Rev Esp Quimioterap, Septiembre 2006; Vol. 19 (Nº 3): 252-247
© 2006 Prous Science, S.A.- Sociedad Española de Quimioterapia
In vitro activity of linezolid
in combination with doxycycline, fosfomycin,
levofloxacin, rifampicin and vancomycin against
methicillin-susceptible Staphylococcus aureus
J.M. Sahuquillo Arce1, E. Colombo Gainza1, A. Gil Brusola1, R. Ortiz Estévez1, E. Cantón2y M. Gobernado1
1Department of Clinical Microbiology, 2Experimental Bacteriology Laboratory, Hospital La Fe, Valencia, Spain
Actividad in vitro de linezolid en combinación con doxiciclina,
fosfomicina, levofloxacino, rifampicina y vancomicina
frente a Staphylococcus aureus sensible a meticilina
R RE ES SU UM
El objetivo de este estudio fue investigar los efectos in vitro de linezolid en combinación con cinco antimicrobianos antiestafilocócicos (doxi-
ciclina, fosfomicina, levofloxacino, rifampicina y vancomicina) en cepas de Staphylococcus aureus sensibles a meticilina (SASM). Se utilizaron
cinco cepas de SASM (hf008, hf095, hf295, hf602 y hf946) aisladas de muestras procedentes de infecciones humanas. Se utilizó el méto-
do del damero para evaluar la sinergia entre linezolid y los cinco antimicrobianos, y se trazaron curvas de tiempo-muerte con las combina-
ciones más activas. El resultado más habitual para las combinaciones de linezolid con rifampicina, vancomicina y doxiciclina fue indiferencia.
La combinación con levofloxacino produjo antagonismo en dos de las cinco cepas. Sin embargo, frente a cuatro cepas se observó sinergia
con la combinación de linezolid y fosfomicina, con un índice de concentración inhibitoria fraccionada (ICIF) >0,5. Ni linezolid ni fosfomicina
en solitario inhibieron el crecimiento a 1/4x CMI, pero la combinación de ambos fármacos a 1/4 de la CMI respectiva mostró un efecto bac-
teriostático sinérgico, un descenso de 2-3 log10respecto al antimicrobiano más activo en solitario. En resumen, la combinación de linezolid y
fosfomicina a concentraciones subinhibitorias se mostró sinérgica, ejerciendo un efecto bacteriostático.
ME EN N
P Pa al la ab br ra as s c cl la av ve e: : Oxazolidinona – Fosfomicina –Sinergia
S SU UM
The objective of this paper was to investigate the in vitro effects of linezolid combined with five antistaphylococcal antibiotics – doxycycline,
fosfomycin, levofloxacin, rifampicin and vancomycin – upon methicillin-susceptible Staphylococcus aureus (MSSA). Five MSSA isolates from
clinical specimens of human infections – hf008, hf095, hf295, hf602 and hf946 – were used in this study. The checkerboard method was
used to assess synergism between linezolid and the five antibiotics, and time-kill curves were carried out with the most active combinations.
Indifference was the most common result achieved by the checkerboard method when linezolid was combined with rifampicin, vancomycin
or doxycycline. The combination with levofloxacin yielded antagonism for two of the five isolates. However, four isolates showed synergy
for the combination of linezolid plus fosfomycin with a fractional inhibitory concentration index (FICI) >0.5. Neither linezolid nor fosfomycin
alone inhibited growth at 1/4x minimum inhibitory concentration (MIC); but the combination of both drugs at 1/4 the respective MIC
showed a synergistic bacteriostatic effect, a 2–3 log10decrease with respect to the most active antibiotic alone. In summary, the combina-
tion of subinhibitory concentrations of linezolid and fosfomycin presented synergism, exerting a bacteriostatic effect.
MMMA AR RY Y
K Ke ey y w wo or rd ds s: : Oxazolidinone – Fosfomycin – Synergism
Linezolid is a synthetic antibiotic belonging to the ox-
azolidinone family. It blocks the protein synthesis at the ri-
bosome preventing the formation of the initiation complex.
It is active against Gram-positive micro-organisms, includ-
ing multiresistant strains, being bacteriostatic against Sta-
phylococcus spp. (1–6). One of the main features of line-
zolid is that it has a high oral bioavailability, which makes
it easy to administer and adhere to the treatment (7–8).
Linezolid has already proven its clinical effectiveness in
processes like pneumonia, soft tissue infections and osteo-
mielytis, among others (9–14). Nowadays, it plays a very
important role against infections caused by multiresistant
Gram-positive pathogens (10, 15–17).
Bone and prosthesis infections, where biofilms are like-
ly to be formed, have a high rate of therapeutic failure
when an antibiotic is used alone due to problems with an-
tibiotic penetration, inoculum effect or stationary-phase or-
ganisms (18). Thus, the search for combinations of antibi-
otics might yield more effective treatment options, shorter
administration periods and fewer major side effects.
The objective of this work was to investigate the in vitro
effects of linezolid combined with five antistaphylococcal
antibiotics – doxycycline, fosfomycin, levofloxacin, rifampi-
cin and vancomycin – upon methicillin-susceptible Staphy-
lococcus aureus (MSSA). This was conducted in two steps:
First, the checkerboard method was utilized with linezolid
plus the other five antibiotics as a screening for synergism;
second, the time-kill curve was performed on the most ac-
tive of them with the aim of strengthening the results by
considering data from two different techniques (19).
MATERIALS AND METHODS
The five MSSA isolates of this investigation – hf008,
hf095, hf295, hf602 and hf946 – were all isolated from
clinical specimens of human infections.
Doxycycline, fosfomycin, levofloxacin, linezolid, rifam-
picin and vancomycin were supplied by the Pharmacy
Department of our hospital as a stock solution of 20 mg/ml,
250 mg/ml, 5 mg/ml, 2 mg/ml, 60 mg/ml and 5 mg/ml, re-
2006; Vol. 19 (Nº 3)
In vitro activity of linezolid in combination against methicillin-susceptible S. aureus
Fosfomycin E-test strips (AB Biodisk N.A. Inc., Solna,
Sweden) were used according to manufacturer’s guidelines.
Minimum inhibitory concentration (MIC) was deter-
mined by microdilution method in Mueller-Hinton broth
(20, 21) and defined as the lowest concentration that pre-
vented growth after 20–24 hours of incubation at 35 ºC.
A 96 well plate (8 rows × 12 columns) was used. Each
well was filled with Mueller-Hinton broth containing line-
zolid at a concentration ranging from 32 mg/l to 0.0625 mg/l
and the other antibiotics from 16× MIC to 1/32× MIC dis-
pensed at two-fold dilutions in a checkerboard manner,
plus the bacterial inoculum to a final volume of 200 µl per
well. Four wells from the first column were used as a steril-
ity control and were filled only with Mueller-Hinton broth;
the other four were used as a growth control and no antibi-
otic was added. The MIC of both linezolid and the other
antibiotic was redetermined every time the checkerboard
was conducted. The inoculum was prepared from a 24-
hour culture on blood agar plates, and then adjusted to a
0.5 McFarland standard (approximately 1 × 108CFU/ml) in
sterile distilled water, subsequently diluted to a final cell
concentration of approximately 5 × 105CFU/ml, which
was confirmed by colony counts in agar plates. The plates
were incubated at 35 ºC for 18 ± 2 hours. The interaction
was measured by the fractional inhibitory concentration in-
dex (FICI) all along the growth–no growth interface by the
formula FICI = (MIC of linezolid in combination/MIC of
linezolid alone) + (MIC of the antibiotic in combination/
MIC of the antibiotic alone). Antagonism was defined as
FICI >4, indifference as FICI >0.5 and >4, and synergism
as FICI <0.5 (10, 29).
Time-kill curves were performed for the combination
of linezolid plus fosfomycin on those isolates that had pre-
sented synergy by the checkerboard method (hf095, hf295,
hf602 and hf946). Linezolid and fosfomycin were tested
alone at 4× and 1/4× MIC of each antibiotic, and both com-
bined at 4× MIC and at 1/4× MIC in a final volume of 5 ml
of Mueller-Hinton broth, using an inoculum of approxi-
mately 1 × 106CFU/ml (22, 23). An antibiotic-free flask
served as a growth control. Surviving bacteria were count-
ed after 0, 3, 6, 24 and 48 hours of incubation at 35 ºC by
J.M. Sahuquillo, E. Colombo, A. Gil, R. Ortiz, E. Cantón and M. Gobernado R E V E S P Q U I M I O T E R A P
subculturing 100 µl serial dilution of every flask on sheep
blood agar plates. The aliquots were deposited as a spot on-
to the agar plates and allowed to soak. After the plate had
dried, spreading was performed; no antibiotic carry-over
was detected. The lower limit of accurate and reproducible
detectable colony count was 100 CFU/ml.
Synergism and indifference were defined as a decrease
>2 log10and <2 log10CFU/ml with respect to the most ac-
tive drug, antagonism as an increase >2 log10.
Bacteriostatic effect was defined as a decrease <3 log10
CFU/ml compared to the initial inoculum and bactericidal
as a decrease >3 log10CFU/ml compared to the initial in-
MICs of the antibiotics are displayed in Table 1. Small
variations of the MIC (±1 doubling dilution) occurred oc-
casionally when the MIC was determined at the time the
checkerboard method was performed.
Indifference was the most common result achieved by
the checkerboard method for doxycycline, rifampicin and
vancomycin, although isolate 095 showed synergism with
rifampicin and linezolid at concentrations of 0.016/0.5
mg/ml respectively, and isolate hf946 showed synergism
for the combination of doxycycline and linezolid at con-
centrations of 0.007/0.5 mg/ml. Isolates hf095 and hf295
presented antagonism for the combination of levofloxacin
plus linezolid at 0.125/1 and 0.125/0.5, respectively. The
other three isolates resulted in indifference (Table 1).
Four isolates showed synergy for the combination of
linezolid plus fosfomycin by the checkerboard method
with a FICI >0.5 (Table 1).
Table 1. MICs by microdilution method and Σ Σ FICIs of the different antibiotics alone and in combination with linezolid by the
Levofloxacin hf 008
AB: current antibiotic; LNZ: linezolid; I: indifference; S: synergism; A: antagonism.
2006; Vol. 19 (Nº 3)
In vitro activity of linezolid in combination against methicillin-susceptible S. aureus
Linezolid alone at 4× MIC exerted a bacteriostatic ef-
fect. However, fosfomycin at 4× MIC had a killing effect
the first six hours; after that, a regrowth occurred. When
both antibiotics were combined at 4× MIC, the time-kill
curve followed that of the linezolid alone.
Neither linezolid nor fosfomycin alone inhibited
growth at 1/4× MIC; but the combination of both drugs at
1/4 the respective MIC showed a synergistic effect, a 2–3
log10decrease with respect to the most active antibiotic
alone, although this decrease was less than 3 log10 with re-
spect to the initial inoculum, thus being bacteriostatic
In order to determine whether the regrowth observed
when fosfomycin was used alone was due to selection of
spontaneous mutants, aliquots of 100 µl were withdrawn
from the flasks onto sheep blood agar plates with a fos-
fomycin E-test strip at the beginning of the experiment and
after 24 hours of incubation. No colonies were observed
within the inhibition zone at the beginning of the experi-
ment. However, colonies emerged inside the inhibition zone
of the cultures from the fosfomycin flasks after 24 hours of
exposure, but not when it was combined with linezolid.
The MIC of the cultures from the 4× MIC fosfomycin
flasks increased more than five doubling dilutions, shifting
from 4 mg/l to 96 mg/l in two isolates, whereas it did not
change in the cultures from the flasks where linezolid was
used in combination.
Linezolid is an antibiotic with a unique mechanism of
action – the inhibition of the initiation process of the protein
transcription, bacteriostatic against Staphylococcus aureus.
It has proven to be a good option for the treatment of a
great variety of infections, such as those of bone or soft tis-
Figure 1. Time-kill curve plots of linezolid and fosfomycin alone and in combination at 1/4x MIC. (?, filled diamond) growth control, (?, filled square) 1/4x
MIC linezolid, (?, filled triangle) 1/4x MIC fosfomycin, (?, filled circle) 1/4x MIC linezolid plus 1/4x MIC fosfomycin.
sue, where Gram-positive micro-organisms play an impor-
tant role. At the present time, combined therapies are being
considered as a way to enhance antibiotic activity, reduce
the duration of the process as well as the duration of the ad-
ministration of drugs, and to avoid the emergence of resis-
In our study, we have tested the interaction of linezolid
with different antibiotics by the checkerboard and time-kill
curve methods. With the checkerboard method we have
observed that indifference was the most frequent effect
when combined with rifampicin, vancomycin and doxycy-
cline, although two isolates presented synergism, one with
rifampicin and another with doxycycline. The combination
with levofloxacin yielded antagonism for two of the five
isolates. Other authors have also reported indifference using
the time-kill curve method for rifampicin and vancomycin
(4, 25), and antagonism for the combination with levoflo-
xacin (17, 26). In contrast, the combination of fosfomycin
and linezolid exhibited synergism in four of the five iso-
lates tested, similar to the findings of Grif et al. (26), who
detected synergism against MSSAand Staphylococcus epi-
With the time-kill curve method, the combination of
both linezolid and fosfomycin at concentrations above the
MIC did not show any enhancement in their killing activi-
ty, linezolid being the leading drug. Still, the combination
of linezolid with fosfomycin prevented the emergence of
resistance observed when fosfomycin was used alone; oth-
er authors found that linezolid in combination with rifampin
or fusidic acid avoided the appearance of resistance to the
partner drug (4, 25, 27). Notably, when combined at subin-
hibitory concentrations, they experience synergism, despite
the fact that both drugs when tested alone were not able to
inhibit growth, the combination accomplished a bacterio-
static effect. This is noteworthy due to the fact that both an-
tibiotics are widely distributed into human tissues and bio-
films (7, 8, 28, 29); consequently, common doses of both
drugs could reach faster and maintain therapeutic levels of
antibiotics at the site of infection in circumstances where it
is difficult to reach drug concentrations similar to those in
In summary, we can presume that linezolid combined
with fosfomycin could perform an important role as an al-
ternative treatment of diseases that are complex to heal and
that have a tendency to become chronic, such as osteoar-
ticular or prosthesis infections (10–12, 28), where biofilms
are likely to be produced by micro-organisms, since the as-
sociation of linezolid with fosfomycin is active at lower
concentrations than the single agents. This combination
J.M. Sahuquillo, E. Colombo, A. Gil, R. Ortiz, E. Cantón and M. GobernadoR E V E S P Q U I M I O T E R A P
could also prevent the emergence of resistance to current
Nevertheless, in vivo studies, as well as analyses on
biofilms, need to be carried out in order to consider this as-
sociation of antibiotics as an option for the treatment of
We thank Julia Hernandez from the Pharmacy Department for tech-
This research was supported in part by a grant from Pfizer, Spain.
Correspondence: J.M. Sahuquillo Arce, Department of Clinical Micro-
biology, Hospital La Fe, Avda. Campanar 21, 46009 Valencia, Spain. Tel.:
+34 96 386 27 44; E-mail: firstname.lastname@example.org
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