy of CLA in a murine model of A. baumannii pneumonia. For this purpose, two clinical
strains, Ab11 and Ab51, were used; CLA MICs for these strains were 2 and 4 mg/liter, respectively, and the
imipenem (IPM) MIC was 0.5 mg/liter for both. A pneumonia model in C57BL/6 mice was used. The CLA
dosage (13 mg/kg of body weight given intraperitoneally) was chosen to reach a maximum concentration of the
drug in serum similar to that in humans and a time during which the serum CLA concentration remained
above the MIC equivalent to 40% of the interval between doses. Six groups (n ? 15) were inoculated with Ab11
or Ab51 and were allocated to IPM or CLA therapy or to the untreated control group. In time-kill experiments,
CLA was bactericidal only against Ab11 whereas IPM was bactericidal against both strains. CLA and IPM both
decreased bacterial concentrations in lungs, 1.78 and 2.47 log10CFU/g (P < 0.001), respectively, in the
experiments with Ab11 and 2.42 and 2.28 log10CFU/g (P < 0.001), respectively, with Ab51. IPM significantly
increased the sterility of blood cultures over that for the controls with both strains (P < 0.005); CLA had the
same effect with Ab51 (P < 0.005) but not with Ab11 (P ? 0.07). For the first time, we suggest that CLA may
be used for the treatment of experimental severe A. baumannii infections.
Acinetobacter baumannii is a gram-negative, nonfermenting,
nonmobile, strictly aerobic, oxidase-negative bacterium that is
able to grow in general culture media without specific require-
ments (4) and is often associated with nosocomial infections
and outbreaks (16, 21, 22). This pathogen can produce differ-
ent types of infections, such as septicemia, endocarditis, men-
ingitis, wound and skin and soft-tissue infections, urinary tract
infections (4), and nosocomial pneumonia, especially in pa-
tients with mechanical ventilation (8, 26). The attributable
mortality of A. baumannii infections ranges from 7.8% to 23%
The standard treatment for infections caused by A. bauman-
nii has been imipenem (IPM). This pathogen exhibits a great
adaptive capacity and a great ability to acquire numerous ef-
fective antibiotic resistance mechanisms, and it may be consid-
ered the paradigm of multiresistant nosocomial bacteria. The
frequent isolation of strains with resistance to the most com-
monly used antimicrobials, including IPM, has prompted the
evaluation of diverse therapeutic alternatives, such as sulbac-
tam and colistin, which have shown efficacy similar to that of
IPM, rifampin (rifampicin), and tigecycline and are under eval-
uation for the treatment of A. baumannii infections, and anti-
microbial peptides, which are still in experimental preclinical
studies. Thus, the absence of an optimal treatment necessitates
the development of new therapeutic alternatives (44).
The commercial ?-lactamase inhibitors (clavulanic acid
[CLA], tazobactam, and sulbactam) generally have low antibi-
otic activity against most microorganisms and therefore are not
used alone as antimicrobial agents. Sulbactam shows good
bactericidal activity in vitro against A. baumannii, and it has
been observed that the efficacy of sulbactam against suscepti-
ble strains is similar to that of IPM in an experimental pneu-
monia model (33). CLA was the first ?-lactamase inhibitor to
be used commercially (35). It is a suicide inhibitor, leading to
an irreversible union with ?-lactamase. This inhibitor has high
affinity for class A ?-lactamases, including TEM, SHV, and
Although the activity of CLA against A. baumannii is lower
than that of sulbactam, two studies with 100 and 115 geneti-
cally different clinical strains have been published recently,
showing a range of CLA MICs from 2 to 256 mg/liter, with
MICs of ?8 mg/liter for 29% and 40.9% of strains, respectively
(3, 27); the MICs at which 50% of isolates were inhibited
(MIC50s) and MIC90s ranged from 16 to 32 and 64 to 512
mg/liter, respectively (3, 27). In another study, the in vitro
activities of various ?-lactams, along with ?-lactamase inhibi-
tors, against 68 strains of Acinetobacter spp. were analyzed,
showing that CLA was more active than other antimicrobials,
such as ceftriaxone, cefotaxime, and piperacillin (41). In an-
other work, nonclassical combinations of ?-lactams and ?-lac-
tamase inhibitors were studied, and CLA increased the efficacy
of the combinations tested, both in vitro and in vivo (45).
The aim of this study was to compare the efficacy of the
* Corresponding author. Mailing address: Servicio de Enfermedades
Infecciosas, Hospitales Universitarios Virgen del Rocío, Avda. Manuel
† A.B. and R.L.-R. contributed equally to this work.
?Published ahead of print on 27 July 2009.
?-lactamase inhibitor CLA with that of IPM in an experimen-
tal murine pneumonia model using two clinical Acinetobacter
(This work was presented in part at the 48th Interscience
Conference on Antimicrobial Agents and Chemotherapy/46th
Annual Meeting of the Infectious Diseases Society of America,
Washington, DC, 25 to 28 October 2008 [29a].)
MATERIALS AND METHODS
Bacterial strains. Two clinical A. baumannii strains with different susceptibility
patterns, Ab11 and Ab51, were selected from 244 Acinetobacter isolates charac-
terized in a previous work (3, 5). The strains were identified phenotypically and
were confirmed by amplified rRNA gene restriction analysis (43). The epidemi-
ological relationships of the two Acinetobacter isolates were determined by re-
petitive extragenic palindromic-PCR (5). The strains were chosen due to their
susceptibility to CLA and their ability to produce infections in the murine
Antimicrobials and susceptibility tests. CLA was obtained from GlaxoSmithKline
(Madrid, Spain), IPM from Merck, Sharp & Dohme (Madrid, Spain), sulbactam
from Pfizer (Orsay, France), and tazobactam from Wyeth Pharmaceuticals (Phil-
adelphia, PA). For these antibiotics, MICs were determined by broth microdi-
lution according to Clinical and Laboratory Standards Institute (CLSI) methods
(9). The activities of CLA and IPM were tested using three different inocula: 105,
106, and 107CFU/ml. For ampicillin, piperacillin, piperacillin-tazobactam, cefo-
taxime, ceftazidime, cefepime, meropenem, ciprofloxacin, trimethoprim-sulfa-
methoxazole, gentamicin, amikacin, and colistin, MICs were determined by Etest
(AB Biodisk, Solna, Sweden) according to the manufacturer’s instructions.
Minimal bactericidal concentrations (MBCs) were determined by subculturing
100-?l aliquots from wells containing antimicrobial concentrations greater than
or equal to the MIC of CLA or IPM onto antimicrobial-free Mueller-Hinton
agar. Plates were incubated at 35°C for 24 to 48 h, and viable colonies were
counted. The MBC was determined as the concentration that killed ?99.9% of
the initial inoculum.
Time-kill curves. The in vitro bactericidal activities of CLA and IPM were
measured using the time-kill method as described by the NCCLS (30). Briefly, 20
ml of MHBCA (Mueller-Hinton broth, cation adjusted; Becton Dickinson Mi-
crobiology Systems, Cockeysville, MD) was incubated at 37°C with a concentra-
tion of antibiotic equivalent to the MIC, twice the MIC, or four times the MIC
(1?, 2?, or 4? MIC) and an inoculum of 5 ? 105CFU of strain Ab11 or
Ab51/ml. As a control, tubes containing 20 ml of medium broth with the inoc-
ulum and without antibiotic were used. The bacterial concentration (expressed in
log10CFU per milliliter) was determined at 0, 2, 4, 8, and 24 h. An antibiotic was
considered bactericidal when it produced a decrease of ?3 log10CFU/ml from
the initial inoculum.
PAE. The in vitro postantibiotic effect (PAE) was determined by exposing both
strains to CLA and IPM at concentrations of 1?, 2?, and 4? MIC for 60 min in
MHBCA. Antibiotic was removed by centrifuging three times at 4,500 ? g for 10
min, removing the supernatant, and resuspending the pellet in prewarmed broth.
The number of CFU per milliliter was counted at 0, 2, 4, 8, and 24 h. A growth
control was performed in the same way without exposure to the antibiotic (12).
In vitro inactivation of CLA. Twenty milliliters of MHBCA with 1?, 2?, and
4? MIC of CLA was inoculated with 5 ? 105CFU/ml of Ab11 or Ab51. The
cultures were incubated at 37°C, and the concentration of CLA was determined
at 0, 2, 4, 8, and 24 h. Aliquots (50 ?l) of each culture were collected and
centrifuged at 4,500 ? g for 10 min, and the bacterium-free supernatant was
analyzed. The CLA concentration was calculated with a bioassay using Klebsiella
pneumoniae ATCC 29665 (31) as an indicator. Two 10-?l aliquots from each
time point were loaded onto blank disks, and the size of the inhibition zone was
measured. Concentrations were estimated by extrapolation from the standard
curve. The intraday and interday variations of the assays are described below.
Studies of AmpC enzymes. For the kinetics experiments, a total-protein extract
was prepared by sonication of each strain. The steady-state kinetics parameters
(Km- and maximum rate of metabolism [Vmax]-like parameters) for CLA were
determined at 25°C using a Nicolette Evolution 300 spectrophotometer (Thermo
Electron Corporation, Waltham, MA). Each rate was determined three times in
phosphate-buffered saline, with quartz cuvettes with a 1-cm path length. The Km
values were calculated as Kivalues in competitive assays with nitrocefin (Oxoid
Ltd., Basingstoke, Hampshire, England). The Vmaxwas calculated by considering
a CLA concentration four times the Km(28).
Animals. Immunocompetent C57BL/6 female mice, weighing 16 to 18 g, were
obtained from the University of Seville; they had a sanitary status of MPF
(murine pathogen free) and were assessed for genetic authenticity. Animals were
housed in regulation cages with food and water ad libitum. The study was
approved by the Ethics and Clinical Research Committee of the University
Hospitals Virgen del Rocío.
Drug pharmacokinetics. Several doses of CLA were assayed in mice in order
to produce serum CLA concentrations similar to the maximum concentration in
serum (Cmax) for humans (data not shown). Finally, the serum pharmacokinetics
of CLA was determined after intraperitoneal administration of a single dose of
13 mg/kg of body weight. The dose of IPM (30 mg/kg given intramuscularly) was
chosen because of its known efficacy against A. baumannii in this experimental
pneumonia model (34). In both cases, after 5, 10, 15, 30, 60, 90, 120, and 240 min,
blood was extracted from the periorbital plexuses of three anesthetized mice per
time point. The serum drug concentrations were calculated by a bioassay using K.
pneumoniae ATCC 29665 for CLA and Micrococcus luteus ATCC 9341 for IPM.
The parameters determined were Cmax(expressed in milligrams per liter), the
area under the concentration-time curve (AUC, expressed in micrograms ? hour/
liter), the terminal half-life (t1/2, expressed in hours) (37), and the time during
which the serum CLA concentration remained above the MIC (TMIC, expressed
in hours), which was estimated by extrapolation from the regression line of serum
elimination using the MIC obtained (20). The intraday and interday variations of
the assays were 3.21% ? 1.28% and 2.96% ? 2.92% for CLA and 2.62% ?
2.44% and 3.22% ? 1.91% for IPM; the linearity (r2) of the assay was 0.99 ? 0.01
and 0.97 ? 0.02, respectively; the lower limits of detection were 0.25 and 0.01
Animal model. An experimental murine pneumonia model (34) was used to
evaluate the efficacies of CLA and IPM against A. baumannii strains. The
animals were anesthetized intraperitoneally with 5% (wt/vol) sodium thiopental
(Braun) and were inoculated with 50 ?l of the bacterial suspension, for which the
bacteria had been grown for 18 h in MHBCA at 37°C and had then been mixed
1:1 with a saline solution of porcine mucin at 10% (wt/vol). The final inoculum
was 8.6 log10CFU/ml for Ab11 and 8.58 log10CFU/ml for Ab51. Treatments
were begun 4 h after inoculation. Prior to the use of the pneumonia model, a
group of 10 uninfected mice were treated with the dose regimen selected for
CLA to evaluate its toxicity.
To ascertain the efficacy of CLA, 45 mice were inoculated with each strain and
were randomly allocated to three groups of 15 animals. The first group did not
receive antimicrobial treatment and was used as a control. The other two groups
were treated with CLA (13 mg/kg, given intraperitoneally) or IPM (30 mg/kg,
given intramuscularly). Because both antimicrobials assayed are ?-lactams, the
dose regimen was calculated to produce serum concentrations above the MIC
during 40% of the interval between doses (11); thus, the intervals between doses
for CLA were 2.5 h and 2 h for strains Ab11 and Ab51, respectively, and the
intervals between doses for IPM were 3.5 h for both strains. The animals were
observed for mortality over 24 h, and all the animals were analyzed immediately
after death. Blood and lung samples were obtained and processed as described
previously (34). The results are expressed as means ? standard deviations of the
log10CFU per gram of lung and as frequencies of sterile blood cultures.
Statistical analysis. The mean log10CFU per gram of lung for the different
treatment groups were compared by analysis of variance (ANOVA). If the
differences were significant, comparisons among groups were made using Dun-
nett and Tukey post hoc tests. Frequencies of sterile tissues, sterile blood cul-
tures, and survival were analyzed by chi-square tests. The SPSS (version 15.0)
statistical package was used (SPSS Inc., Chicago, IL).
In vitro studies. The MICs and MBCs of CLA and IPM
against Ab11 and Ab51 are shown in Table 1. For both strains
and both antibiotics, a low inoculum effect was observed.
Sulbactam MICs were 1 and 2 mg/liter, respectively. Tazo-
bactam had a MIC of 16 mg/liter against both strains. For these
two ?-lactamase inhibitors, only one inoculum (105) was
acillin-tazobactam, meropenem, and colistin and were resistant
to ampicillin, cefotaxime, and cefepime. Moreover, against
ceftazidime, ciprofloxacin, trimethoprim-sulfamethoxazole, gen-
VOL. 53, 2009 CLAVULANIC ACID AGAINST A. BAUMANNII4299
tamicin, and amikacin, Ab11 was susceptible and Ab51 was re-
Time-kill curves are represented in Fig. 1. CLA at 4? MIC
showed bactericidal activity at 8 h against Ab11; against Ab51,
CLA was not bactericidal. IPM was bactericidal at 1?, 2?, and
4? MIC against Ab11 at 8 h; against Ab51, IPM was bacteri-
cidal at 1?, 2?, and 4? MIC at 24 h. No PAE of CLA or IPM
was observed with either of the strains.
The increase in the growth of strain Ab51 at 24 h in the
time-kill curve experiment with CLA was remarkable, reaching
the growth level of the antibiotic-free control (Fig. 1). Figure 2
shows the decrease in the CLA concentration in the culture
medium during the assay with strain Ab51 but not with strain
Ab11. As stated above, one possible explanation is that some-
how CLA is either sequestered or inactivated by ?-lactamase
enzymes. In this respect, the relative enzymatic efficiency
(Vmax/Km-like) parameters of the Ab51 AmpC enzyme
(0.02597 min?1) were twice that of the Ab11 AmpC enzyme
(0.01255 min?1), which can explain the decrease in the CLA
drug concentration in MHBCA along the time-kill curves. On
the other hand, the presence of additional ?-lactamases was
discarded by isoelectric focusing gel experiments (data not
In vivo studies. (i) Pharmacokinetics/pharmacodynamics.
The t1/2values of CLA and IPM after doses of 13 mg/kg and 30
mg/kg, respectively, were 0.24 and 0.26 h. The TMICs were 0.93
and 0.79 h for CLA with Ab11 and Ab51, respectively, and
1.27 h for IPM with both strains. The pharmacokinetics curves
are shown in Fig. 3. The calculated pharmacokinetic/pharma-
codynamic parameters are detailed in Table 2.
(ii) Efficacy of treatment in experimental pneumonia. In
groups inoculated with strain Ab11, both treatments (CLA and
IPM) were effective compared to the untreated control group,
decreasing the bacterial load by 1.78 log10CFU/g lung and 2.47
TABLE 1. MICs and MBCs of CLA and IPM against two strains of
A. baumannii at three different inocula
and inoculum (CFU/ml)
MIC (mg/liter)MBC (mg/liter)
FIG. 1. Time-kill curves of CLA and IPM against strains Ab11 and Ab51. The control has no antibiotic.
4300 BECEIRO ET AL.ANTIMICROB. AGENTS CHEMOTHER.
log10CFU/g lung, respectively (P ? 0.001). The frequency of
sterile blood cultures increased to 80% in the IPM group (P ?
0.005) and 60% in the CLA group (P ? 0.07) compared with
the control group (26.67%). In the groups inoculated with
Ab51, both treatments (CLA and IPM) were effective com-
pared to the control group, decreasing the bacterial load by
2.41 log10CFU/g lung (P ? 0.001) and 2.28 log10CFU/g lung,
respectively (P ? 0.001). The IPM and CLA groups had higher
frequencies of sterile blood cultures (100% and 93.3%, respec-
tively; P ? 0.001) than the control group (20%). The results
are detailed in Table 3 and Fig. 4. No toxicity was observed
with CLA in the group of uninfected mice.
The results of the present study show, for the first time, that
CLA is effective in the treatment of A. baumannii in experi-
mental murine pneumonia, in terms of reduction of lung bac-
terial concentrations, sterilization of blood cultures, and sur-
vival, with the last two effects being strain dependent. The
results with CLA are similar to those with IPM, which is the
gold standard for the treatment of clinical A. baumannii infec-
tions and has shown high efficacy in the treatment of experi-
mental pneumonia with this bacterium (34), although CLA was
slightly less efficacious in the sterilization of blood cultures
than IPM with one of the strains assayed.
Due to the high frequency of antimicrobial multiresistance
among clinical isolates of A. baumannii, new therapeutic alter-
natives are being evaluated (44). Among the ?-lactamase in-
hibitors, sulbactam has shown good efficacy in treating A. bau-
mannii infections, both in experimental murine pneumonia
and in clinical infections (10, 29, 33), but less is known about
the activities of the other ?-lactamase inhibitors, such as tazo-
bactam or CLA. CLA has wide antibacterial activity (19), es-
pecially against pathogens such as Neisseria spp., Chlamydia
spp., Legionella pneumophila, and Enterobacteriaceae. In an in
vitro study with 68 isolates of Acinetobacter spp., CLA was
FIG. 3. Serum CLA and IPM concentrations. CLA was adminis-
tered at 13 mg/kg, and IPM was administered at 30 mg/kg.
FIG. 2. CLA concentrations during the time-kill curves with strains Ab11 and Ab51. The control has no bacteria and 8 mg of CLA/liter.
VOL. 53, 2009 CLAVULANIC ACID AGAINST A. BAUMANNII 4301
more active than other ?-lactams, such as ceftriaxone, cefo-
taxime, and piperacillin; the MIC50and MCI90of CLA were
15.4 and 28.4 mg/liter, respectively (41). In two studies with 100
and 115 epidemiologically defined strains of Acinetobacter spp.,
the CLA MIC was ?8 mg/liter for 29% and 40.9% of the
strains, respectively, and CLA MIC50s were 32 and 16 mg/liter
In the time-kill curves, a bactericidal effect was observed
against strain Ab11, but not against Ab51, at 4? MIC. To
explain this unexpected result, the CLA concentration in the
culture medium was determined, and a significant decrease
was observed with strain Ab51, from 16 to 2.5 mg/liter after
24 h of incubation at 4? MIC, but not with Ab11. This effect
could be due to hydrolysis or a sequestration-like effect of the
AmpC enzyme that decreases the CLA concentration in the
medium. Indeed, it has been shown previously that CLA can
inhibit AmpC from A. baumannii, which demonstrates the
interaction of this inhibitor with the naturally occurring AmpC
enzyme from this microorganism (6). Another possible expla-
nation could be an increase in CLA MICs during the time-kill
experiments; however, we determined CLA MICs against both
strains before and after a 24-h incubation with a CLA concen-
tration of 8 mg/liter, and we observed no changes in the MICs
(data not shown).
As occurs with other ?-lactams against gram-negative bacilli,
except in the case of IPM (39), we have found absent or
minimal PAE with CLA against the strains studied. Previously,
it has been shown that the addition of CLA to amoxicillin
(amoxicilline) produced an extended PAE against ?-lacta-
mase-negative strains of Staphylococcus aureus, Streptococcus
pneumoniae, Streptococcus pyogenes, Haemophilus influenzae,
Moraxella catarrhalis, and Escherichia coli (24). Other authors
(42) found a lack of enhancement of the amoxicillin PAE by
CLA, and they explained the findings of the previous work (24)
by the high concentrations of CLA used, which have antibac-
terial activity per se. We have found no PAE with IPM, in spite
of the results of other authors (39).
Different in vitro studies of pathogens such as S. pneumoniae
(15, 36), L. pneumophila (40), E. coli (25), and Acinetobacter
spp. (39) suggest that CLA has affinity for penicillin-binding
proteins, although the activity can differ greatly depending on
the microorganism. It seems that an important factor in con-
sidering the activity of CLA, especially when it is combined
with other ?-lactams, such as amoxicillin or ticarcillin, is the
interaction with the immune system. In two works in which
Klebsiella pneumoniae infections in renal transplant patients
(13) and patients with chronic hemodialysis (14) were studied,
addition of amoxicillin-clavulanate at 0.5? MIC restored the
activity of human polymorphonuclear cells to levels similar to
those in healthy subjects. In agreement with these results, the
association of amoxicillin and CLA resulted in a synergistic
potentiation of the activity of both drugs on polymorphonu-
clear cell activity against S. pneumoniae in such a manner that
the bacteria became more susceptible either to phagocytosis or
to the microbicidal activities of phagocytes (15). The use of
subinhibitory concentrations of amoxicillin-clavulanate may in-
crease the expression of the proinflammatory cytokines inter-
leukin-8 and interleukin-1? (32). The interaction between
amoxicillin-clavulanate and the immune cells seems clear, but
further studies are necessary to determine the involvement of
CLA in this interaction.
Animal infection models are very useful for the observation
of new potential uses of antibiotics already established in clin-
ical practice. For example, good in vitro activity of CLA was
observed against Chlamydia trachomatis (7). Later, the efficacy
of treatment with CLA was demonstrated in experimental mu-
rine pneumonia caused by C. trachomatis, in which CLA pro-
tected 75% of mice, amoxicillin protected 90%, and the com-
bination of the two protected 100% (2). In the treatment of L.
pneumophila in neutropenic rats, CLA was highly efficacious,
TABLE 2. Pharmacokinetic/pharmacodynamic parameters of CLA (13 mg/kg) and IPM (30 mg/kg)
Pharmacokinetic parameterPharmacodynamic parameter
(mg ? h/liter)
aThe IPM MIC is the same for strains Ab11 and Ab51, and consequently the pharmacodynamic parameters for the two strains are the same.
TABLE 3. Effects of antibiotic therapy on the clearance of Ab11 and Ab51 strains from mouse lungs, the frequency of
sterile blood cultures, and mortality
Bacterial load (mean
lung ? SD)
Bacterial load (mean
lung ? SD)
8.28 ? 1.40
5.81 ? 1.13a
6.50 ? 0.81a
7.75 ? 1.07
5.46 ? 1.55a
5.33 ? 1.88a
aP ? 0.01 relative to the control group (by ANOVA, post hoc Tukey and Dunnett tests, and chi-square tests).
4302 BECEIRO ET AL.ANTIMICROB. AGENTS CHEMOTHER.
similar to the gold standard, erythromycin; amoxicillin-clavu-
lanate was not more effective, while amoxicillin alone was
ineffective (38). This correlates with the results obtained in
vitro, in which the CLA MIC was 0.1 to 0.25 mg/liter (40).
In the murine model, both CLA and IPM treatments were
efficacious in pulmonary bacterial clearance, as determined by
comparison with clearance in the group without treatment, and
increased the number of sterile blood cultures. There were no
significant differences between two treatments in these param-
eters, although the number of sterile blood cultures was higher
(and significantly different from that for the control) with IPM
than with CLA. The model was not used beyond 24 h because
of the high number of doses that had to be administered to the
animals to reach a serum concentration above the MIC for at
least 40% of the time between doses. In spite of this, CLA
decreased the mortality rate in the model with strain Ab51,
which produced a mortality rate of 93.3% in the control group.
In an experimental A. baumannii pneumonia model in neu-
tropenic mice, the addition of CLA to ticarcillin, using a strain
for which ticarcillin and ticarcillin-clavulanate MICs were 32
mg/liter, decreased the lung bacterial concentration, whereas
ticarcillin alone was not efficacious in comparison with the
control group. In this work, IPM alone was also efficacious in
comparison with the control group, showing no differences
from ticarcillin plus CLA (45).
Data about the pharmacokinetics of CLA in humans are
scarce. In the present experiments, the dose of CLA was cho-
sen to produce a Cmaxsimilar to that in humans. Thus, the
Cmaxof CLA after a dose of 200 mg in humans is 11.4 mg/liter
(1), and in the mice it was 13.38 mg/liter. The t1/2in humans
receiving the same dose is 0.8 to 1 h (1), higher than the t1/2
found in our experiments with mice, which was 0.24 h, as is the
rule for small animals. Also, in human bronchial mucosae, the
levels of CLA are 118% of the corresponding levels in serum
(23). These data suggest that CLA may be used to treat human
lung infections with A. baumannii strains for which the CLA
MIC is ?8 mg/liter, such as those in the present study, taking
into account that a similar Cmaxis obtained with a 200-mg dose
and that the interval between doses may be the same as that
obtained when CLA is used as a ?-lactamase inhibitor because
of its higher t1/2in humans than in mice.
In summary, the present study suggests that CLA is effective
in the treatment of experimental murine pneumonia caused by
A. baumannii and that it thus warrants future assessment of its
efficacy with A. baumannii strains showing higher CLA MICs
than those in the present study. Studies of humans, such as
those performed with sulbactam, are necessary to confirm
This study was partially supported by the Spanish Network for Re-
search in Infectious Diseases (REIPI RD06/0008), the Ministerio de
Ciencia e Innovacio ´n, Instituto de Salud Carlos III–FEDER, Fondo
de Investigacio ´n Sanitaria (PI061368, PI081613), and Conselleria de
Sanidad PS07/90. A.B. has an Angeles Alvarin ˜o research contract from
Xunta de Galicia.
None of the authors has a conflict of interest to declare.
1. Bamberger, D. M., J. W. Foxworth, D. L. Bridwell, C. S. Shain, and D. N.
Gerdin. 2005. Extravascular antimicrobial distribution and the respective
blood and urine concentrations in humans, p. 719–814. In V. Lorian (ed.),
Antibiotics in laboratory medicine. Lippincott Williams & Wilkins, Philadel-
2. Beale, A. S., E. Faulds, S. E. Hurn, J. Tyler, and B. Slocombe. 1991. Com-
parative activities of amoxycillin, amoxycillin/clavulanic acid and tetracycline
against Chlamydia trachomatis in cell culture and in an experimental mouse
pneumonitis. J. Antimicrob. Chemother. 27:627–638.
3. Beceiro, A., F. Fernandez-Cuenca, A. Ribera, L. Martinez-Martinez, A. Pas-
cual, J. Vila, J. Rodriguez-Bano, J. M. Cisneros, J. Pachon, and G. Bou.
2008. False extended-spectrum beta-lactamase detection in Acinetobacter
spp. due to intrinsic susceptibility to clavulanic acid. J. Antimicrob. Che-
4. Bergogne-Berezin, E., and K. J. Towner. 1996. Acinetobacter spp. as noso-
comial pathogens: microbiological, clinical, and epidemiological features.
Clin. Microbiol. Rev. 9:148–165.
5. Bou, G., G. Cervero, M. A. Dominguez, C. Quereda, and J. Martinez-Bel-
tran. 2000. PCR-based DNA fingerprinting (REP-PCR, AP-PCR) and
pulsed-field gel electrophoresis characterization of a nosocomial outbreak
caused by imipenem- and meropenem-resistant Acinetobacter baumannii.
Clin. Microbiol. Infect. 6:635–643.
6. Bou, G., and J. Martinez-Beltran. 2000. Cloning, nucleotide sequencing, and
analysis of the gene encoding an AmpC beta-lactamase in Acinetobacter
baumannii. Antimicrob. Agents Chemother. 44:428–432.
7. Bowie, W. R. 1986. In vitro activity of clavulanic acid, amoxicillin, and
ticarcillin against Chlamydia trachomatis. Antimicrob. Agents Chemother.
8. Cefai, C., J. Richards, F. K. Gould, and P. McPeake. 1990. An outbreak of
Acinetobacter respiratory tract infection resulting from incomplete disinfec-
tion of ventilatory equipment. J. Hosp. Infect. 15:177–182.
9. Clinical and Laboratory Standards Institute. 2007. Performance standards for
antimicrobial susceptibility testing; 17th informational supplement. CLSI docu-
ment M100–S17. Clinical and Laboratory Standards Institute, Wayne, PA.
10. Corbella, X., J. Ariza, C. Ardanuy, M. Vuelta, F. Tubau, M. Sora, M. Pujol,
and F. Gudiol. 1998. Efficacy of sulbactam alone and in combination with
ampicillin in nosocomial infections caused by multiresistant Acinetobacter
baumannii. J. Antimicrob. Chemother. 42:793–802.
11. Craig, W. A. 1998. Pharmacokinetic/pharmacodynamic parameters: rationale
for antibacterial dosing of mice and men. Clin. Infect. Dis. 26:1–10.
12. Craig, W. A., and S. Gudmundsson. 1991. Postantibiotic effect, p. 403–431.
In V. Lorian (ed.), Antibiotics in laboratory medicine. Williams & Wilkins,
13. Cuffini, A. M., V. Tullio, F. Giacchino, A. Bonino, N. Mandras, N. Bianchi,
J. Roana, D. Scalas, F. Bonello, and N. A. Carlone. 2001. Improved phago-
cyte response by co-amoxiclav in renal transplant recipients. Transplantation
14. Cuffini, A. M., V. Tullio, F. Giacchino, N. Mandras, D. Scalas, P. Belardi, C.
Merlino, and N. A. Carlone. 2001. Impact of co-amoxiclav on polymorpho-
nuclear granulocytes from chronic hemodialysis patients. Am. J. Kidney Dis.
15. Cuffini, A. M., V. Tullio, A. Ianni Palarchio, A. Bonino, G. Paizis, and N. A.
Carlone. 1998. Enhanced effects of amoxycillin/clavulanic acid compared
FIG. 4. Effect of antibiotic therapy with CLA or IPM on the clear-
ance of A. baumannii from mouse lungs.*, P ? 0.001 relative to the
control group (by ANOVA and Tukey and Dunnett post hoc tests).
VOL. 53, 2009CLAVULANIC ACID AGAINST A. BAUMANNII4303
with amoxycillin and clavulanic acid alone on the susceptibility to immuno-
defenses of a penicillin-resistant strain of Streptococcus pneumoniae. Drugs
Exp. Clin. Res. 24:173–184.
16. Dijkshoorn, L., H. M. Aucken, P. Gerner-Smidt, M. E. Kaufmann, J. Ursing,
and T. L. Pitt. 1993. Correlation of typing methods for Acinetobacter isolates
from hospital outbreaks. J. Clin. Microbiol. 31:702–705.
17. Falagas, M. E., I. A. Bliziotis, and I. I. Siempos. 2006. Attributable mortality
of Acinetobacter baumannii infections in critically ill patients: a systematic
review of matched cohort and case-control studies. Crit. Care 10:R48.
18. Falagas, M. E., and P. I. Rafailidis. 2007. Attributable mortality of Acineto-
bacter baumannii: no longer a controversial issue. Crit. Care 11:134.
19. Finlay, J., L. Miller, and J. A. Poupard. 2003. A review of the antimicrobial
activity of clavulanate. J. Antimicrob. Chemother. 52:18–23.
20. Frimodt-Moller, N., M. W. Bentzon, and V. F. Thomsen. 1986. Experimental
infection with Streptococcus pneumoniae in mice: correlation of in vitro
activity and pharmacokinetic parameters with in vivo effect for 14 cephalo-
sporins. J. Infect. Dis. 154:511–517.
21. Gerner-Smidt, P., and W. Frederiksen. 1993. Acinetobacter in Denmark: I.
Taxonomy, antibiotic susceptibility, and pathogenicity of 112 clinical strains.
22. Gerner-Smidt, P., and I. Tjernberg. 1993. Acinetobacter in Denmark. II.
Molecular studies of the Acinetobacter calcoaceticus-Acinetobacter baumannii
complex. APMIS 101:826–832.
23. Gould, I. M., G. Harvey, D. Golder, T. M. Reid, S. J. Watt, J. A. Friend, J. S.
Legge, and J. G. Douglas. 1994. Penetration of amoxycillin/clavulanic acid
into bronchial mucosa with different dosing regimens. Thorax 49:999–1001.
24. Gould, I. M., K. Milne, and C. Jason. 1991. Post antibiotic effect of coamoxy-
clav, abstr. 1360. Progr. abstr. 5th Eur. Cong. Clin. Microbiol. Infect. Dis.
25. Greenwood, D., F. O’Grady, and P. Baker. 1979. An in vitro evaluation of
clavulanic acid, a potent, broad-spectrum beta-lactamase inhibitor. J. Anti-
microb. Chemother. 5:539–547.
26. Hartstein, A. I., A. L. Rashad, J. M. Liebler, L. A. Actis, J. Freeman, J. W.
Rourke, Jr., T. B. Stibolt, M. E. Tolmasky, G. R. Ellis, and J. H. Crosa. 1988.
Multiple intensive care unit outbreak of Acinetobacter calcoaceticus subspe-
cies anitratus respiratory infection and colonization associated with contam-
inated, reusable ventilator circuits and resuscitation bags. Am. J. Med. 85:
27. Higgins, P. G., H. Wisplinghoff, D. Stefanik, and H. Seifert. 2004. In vitro
activities of the beta-lactamase inhibitors clavulanic acid, sulbactam, and
tazobactam alone or in combination with beta-lactams against epidemiolog-
ically characterized multidrug-resistant Acinetobacter baumannii strains. An-
timicrob. Agents Chemother. 48:1586–1592.
28. Hujer, K. M., N. S. Hamza, A. M. Hujer, F. Perez, M. S. Helfand, C. R.
Bethel, J. M. Thomson, V. E. Anderson, M. Barlow, L. B. Rice, F. C. Tenover,
and R. A. Bonomo. 2005. Identification of a new allelic variant of the Acin-
etobacter baumannii cephalosporinase, ADC-7 beta-lactamase: defining a
unique family of class C enzymes. Antimicrob. Agents Chemother. 49:2941–
29. Jimenez-Mejias, M. E., J. Pachon, B. Becerril, J. Palomino-Nicas, A. Ro-
driguez-Cobacho, and M. Revuelta. 1997. Treatment of multidrug-resistant
Acinetobacter baumannii meningitis with ampicillin/sulbactam. Clin. Infect.
29a.López-Rojas, R., A. Beceiro, J. Domínguez-Herrera, F. Docobo-Pérez, G.
Bou, and J. Pachón. 2008. In vitro activity and in vivo efficacy of clavulanic
acid against Acinetobacter baumannii, poster B-066. Abstr. 48th Annu. In-
tersci. Conf. Antimicrob. Agents Chemother. American Society for Micro-
biology, Washington, DC.
30. National Committee for Clinical Laboratory Standards. 1999. Methods for
determining bactericidal activity of antimicrobial agents. National Commit-
tee for Clinical Laboratory Standards, Wayne, PA.
31. Reading, C., and M. Cole. 1977. Clavulanic acid: a beta-lactamase-inhibiting
beta-lactam from Streptomyces clavuligerus. Antimicrob. Agents Chemother.
32. Reato, G., A. M. Cuffini, V. Tullio, A. I. Palarchio, A. Bonino, R. Foa, and
N. A. Carlone. 1999. Co-amoxiclav affects cytokine production by human
polymorphonuclear cells. J. Antimicrob. Chemother. 43:715–718.
33. Rodriguez-Hernandez, M. J., L. Cuberos, C. Pichardo, F. J. Caballero, I.
Moreno, M. E. Jimenez-Mejias, A. Garcia-Curiel, and J. Pachon. 2001.
Sulbactam efficacy in experimental models caused by susceptible and inter-
mediate Acinetobacter baumannii strains. J. Antimicrob. Chemother. 47:479–
34. Rodriguez-Hernandez, M. J., J. Pachon, C. Pichardo, L. Cuberos, J. Ibanez-
Martinez, A. Garcia-Curiel, F. J. Caballero, I. Moreno, and M. E. Jimenez-
Mejias. 2000. Imipenem, doxycycline and amikacin in monotherapy and in
combination in Acinetobacter baumannii experimental pneumonia. J. Anti-
microb. Chemother. 45:493–501.
35. Rolinson, G. N. 1994. A review of the microbiology of amoxycillin/clavulanic
acid over the 15 year period 1978–1993. J. Chemother. 6:283–318.
36. Severin, A., E. Severina, and A. Tomasz. 1997. Abnormal physiological
properties and altered cell wall composition in Streptococcus pneumoniae
grown in the presence of clavulanic acid. Antimicrob. Agents Chemother.
37. Shumaker, R. C. 1986. PKCALC: a BASIC interactive computer program
for statistical and pharmacokinetic analysis of data. Drug Metab. Rev. 17:
38. Smith, G. M., K. H. Abbott, and R. Sutherland. 1992. Bactericidal effects of
co-amoxiclav (amoxycillin clavulanic acid) against a Legionella pneumophila
pneumonia in the immunocompromised weanling rat. J. Antimicrob. Che-
39. Spivey, J. M. 1992. The postantibiotic effect. Clin. Pharm. 11:865–875.
40. Stokes, D. H., B. Slocombe, and R. Sutherland. 1989. Bactericidal effects of
amoxycillin/clavulanic acid against Legionella pneumophila. J. Antimicrob.
41. Suh, B., T. Shapiro, R. Jones, V. Satishchandran, and A. L. Truant. 1995. In
vitro activity of beta-lactamase inhibitors against clinical isolates of Acineto-
bacter species. Diagn. Microbiol. Infect. Dis. 21:111–114.
42. Thorburn, C. E., S. J. Molesworth, R. Sutherland, and S. Rittenhouse. 1996.
Postantibiotic and post-beta-lactamase inhibitor effects of amoxicillin plus
clavulanate. Antimicrob. Agents Chemother. 40:2796–2801.
43. Vaneechoutte, M., L. Dijkshoorn, I. Tjernberg, A. Elaichouni, P. de Vos, G.
Claeys, and G. Verschraegen. 1995. Identification of Acinetobacter genomic
species by amplified ribosomal DNA restriction analysis. J. Clin. Microbiol.
44. Vila, J., and J. Pachon. 2008. Therapeutic options for Acinetobacter bauman-
nii infections. Expert Opin. Pharmacother. 9:587–599.
45. Wolff, M., M. L. Joly-Guillou, R. Farinotti, and C. Carbon. 1999. In vivo
efficacies of combinations of beta-lactams, beta-lactamase inhibitors, and
rifampin against Acinetobacter baumannii in a mouse pneumonia model.
Antimicrob. Agents Chemother. 43:1406–1411.
4304BECEIRO ET AL.ANTIMICROB. AGENTS CHEMOTHER.