1098 Clinical Microbiology and Infection, Volume 10 Number 12, December 2004
REFERENCES
1. Waites KB, Thacker LW, Talkington DF. The value of
culture and serology for detection of Mycoplasma
pneumoniae in the clinical laboratory in the age of
molecular diagnostics. Clin Microbiol Newslett 2001; 23:
123–129.
2. Daxboeck F, Krause R, Wenisch C. Laboratory diagnosis of
Mycoplasma pneumoniae infection. Clin Microbiol Infect 2003;
9: 263–273.
3. Cimolai N, Cheong ACH. An assessment of a new diag-
nostic indirect enzyme immunoassay for the detection of
anti-Mycoplasma pneumoniae IgM. Am J Clin Pathol 1996;
105: 205–209.
4. Waris ME, Toikka P, Saarinen T et al. Diagnosis of Myco-
plasma pneumoniae pneumonia in children. J Clin Microbiol
1998; 36: 3155–3159.
5. Dorigo-Zetsma JW, Zaat SAJ, Wertheim-van Dillen PME
et al. Comparison of PCR, culture, and serological tests for
diagnosis of Mycoplasma pneumoniae respiratory tract
infection in children. J Clin Microbiol 1999; 37: 14–17.
6. Petitjean J, Vabret A, Gouarin S, Freymuth F. Evaluation
of four commercial immunoglobulin G (IgG)- and IgM-
specific enzyme immunoassays for diagnosis of Myco-
plasma pneumoniae infections. J Clin Microbiol 2002; 40:
165–171.
7. Thacker WL, Talkington DF. Analysis of complement fix-
ation and commercial enzyme immunoassays for detection
of antibodies to Mycoplasma pneumoniae in human serum.
Clin Diagn Lab Immunol 2000; 7: 778–780.
8. Sillis M. The limitations of IgM assays in the serological
diagnosis of Mycoplasma pneumoniae infections. J Med
Microbiol 1990; 33: 253–258.
9. Granström M, Holme T, Sjögren AM, Örtqvist A, Kalin M.
The role of IgA determination by ELISA in the early
serodiagnosis of Mycoplasma pneumoniae infection, in rela-
tion to IgG and l-capture IgM methods. J Med Microbiol
1994; 40: 288–292.
10. Segev JS, Sedmak GV, Kurup VP. Isotype-specific antibody
responses to acute Mycoplasma pneumoniae infection. Ann
Allergy Asthma Immunol 1996; 77: 67–73.
11. Watkins-Riedel T, Stanek G, Daxboeck F. Comparison of
SeroMP IgA with four other commercial assays for sero-
diagnosis of Mycoplasma pneumoniae pneumonia. Diagn
Microbiol Infect Dis 2001; 40: 21–25.
12. Lieberman D, Lieberman D, Ben-Yaakov M et al. Serolog-
ical evidence of Mycoplasma pneumoniae infection in acute
exacerbation of COPD. Diagn Microbiol Infect Dis 2002;
44: 1–6.
 2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 1089–1104
RESEARCH NOTE
Synergic in-vitro activity of imipenem and
sulbactam against Acinetobacter baumannii
J. Y. Choi1, Y. Soo Park1, C. H. Cho1, Y. Seon
Park1, S. Y. Shin1, Y. G. Song1, D. Yong2,
K. Lee2 and J. M. Kim1
1
Department of Internal Medicine and AIDS
Research Institute and 2Department of Laborat-
ory Medicine and Research Institute of Bacterial
Resistance, BK21 Project for Medical Sciences,
Yonsei University College of Medicine, Seoul,
Korea
ABSTRACT
The interaction between sulbactam and imipenem
was evaluated with four clinical isolates of Acine-
tobacter baumannii, including two isolates resistant
to imipenem, one of which produced IMP-1
metallo-b-lactamase. Two isolates (one of which
was imipenem-resistant) were sulbactam-resist-
ant by undefined mechanisms. MICs were deter-
mined by standard broth microdilution methods.
Time-kill assays with imipenem and sulbactam,
alone or in combination at 0.5 · MIC and
1 · MIC, showed a synergic effect in all four
isolates of A. baumannii after incubation for 0, 4,
8 and > 24 h at 35
C.
Keywords Acinetobacter baumannii, imipenem, sulbac-
tam, synergy
Original Submission: 16 February 2004; Revised
Submission: 13 April 2004; Accepted: 10 May 2004
Clin Microbiol Infect 2004; 10: 1098–1101
10.1111/j.1469-0691.2004.00987.x
Acinetobacter baumannii has emerged worldwide
as an important nosocomial pathogen that is
difficult to treat because of its multiple antibiotic
resistance [1]. Most nosocomial isolates of
A. baumannii are now resistant to a wide variety
of antibiotics, with carbapenems being the main
Corresponding author and reprint requests: J. M. Kim,
Department of Internal Medicine, Yonsei University College
of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-
752, Korea
E-mail: jmkim@yumc.yonsei.ac.kr
 Research Note 1099

remaining therapeutic choice [2,3]. In the hospital an inoculum of 5 · 105 CFU ⁄ mL in Mueller–
setting, overuse of imipenem has been associated Hinton broth, and results were read after incuba-
with several outbreaks caused by carbapenem- tion for 20 h at 35
C. The antibiotics were diluted
resistant strains, which leaves polymyxin, and in two-fold steps in Mueller–Hinton broth to
perhaps sulbactam, as the only antibiotics with give concentrations of 0.008–128 mg ⁄ L. Time-kill
in-vitro activity against these organisms [2,4]. assays were performed with 1.5 · 106 CFU ⁄ mL in
Therapy in such cases is a serious challenge, and trypticase soy broth and antibiotic concentrations
consequently, the activity of sulbactam against of 0.5· and 1· MIC. Imipenem and sulbactam
the genus Acinetobacter is receiving renewed were tested alone and in combination, with
attention [5]. However, resistance to sulbactam incubation for 0, 4, 8 and 24 h at 35
C. Following
has already been noted in imipenem-resistant incubation, aliquots (0.1 mL) were diluted ten-
strains of A. baumannii, leaving polymyxin as the fold in sterile saline and plated on to trypticase
only treatment alternative [6,7]. Levin et al. [8] soy agar plates; colonies were counted on plates
reported the outcomes of 60 nosocomial infec- yielding 10–100 colonies after incubation for 24 h
tions, including bacteraemia, caused by A. bau- at 35
C. All time-kill experiments were performed
mannii and Pseudomonas aeruginosa isolates that in triplicate. Synergy and additivity ⁄ indifference
were resistant to all commercially available anti- were defined, respectively, as a ‡ 2 log10 CFU ⁄ mL
microbial agents. These infections were treated decrease, and a < 2 log10 CFU ⁄ mL change in the
with colistin, although colistin can have import- viable count after incubation with the combina-
ant adverse effects such as nephrotoxicity or tion for 24 h, compared with the viable count in
neurotoxicity. Other therapeutic options are the presence of the more active of the two
required urgently. antibiotics used.
A few reports have described the interactions MICs of imipenem and sulbactam for the four
between carbapenems and sulbactam in A. bau- A. baumannii strains are listed in Table 1. The
mannii. Ko et al. [9] reported that meropenem MICs of imipenem ranged from 0.12 to 16 mg ⁄ L,
combined with sulbactam exhibited more potent and those for sulbactam from 1 to 64 mg ⁄ L. Fig. 1
antimicrobial activity against multiresistant shows the killing activities of imipenem and
A. baumannii than did meropenem or sulbactam sulbactam, alone and in combination, expressed
alone. Wolff et al. [10] found that imipenem and as log10 CFU ⁄ mL changes in the number of
sulbactam did not provide true bactericidal effects surviving bacteria after incubation for 0, 4, 8 and
against A. baumannii in a mouse pneumonia 24 h. The combination of imipenem and sulbac-
model, but the interactions between imipenem tam produced a synergic effect against all four
and sulbactam against A. baumannii remained A. baumannii strains.
unclear. Sulbactam is a b-lactamase inhibitor with low
The present study investigated the in-vitro antibacterial activity against most microorgan-
antibacterial activity of imipenem combined with isms except Neisseria gonorrhoeae, Neisseria menin-
sulbactam against four clinical isolates of A. bau- gitidis, Burkholderia cepacia and Acinetobacter spp.
mannii selected on the basis of their resistance [14,15]. However, in addition to anti-b-lactamase
phenotypes. Strain YMC01 ⁄ 7 ⁄ R234 produced
PER-1 extended-spectrum b-lactamase, while
YMC01 ⁄ 7 ⁄ P40 produced IMP-1 metallo-b-lacta-
Table 1. MICs of imipenem and sulbactam for Acineto-
mase, as confirmed by PCR-based detection of the bacter baumannii and the killing activity of sulbactam
blaPER-1 and blaIMP-1 genes, respectively [11,12]. combined with imipenem against A. baumannii
Strains YMC01 ⁄ 7 ⁄ R520 and YMC01 ⁄ 7 ⁄ R300 were
Killing activitya of sulbactam
susceptible to imipenem. Strains YMC01 ⁄ 7 ⁄ R234 MIC (mg ⁄ L) combined with imipenem
and YMC01 ⁄ 7 ⁄ R520 were resistant to sulbactam Strain Imipenem Sulbactam 0.5· MIC 1· MIC
by undefined mechanisms. The strains were not
YMC01 ⁄ 7 ⁄ R234 16 64 ) 6.63 ± 0.08 S ) 4.62 ± 0.06 S
clonally related. YMC01 ⁄ 7 ⁄ P40 16 8 ) 5.82 ± 0.11 S ) 6.61 ± 0.02 S
The antibiotics used for the tests were imipe- YMC01 ⁄ 7 ⁄ R520 1 64 ) 5.24 ± 0.10 S ) 1.24 ± 0.06 A
YMC01 ⁄ 7 ⁄ R300 0.12 1 ) 4.74 ± 0.05 S ) 3.12 ± 0.06 S
nem (MSD, Seoul, Korea) and sulbactam (Pfizer,
a
Seoul, Korea). MICs were determined with the Expressed as a change in log10 CFU ⁄ mL at 24 h produced by the combination,
compared with that produced by the most active agent used alone.
standard broth microdilution method [13], with S, synergic; A, additive ⁄ indifferent.

 2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 1089–1104
1100 Clinical Microbiology and Infection, Volume 10 Number 12, December 2004

(a) (b)10
10 9
9 8
8

log10CFU/mL
7
log10CFU/mL

7 6
6
5 5
4 4
3 3
2 2
1 1
0 0
0 4 8 24 0 4 8 24
Time (h) Time (h)

(c) 10 (d)10
9 9
8 8

log10CFU/mL
log10CFU/mL

7 7
6 6
5 5
4 4
3 3
2 2
1 1
0 0
0 4 8 24 0 4 8 24
Time (h) Time (h)

(e) 10 (f) 10
9 9
8 8
log10CFU/mL
log10CFU/mL

7 7
6 6
5 5
4 4
3 3
2 2
1 1
0 0
0 4 8 24 0 4 8 24
Time (h) Time (h)
(g) 10 (h) 10
9 9
8 8
log10CFU/mL

log10CFU/mL

7 7
6 6
5 5
4 4
3 3
2 2
1 1
0 0
0 4 8 24 0 4 8 24
Time (h) Time (h)

Fig. 1. Bactericidal activity of imipenem and sulbactam alone or combined against four strains of Acinetobacter baumannii:
diamonds, imipenem; squares, sulbactam; triangles, imipenem + sulbactam. (a) Strain YMC01 ⁄ 7 ⁄ R234 at 1· MIC. (b)
Strain YMC01 ⁄ 7 ⁄ R234 at 0.5x MIC. (c) Strain YMC01 ⁄ 7 ⁄ P40 at 1· MIC. (d) Strain YMC01 ⁄ 7 ⁄ P40 at 0.5x MIC. (e) Strain
YMC01 ⁄ 7 ⁄ R520 at 1· MIC. (f) Strain YMC01 ⁄ 7 ⁄ R520 at 0.5x MIC. (g) Strain YMC01 ⁄ 7 ⁄ R300 at 1· MIC. (h) Strain
YMC01 ⁄ 7 ⁄ R300 at 0.5x MIC.

activity, sulbactam can bind to bacterial penicil-
lin-binding protein 2 (PBP2). Since the activity of
most penicillins and cephalosporins results from
binding to PBP1 and PBP3, the combination of
sulbactam with a penicillin or a cephalosporin
may result in a synergic effect [14].
Imipenem was the first commercially available
carbapenem and has an excellent spectrum of
antimicrobial activity. It rapidly penetrates the
outer-membrane of Gram-negative bacteria, and
has excellent stability to class C and A serine
b-lactamases, including extended-spectrum b-lac-
tamases [16]. Imipenem and meropenem possess
important activity against most Gram-negative
bacteria because of their high affinity for PBPs
such as PBP2 and ⁄ or PBP3.

 2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 1089–1104

In the present study, sulbactam and imipenem
were found to act synergically against four strains
of A. baumannii, including two imipenem-resistant
A. baumannii strains. The affinity of sulbactam for
PBP2 and of imipenem for PBP3 may be the cause
of such synergic interactions. However, imipenem
has good affinity for the PBP2 of Gram-negative
bacteria, and it has been reported that reduced
expression of PBP2 is related to reduced suscep-
tibility or resistance to carbapenems [17]. There-
fore, it seems possible that another mechanism
might be involved in the synergic interactions
observed in the present study. Further studies
with a larger number of strains are required to
confirm these observations. In addition, the
expression of PBPs was not investigated, and
thus it was not possible to evaluate the precise
mechanism(s) of synergy. It will also be necessary
to determine the mechanisms responsible for
resistance in the strains tested.
ACKNOWLEDGEMENTS
This study was supported by the BK21 Project for Medical
Sciences, Yonsei University.
REFERENCES
1. Urban C, Segal-Maurer S, Rahal JJ. Considerations in
control and treatment of nosocomial infections due to
multidrug-resistant Acinetobacter baumannii. Clin Infect Dis
2003; 36: 1268–1274.
2. Corbella X, Ariza J, Ardanuy C et al. Efficacy of sulbactam
alone and in combination with ampicillin in nosocomial
infections caused by multiresistant Acinetobacter baumannii.
J Antimicrob Chemother 1998; 42: 793–802.
3. Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as
nosocomial pathogens: microbiological, clinical, and epi-
demiological features. Clin Microbiol Rev 1996; 9: 148–165.
4. Cisneros JM, Reyes MJ, Pachon J et al. Bacteremia due to
Acinetobacter baumannii: epidemiology, clinical findings,
and prognostic features. Clin Infect Dis 1996; 22: 1026–1032.
5. Levin AS. Multiresistant Acinetobacter infections: a role for
sulbactam combinations in overcoming an emerging
worldwide problem. Clin Microbiol Infect 2002; 8: 144–153.
 2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 1089–1104
Research Note 1101
6. Cisneros JM, Rodrı́quez-Baño J. Nosocomial bacteremia
due to Acinetobacter baumannii: epidemiology, clinical fea-
tures and treatment. Clin Microbiol Infect 2002; 8: 687–693.
7. Wood CA, Reboli AC. Infections caused by imipenem-
resistant Acinetobacter calcoaceticus biotype anitratus. J Infect
Dis 1993; 167: 448–451.
8. Levin AS, Barone AA, Penço J et al. Intravenous colistin as
therapy for nosocomial infections caused by multidrug-
resistant Pseudomonas aeruginosa and Acinetobacter bau-
mannii. Clin Infect Dis 1999; 28: 1008–1011.
9. Ko WC, Lee HC, Chiang SR et al. In vitro and in vivo
activity of meropenem and sulbactam against a multidrug-
resistant Acinetobacter baumannii strain. J Antimicrob
Chemother 2004; 53: 393–395.
10. Wolff M, Joly-Guillou ML, Farinotti R, Carbon C. In vivo
efficacies of combinations of b-lactams, b-lactamase
inhibitors, and rifampin against Acinetobacter baumannii in
a mouse pneumonia model. Antimicrob Agents Chemother
1999; 43: 1406–1411.
11. Yong D, Shin JH, Kim S et al. High prevalence of PER-1
extended-spectrum b-lactamase producing Acinetobacter
spp. in Korea. Antimicrob Agents Chemother 2003; 47: 1749–
1751.
12. Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH.
Modified Hodge test and EDTA-disk synergy tests to
screen metallo-b-lactamase-producing strains of Pseudo-
monas and Acinetobacter species. Clin Microbiol Infect 2001;
7: 88–89.
13. National Committee for Clinical Laboratory Standards.
Methods for dilution antimicrobial susceptibility testing for
bacteria that grow aerobically, 5th edn. Approved standard
M7-A5. Wayne, PA: NCCLS, 2000.
14. Fu W, Demei Z, Shi W, Fupin H, Yingyuan Z. The
susceptibility of non-fermentative Gram-negative bacilli
to cefoperazone and sulbactam compared with other
antibacterial agents. Int J Antimicrob Agents 2003; 22: 444–
448.
15. Jacoby GA, Sutton L. Pseudomonas cepacia susceptibility
to sulbactam. Antimicrob Agents Chemother 1989; 33: 583–
584.
16. Yang Y, Bhachech N, Bush K. Biochemical comparison of
imipenem, meropenem and biapenem: permeability,
binding to penicillin-binding proteins, and stability to
hydrolysis by b-lactamases. J Antimicrob Chemother 1995;
35: 75–84.
17. Fernández-Cuenca F, Martı́nez L, Conejo MC, Ayala JA,
Perea EJ, Pascual A. Relationship between b-lactamase
production, outer membrane protein and penicillin-bind-
ing protein profiles on the activity of carbapenems against
clinical isolates of Acinetobacter baumannii. J Antimicrob
Chemother 2003; 51: 565–574.