Eur J Clin Microbiol Infect Dis (2012) 31:695701

DOI 10.1007/s10096-011-1360-5

ARTICLE

Synergy of fosfomycin with carbapenems, colistin, netilmicin,

and tigecycline against multidrug-resistant Klebsiella
pneumoniae, Escherichia coli, and Pseudomonas aeruginosa
clinical isolates
G. Samonis & S. Maraki & D. E. Karageorgopoulos &
E. K. Vouloumanou & M. E. Falagas

Received: 25 May 2011 / Accepted: 25 June 2011 / Published online: 31 July 2011
# Springer-Verlag 2011

Abstract Fosfomycin represents a potential last-resort

treatment option for infections with certain multidrugresistant (MDR) Gram-negative pathogens. We evaluated
double-drug combinations of fosfomycin with imipenem,
meropenem, doripenem, colistin, netilmicin, and tigecycline
for in vitro synergy against 100 MDR Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa
clinical isolates, using the Etest method. Synergy was
defined as a fractional inhibitory concentration index 0.5.
The isolates were consecutively collected at a university
hospital in Greece from various clinical specimens.
Against 50 serine carbapenemase-producing K. pneumoniae isolates, synergy of fosfomycin with imipenem,
meropenem, doripenem, colistin, netilmicin, and tigecy-

cline was observed for 74.0%, 70.0%, 74.0%, 36.0%,

42.0%, and 30.0% of the isolates, respectively. Against 14
extended-spectrum -lactamase (ESBL)-producing K.
pneumoniae isolates, synergy of fosfomycin with imipenem, meropenem, doripenem, colistin, netilmicin, and
tigecycline was observed for 78.6%, 42.9%, 42.9%,
7.1%, 42.9%, and 21.4%, respectively; for 20 ESBLproducing E. coli isolates, the corresponding values were
55.0%, 25.0%, 30.0%, 15.0%, 25.0%, and 25.0%; and for
15 MDR P. aeruginosa isolates, the corresponding values
were 46.7%, 53.3%, 73.3%, 13.3% , 13.3%, and 13.3%.
Antagonism was not observed for any of the combinations
tested. Further studies are needed in order to confirm the
clinical relevance of the above findings.

G. Samonis
Department of Medicine, University Hospital of Heraklion,
Heraklion, Crete, Greece

Introduction

S. Maraki
Department of Clinical Bacteriology, University Hospital
of Heraklion,
Heraklion, Crete, Greece
D. E. Karageorgopoulos : E. K. Vouloumanou : M. E. Falagas (*)
Alfa Institute of Biomedical Sciences (AIBS),
9 Neapoleos Street,
151 23 Marousi, Athens, Greece
e-mail: m.falagas@aibs.gr
M. E. Falagas
Department of Medicine, Henry Dunant Hospital,
Athens, Greece
M. E. Falagas
Department of Medicine, Tufts University School of Medicine,
Boston, MA, USA

Since the era of pandrug resistance for Gram-negative

pathogens may not be a scenario for the distant future, and
the pace of development of new antimicrobial agents for
clinical use is far from encouraging, the identification of
novel therapeutic options among the currently available
agents seems imperative [1, 2]. Fosfomycin, an antimicrobial agent known for over 40 years, could represent one
such option [3, 4]. Fosfomycin appears to have retained
antimicrobial activity against Enterobacteriaceae with
advanced resistance patterns, particularly Escherichia coli
producing extended-spectrum -lactamases (ESBLs), and
even carbapenem-resistant Klebsiella pneumoniae [5, 6].
Fosfomycin may also show antimicrobial activity against a
minor but considerable subset of multidrug-resistant
(MDR) Pseudomonas aeruginosa.

696

Although fosfomycin has found clinical use over the

years mainly in the treatment of uncomplicated urinary tract
infections, the relatively limited published clinical evidence
regarding its use for systemic infections appears to be
favorable [7]. The major consideration against the use of
fosfomycin for systemic infections refers to the potential for
the emergence of resistance during therapy, related to a high
mutation frequency to fosfomycin resistance observed in
vitro [8]. The above possibility could be overcome with the
use of fosfomycin in combination with other active
antibiotics [9]. Apart from the prevention of the emergence
of resistance, another desired feature of such combination
antimicrobial therapy is the potential for synergy between
the agents used. This could be particularly relevant for
fosfomycin, as it might be used as a last resort option in
cases where traditional antimicrobial regimens are not
active, have failed, or are contraindicated. In this regard,
we sought to study the in vitro synergistic activity between
fosfomycin and other relevant antimicrobial agents against
MDR Gram-negative clinical isolates.

Materials and methods

We studied 100 consecutive MDR clinical isolates of E.
coli, K. pneumoniae, or P. aeruginosa. The isolates were
collected between August 2009 and May 2010 from clinical
specimens that were submitted for culture to the microbiological laboratory of the tertiary-care, 700-bed, University
Hospital of Heraklion, Crete, Greece. Multidrug resistance
was defined as resistance to at least three classes of
antimicrobial agents among those that are considered as
potentially effective against wild-type pathogens [10].
Species identification was done by standard biochemical
methods, the API system, or the Vitek2 automated system
(bioMrieux SA, Marcy lEtoile, France [11]). The minimum inhibitory concentration (MIC) for fosfomycin and 17
other antimicrobial agents were determined by the gradient
diffusion method using the Etest (AB bioMrieux, Solna,
Sweden), following the manufacturers recommendations.
Regarding specifically susceptibility testing to fosfomycin,
Etest strips of this agent containing 25 mg/L glucose-6phosphate were used.
The identification of ESBL production was performed
by phenotypic testing based on synergy between
clavulanic acid and extended-spectrum cephalosporins
[12]. The detection of carbapenemase production was
done using the modified Hodge test [13]. The detection of
metallo--lactamase (MBL) production was done using
phenotypic methods based on a three-fold or greater
decrease in the imipenem MICs in the presence of
ethylenediaminetetraacetic acid (EDTA). Isolates that
tested positive with the modified Hodge test and negative

Eur J Clin Microbiol Infect Dis (2012) 31:695701

with the EDTA test were considered to produce a serine

carbapenemase [14, 15].
The United States Food and Drug Administration (FDA)
breakpoints were used to interpret tigecycline MIC results,
while the European Union Committee on Antimicrobial
Susceptibility Testing (EUCAST) breakpoints were used to
interpret those of doripenem and colistin. The Clinical and
Laboratory Standards Institute (CLSI) M100-S20 interpretative breakpoints were used to interpret the MIC results for
all other antimicrobial agents studied [16]. Particularly for
fosfomycin, the CLSI breakpoints that apply to E. coli
urinary isolates were used as provisional breakpoints for the
Gram-negative pathogens included in our study. The
identification of the production of ESBLs or carbapenemases was also taken into consideration in the determination of the susceptibility of the studied isolates to antibiotics
affected by the presence of such mechanisms.
Antimicrobial synergy testing was performed using the
relevant Etest method [17]. The combinations tested against
each isolate were fosfomycin plus imipenem, meropenem,
doripenem, colistin, netilmicin, and tigecycline. The Etest
strips of fosfomycin and the other test antimicrobials were
crossed at a 90 angle at their predetermined MIC values
for each of the studied isolates, on an inoculated Mueller
Hinton agar plate. The plates were then incubated at 35C
for 18 h. The MIC of each antimicrobial in the combination
was interpreted as the value at which the zone of inhibition
intersects the Etest strip. All study tests were performed in
duplicate.
The fractional inhibitory concentration index (FICI) for a
given isolate was calculated according to the formula: FICI=
(MIC of fosfomycin in combination/MIC of fosfomycin
alone)+(MIC of the other antibiotic in combination/MIC of
the other antibiotic alone). Synergy was defined as an
FICI of 0.5; indifference was defined as an FICI >0.5 but 4;
and antagonism was defined as an FICI of >4 [18].

Results
The 100, in total, isolates that we studied included 65K.
pneumoniae, 20 E. coli, and 15 P. aeruginosa isolates. Of the
65K. pneumoniae isolates, 50 (76.9%) were producers of
serine carbapenemase, 1 (1.5%) of MBL, and the remaining
14 (21.5%) were ESBL producers. All of the 20 E. coli
isolates were ESBL producers. The above isolates were
derived from a total of 97 individual patients. Two patients
provided two isolates of different species (K. pneumoniae
and P. aeruginosa), and another patient provided two isolates
of the same species (K. pneumoniae), but with a different
antibiotype. Table 1 presents data on the hospital location of
the patients who provided the isolates studied, and the type
of the culture specimens used.

Eur J Clin Microbiol Infect Dis (2012) 31:695701

697

Table 1 Characteristics of the

studied isolates

Klebsiella pneumoniae

Escherichia coli

Pseudomonas aeruginosa

Origin of patients
Surgical wards

18 (27.6)

8 (40.0)

4 (26.6)

Medical wards

24 (36.9)

1 (5.0)

4 (26.6)

Outpatient units
Intensive care unit

3 (4.6)
20 (30.7)

10 (50.0)
1 (5)

0
7 (46.6)

16 (24.6)
23 (35.3)

0
17 (85.0)

3 (20.0)
2 (13.3)

Culture specimens
Blood
Urine
Bronchoalveolar lavage fluid

10 (15.3)

1 (5.0)

3 (20.0)

Pus
Arterial catheter

5 (7.6)
4 (6.1)

2 (10.0)
0

4 (26.6)
2 (13.3)

Sputum

1 (1.5)

1 (6.6)

Bile
Transplant tissue

1 (1.5)
1 (1.5)

0
0

0
0

Peritoneal fluid

3 (4.6)

Abdominal drainage fluid

1 (1.5)

Antimicrobial susceptibility
Table 2 provides data on the susceptibility pattern of the
isolates studied to the antimicrobials tested. In summary, all
of the isolates were susceptible to fosfomycin, except for
one K. pneumoniae isolate that was intermediately susceptible and one P. aeruginosa isolate that was resistant to this

agent. Of the 65 MDR K. pneumoniae isolates studied,

84.6% were susceptible to tigecycline, 75.4% to colistin,
21.5% to carbapenems, and 9.2% to netilmicin. Of the 20
ESBL E. coli isolates, all were susceptible to carbapenems
and colistin, 95% were susceptible to tigecycline, and 55%
were susceptible to netilmicin. Finally, of the 15 P.
aeruginosa isolates, all were susceptible to colistin, 40%

Table 2 Susceptibility of the studied isolates to the antibiotics tested

Antibiotics

Klebsiella pneumoniae (N=65)

Escherichia coli (N=20)

Pseudomonas aeruginosa (N=15)

S (%)

S (%)

I (%)

S (%)

I (%)

I (%)

Aztreonam

3 (20.0)

Cefotaxime
Ceftazidime
Cefepime
Piperacillintazobactam
Ertapenem

0
0
0
0
14 (21.5)

0
0
0
0
2 (3.1)

0
0
0
0
20 (100)

0
0
0
0
0

0
0
0
0
0

0
0
0
0
0

Imipenem
Meropenem
Doripenem
Colistin
Chloramphenicol
Ciprofloxacin
Fosfomycin
Gentamicin
Netilmicin
Tetracycline
Tigecycline
Trimethoprimsulfamethoxazole

14 (21.5)
14 (21.5)
14 (21.5)
49 (75.4)
2 (3.1)
3 (4.6)
63 (96.9)
43 (66.2)
6 (9.2)
5 (7.7)
55 (84.6)
6 (9.2)

6 (9.2)
8 (12.3)
2 (3.1)
0
4 (6.2)
0
1 (1.5)
18 (27.7)
2 (3.1)
11 (16.9)
5 (7.7)
0

20 (100)
20 (100)
20 (100)
20 (100)
12 (60.0)
2 (10.0)
20 (100)
12 (60.0)
11 (55.0)
3 (15.0)
19 (95.0)
7 (35.0)

0
0
0
0
1
0
0
2
2
0
1
0

3 (20.0)
6 (40.0)
3 (20.0)
15 (100)
0
0
14 (93.3)
2 (13.3)
2 (13.3)
0
0
0

2 (13.3)
0
0
0
0
0
0
0
0
0
0
0

Abbreviations: I: intermediately susceptible; S: susceptible

(5.0)

(10.0)
(10.0)
(5.0)

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Eur J Clin Microbiol Infect Dis (2012) 31:695701

combinations tested, synergy against this isolate was only

observed between fosfomycin and imipenem. Regarding
the 14 ESBL-producing K. pneumoniae isolates, fosfomycin was synergistic with imipenem against 78.6% of the
isolates, whereas synergy between fosfomycin and meropenem or doripenem was seen against 42.9% of the
isolates for both agents.
Against the 20 ESBL-producing E. coli isolates, synergy
was more frequently observed between fosfomycin and
imipenem (55.0%), followed by doripenem (30.0%),
meropenem, tigecycline, and netilmicin (25.0% for each,
respectively), and colistin (15.0%). With regard to the
susceptibility status of these isolates to netilmicin, synergy
between fosfomycin and netilmicin was observed against 2
of the 7 (28.6%) netilmicin-susceptible isolates and 3 of the
11 (27.3%) netilmicin-resistant isolates. Finally, against the
15 MDR P. aeruginosa isolates, synergy was more
frequently observed between fosfomycin and doripenem
(73.3%), followed by meropenem (53.3%), imipenem
(46.7%), colistin, netilmicin, and tigecycline (13.3% for
each, respectively).

were susceptible to meropenem (the most active carbapenem for this group), and 13.3% to netilmicin.
Synergy
Table 3 presents data on the synergistic properties of
fosfomycin in combination with the six other antimicrobial
agents tested against the isolates evaluated. Antagonism
was not observed in any of the experiments. Against the 65
K. pneumoniae isolates studied, the most synergistic
fosfomycin combination was the one with imipenem
(synergy for 75.4% of the isolates), followed by doripenem
(66.2%), meropenem (63.1%), netilmicin (41.5%), colistin
(29.2%), and tigecycline (27.7%). Against specifically the
50 serine carbapenemase-producing K. pneumoniae isolates, the three different carbapenems tested showed similar
synergistic activity with fosfomycin (70.074.0% of the
isolates). Synergy between fosfomycin and the other agents
tested against these 50 serine carbapenemase-producing
isolates was seen for 42% of the isolates for netilmicin,
36% for colistin, and 30% for tigecycline.
Table 4 presents detailed data on the synergistic
properties between fosfomycin and the other antimicrobial
agents tested for the 50 serine carbapenemase-producing K.
pneumoniae isolates, with regard to the susceptibility
pattern for each agent. Fosfomycin was synergistic in
combination with doripenem against 77.1% of the 35 serine
carbapenemase-producing isolates that had a doripenem
MIC of more than 8 mg/L; with imipenem against 74.4% of
the 43 isolates with an imipenem MIC of more than 8 mg/
L; and with meropenem against 68.6% of the 35 isolates
with a meropenem MIC of more than 8 mg/L. Fosfomycin
was also synergistic in combination with netilmicin against
45.6% of the 46 netilmicin-resistant isolates.
The MBL-producing K. pneumoniae isolate studied had
an MIC of more than 8 mg/L for all of the carbapenems
tested, and it was susceptible to both colistin and
tigecycline, but resistant to netilmicin. Of all six fosfomycin

Discussion
The main finding of our study is that the combination of
fosfomycin with a carbapenem (imipenem, meropenem, or
doripenem) can be synergistic against a substantial proportion of, primarily, MDR K. pneumoniae and, also, MDR P.
aeruginosa, and ESBL E. coli clinical isolates. Furthermore, fosfomycin exhibits synergy in combination with
carbapenems against more than two-thirds of the serine
carbapenemase-producing K. pneumoniae isolates, including those with elevated carbapenem MICs. Between the
carbapenems evaluated, imipenem appeared to be most
frequently synergistic in combination with fosfomycin
against the ESBL-producing K. pneumoniae isolates, as
well against the ESBL E. coli isolates; doripenem appeared

Table 3 Synergy of fosfomycin combinations against the isolates studied

Antibiotic in
combination with
fosfomycin

Klebsiella
pneumoniae, all
isolates (N=65)

ESBL-producing
Klebsiella
pneumoniae (N=14)

Serine carbapenemaseproducing Klebsiella

pneumoniae (N=50)

ESBL-producing
Escherichia coli
(N=20)

MDR Pseudomonas
aeruginosa (N=15)

Isolates exhibiting synergy, n (%)

Imipenem
49 (75.4)
Meropenem
41 (63.1)
Doripenem
43 (66.2)
Colistin
19 (29.2)
Netilmicin
27 (41.5)

11 (78.6)
6 (42.9)
6 (42.9)
1 (7.1)
6 (42.9)

37
35
37
18
21

11 (55.0)
5 (25.0)
6 (30.0)
3 (15.0)
5 (25.0)

7 (46.7)
8 (53.3)
11 (73.3)
2 (13.3)
2 (13.3)

Tigecycline

3 (21.4)

15 (30.0)

5 (25.0)

2 (13.3)

18 (27.7)

(74.0)
(70.0)
(74.0)
(36.0)
(42.0)

Abbreviations: ESBL: extended-spectrum -lactamase; MDR: multidrug-resistant

Eur J Clin Microbiol Infect Dis (2012) 31:695701

Table 4 Synergy of fosfomycin
combinations against the 50 serine
carbapenemase-producing Klebsiella pneumoniae isolates studied
according to the minimum inhibitory concentration (MIC) of each
of the carbapenems tested or the
susceptibility status to the other
antibiotics tested

Antibiotic in combination with fosfomycin

*Percentages are given only

when the denominator is at least
10

Isolates exhibiting synergy, n/N (%)*

Carbapenem MIC, mg/L

Imipenem
Meropenem
Doripenem

Abbreviations: I: intermediately
susceptible; S: susceptible; R:
resistant

699

4<MIC8

>8

1/3
2/5

4/4
9/10 (90.0)

32/43 (74.4)
24/35 (68.6)

7/11 (63.6)

3/4

27/35 (77.1)

Susceptibility status
S
I

Colistin

13/36 (36.1)

0/0

5/14 (35.7)

Netilmicin
Tigecycline

0/4
13/43 (30.2)

0/0
1/3

21/46 (45.6)
1/4

to be most frequently synergistic with fosfomycin against

the MDR P. aeruginosa isolates (most of which were
carbapenem-resistant). Regarding the other fosfomycin
combinations studied, the most noteworthy one is with
netilmicin, which showed to be synergistic against more
than 40% of the MDR K. pneumoniae isolates, including
the netilmicin-resistant carbapenemase-producing K. pneumoniae isolates. Importantly, antagonism was not observed
for any of the fosfomycin combinations against any isolate
tested.
Data from other studies for the in vitro synergistic
properties of fosfomycin in combination with other antibiotics
against Gram-negative pathogens are relatively scarce [3, 19];
the majority of these data refer to P. aeruginosa [20, 21].
Although synergy between fosfomycin and -lactam antibiotics, including carbapenems, has frequently been observed, these findings have been variable[22]. Moreover, a
few studies have even reported antagonism between fosfomycin and -lactams. This might be more relevant when low
fosfomycin concentrations are used [23].
The synergistic activity between fosfomycin and carbapenems against Gram-negative pathogens observed in our study
could be explained by the fact that these two classes of
antimicrobial agents act on different steps of the same bacterial
process (i.e., cell-wall synthesis [24]). Specifically, fosfomycin
inhibits the first committed enzymatic step in peptidoglycan
synthesis by binding to the enzyme UDP-N-acetylglucosamine
enolpyruvyl transferase (MurA) and, thereby, inhibiting the
formation of N-acetylmuramic acid by N-acetylglucosamine
and phosphoenolpyruvate [25]. -Lactams mainly act on the
latest stage in cell-wall synthesis by preventing the transpeptidation of peptidoglycan.
A more specific interaction between fosfomycin and
-lactams could supposedly relate to modification of the
pattern of expression of penicillin-binding proteins by
fosfomycin[26]. This mechanism has mainly been observed
for Gram-positive pathogens and could account for the
synergy between fosfomycin and -lactams against

methicillin-resistant Staphylococcus aureus or penicillinresistant Streptococcus pneumoniae that has been observed
in some in vitro studies [26, 27]. Still, -lactam resistance in
these Gram-positive cocci is primarily a result of differences
in the expression of penicillin-binding proteins or their affinity
to -lactams, rather than the presence of -lactamases.
Our study findings showing that fosfomycin is frequently
synergistic in vitro against MDR Gram-negative pathogens
with other clinically relevant antimicrobial agents could have
therapeutic implications. Regarding specifically the
carbapenemase-producing K. pneumoniae infections, the
clinical evidence regarding the appropriate treatment options
is still rather limited [28, 29]. However, it seems probable
that the use of carbapenems in combination with other in
vitro active antimicrobial agents can be helpful, particularly for pathogens with relatively low carbapenem
MICs (4 mg/L) [30]. Our study suggests that fosfomycin
can be used in combination with carbapenems to maximize their activity. It is uncertain, though, if the use of a
double-drug combination regimen consisting of a carbapenem
with fosfomycin for the treatment of carbapenemaseproducing K. pneumoniae infections is adequate to
prevent the emergence of resistance to fosfomycin during
therapy.
The use of triple-drug combinations with the addition to
the above regimen of another active agent, such as colistin,
tigecycline, or an aminoglycoside, could also be considered
in this regard [31]. The inclusion of fosfomycin in such
triple-drug combination regimens might not result in
increased toxicity, as it is a generally safe drug, and it
could even reduce the nephrotoxicity from drugs like
aminoglycosides [4]. Alternatively, fosfomycin could be
combined in a double-drug combination regimen with one
of the non-carbapenem, microbiologically active agents,
such as the ones mentioned above, particularly in the case
that synergy exists, which can be observed for approximately 30% of such isolates, according to our study
findings. Between the different aminoglycosides, the

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Eur J Clin Microbiol Infect Dis (2012) 31:695701

combination of fosfomycin with gentamicin against

carbapenemase-producing K. pneumoniae requires further
study, as gentamicin had higher antimicrobial activity for
these isolates compared with netilmicin in our study.
The findings of our study regarding the synergistic
activity of fosfomycin in combination with other clinically
relevant antimicrobial agents against MDR Gram-negative
infections should be confirmed with a methodologically
more robust method, such as timekill studies [32]. The
detection of synergy using the Etest method might be less
accurate, although it may be clinically relevant, as it can be
easily performed in the clinical microbiological laboratory
[33]. Our study also did not evaluate the studied isolates for
clonality. Instead, we assessed a relatively large number of
isolates, with several resistance mechanisms, collected from
patients in different hospital locations, and from various
types of specimens. Finally, we did not study molecularly
the presence and type of specific -lactamases and
carbapenemases. We should mention, though, that the
KPC-2 carbapenemase is the one predominantly found
among K. pneumoniae isolates in our region [34].
In conclusion, fosfomycin exhibited in vitro synergy with
carbapenems against a substantial percentage of the serine
carbapenemase-producing K. pneumoniae clinical isolates
evaluated in our study. Synergy was also observed against
some of these isolates between fosfomycin and other agents,
such as aminoglycosides, colistin, and tigecycline, which
may be used in the treatment of infections with the above
pathogens. The fosfomycin combinations tested, particularly
those with the carbapenems, were also often synergistic
against the ESBL-producing K. pneumoniae and ESBLproducing E. coli isolates evaluated, as well as against the
MDR P. aeruginosa ones. Although the above findings need
to be further established in additional studies, they could be
considered in the treatment of infections by extensively drugresistant Gram-negative pathogens, for which the relevant
clinical evidence is limited and most therapeutic regimens
represent last-resort options.

Funding

None.

Conflict of interest None.

Transparency declarations

None to declare.

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