Skip Navigation



JAC Advance Access published online on May 31, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm136
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
60/2/317    most recent
dkm136v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Song, J. Y.
Right arrow Articles by Cheong, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Song, J. Y.
Right arrow Articles by Cheong, H. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

In vitro activities of carbapenem/sulbactam combination, colistin, colistin/rifampicin combination and tigecycline against carbapenem-resistant Acinetobacter baumannii

Joon Young Song1, Sae Yoon Kee1, In Sook Hwang2, Yu Bin Seo1, Hye Won Jeong1, Woo Joo Kim1 and Hee Jin Cheong1,*

1 Division of Infectious Diseases, Department of Internal Medicine, College of Medicine, Korea University, Seoul, Republic of Korea 2 Graduate School of Life Science and Biotechnology, Korea University, Seoul, Republic of Korea

* Correspondence address. Division of Infectious Disease, Department of Internal Medicine, Korea University Guro Hospital, 97 Guro Dong-Gil, Guro Gu, 152-703 Seoul, Republic of Korea. Tel: +82-2-818-6649; Fax: +82-2-866-1643; E-mail: heejinmd{at}medimail.co.kr

Received 22 December 2006; returned 1 March 2007; revised 5 April 2007; accepted 11 April 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Objectives: To determine the in vitro activities and interactions of imipenem, colistin and tigecycline with old antibacterial agents against carbapenem-resistant Acinetobacter baumannii.

Methods: Forty-three carbapenem-resistant A. baumannii isolates from the intensive care unit of a university hospital were collected and their MICs of imipenem, colistin and tigecycline were determined. With eight randomly selected carbapenem-resistant isolates, an in vitro time–kill study was performed for the evaluation of antibacterial activity of colistin, tigecycline, imipenem/sulbactam and colistin/rifampicin.

Results: The time–kill study of colistin demonstrated bactericidal activity against A. baumannii at concentrations of 4x MIC and 8x MIC, whereas tigecycline showed bacteriostatic activity at all concentrations. The combination regimens of imipenem/sulbactam and colistin/rifampicin were synergistic and bactericidal at 1x MIC.

Conclusions: Imipenem/sulbactam combination, colistin and tigecycline showed good in vitro activities against carbapenem-resistant A. baumannii isolates. Even though colistin is bactericidal against carbapenem-resistant A. baumannii, the colistin/rifampicin combination is more warranted in order to be certain.

Key Words: A. baumannii , synergy , time–kill assay


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Acinetobacter baumannii has emerged as one of the most important nosocomial pathogens, especially in patients admitted to an intensive care unit (ICU). A. baumannii can colonize multiple body sites of hospitalized patients and survive for a long time on inanimate surfaces.1,2 Both these aforementioned characteristics may have contributed to the prominent role of A. baumannii in nosocomial infections.

Fluoroquinolones, ceftazidime, trimethoprim/sulfamethoxazole and carbapenems have been considered active against nosocomial A. baumannii strains. However, treatment of this organism has become a medical challenge because of the emergence of multidrug-resistant isolates, including resistance to carbapenems, with increasing frequency.3,4 Therefore, new agents or combination regimens of old agents known to be effective against multidrug-resistant A. baumannii are attracting more attention. Meropenem/sulbactam combinations have shown synergistic effects in multidrug-resistant strains of A. baumannii.5,6 An old drug, colistin, which was abandoned since the 1960s due to nephrotoxicity, gained new interest for its activity against A. baumannii and one study, although small in sample size, showed a good outcome with a combination regimen of colistin and rifampicin.7,8 Tigecycline showed good in vitro bacteriostatic activity against A. baumannii strains that showed different susceptibilities to imipenem.9

The objective of the present study was to demonstrate in vitro activities of carbapenem/sulbactam combination, colistin, colistin/rifampicin combination and tigecycline against carbapenem-resistant A. baumannii.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Transparency declarations
 References
 
Forty-three clinical isolates of carbapenem-resistant A. baumannii were collected from routine clinical isolates from patients admitted to the ICU during the period of January 2002 to June 2006. Isolates were selected randomly from different patients.

For the 43 isolates, MICs of imipenem, colistin (colistimate sodium), tigecycline, sulbactam and rifampicin were determined by a broth microdilution method in Mueller–Hinton broth by geometric 2-fold serial dilutions. According to CLSI (formerly NCCLS) standards, strains were considered resistant to carbapenems if the MICs of imipenem were ≥32 mg/L.10 The following concentrations were considered as the susceptibility breakpoints of other tested antimicrobials: colistin, 4 mg/L; tigecycline, 4 mg/L; sulbactam, 8 mg/L; rifampicin, 2 mg/L.10–12

In vitro bactericidal activities and synergistic effects were evaluated using time–kill assays. Eight imipenem-resistant isolates were randomly selected from the 43 clinical isolates.

Aliquots of A. baumannii strains were cultured at 35°C in tubes containing Trypticase soy broth, bacterial inoculum at an approximate size of 105 cfu/mL and antibiotics to be tested. The concentrations used for colistin and tigecycline were 1x, 4x and 8x MIC. For the evaluation of synergistic effects, imipenem and sulbactam were combined at 1x MIC each and colistin and rifampicin at 1x MIC each. At 0, 4, 8 and 24 h of incubation at 35°C, aliquots of 100 µL were obtained from each tube and 10-fold dilutions were made and cultured on Trypticase soy agar plates for 24 h at 35°C. The number of colonies formed was counted. Bactericidal activity was defined as a 3 log10 decrease in cfu/mL, and synergy was defined as a ≥2 log10 decrease in cfu/mL between the combination and its most active constituent.10,13

Antibiotic powders were obtained from their respective manufacturers: colistin methanesulphonate (X-gen Pharmaceuticals Inc., NY, USA), imipenem cilastatin (Choong-Wae Pharm, Seoul, Korea), sulbactam pivoxil (Keun-hwa Pharm, Seoul, Korea), tigecycline (Wyeth Co., MA, USA) and rifampicin (Donginbiotech Co, Seoul, Korea).

As for the above eight imipenem-resistant isolates, PCR was carried out to detect Ambler class B carbapenemase blaIMP-1, blaIMP-2, blaVIM-1 and blaVIM-2 and Ambler class D OXA-type carbapenemases. The primers used in this study are shown in Table 1. Each reaction mixture (20 µL) contained 1 µL of genomic DNA, 10 pmol of each primer, 1 U of Taq DNA polymerase, 0.25 mM dNTP, 10 mM Tris–HCl (pH 9.0), 40 mM KCl and 1.5 mM MgCl2. For Ambler class B carbapenemases, the mixture was heated to 94°C for 5 min and then subjected to 30 cycles of 94°C for 30 s, at each specific annealing temperature (Table 1) for 1 min and 72°C for 1 min, followed by a final extension for 7 min at 72°C. PCR reactions for OXA-type carbapenemases were carried out with heating to 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 52°C for 40 s and 72°C for 50 s, and final extension for 7 min at 72°C.


View this table:
[in this window]
[in a new window]

  		Table 1.. PCR primers, conditions and product sizes for the detection of carbapenemases among A. baumannii strains

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Transparency declarations
 References
 
MICs of imipenem, colistin, tigecycline, sulbactam and rifampicin are shown in Table 2. All the isolates were resistant to imipenem but susceptible to colistin and tigecycline.


View this table:
[in this window]
[in a new window]

  		Table 2.. Susceptibilities of 43 A. baumannii isolates

 
In time–kill studies, colistin showed bactericidal activity against all carbapenem-resistant strains at 4x and 8x MIC concentrations, usually beginning at 8 h after inoculation (Table 3). In comparison, tigecycline was not bactericidal, but showed good bacteriostatic activity against all carbapenem-resistant strains at any tested concentration (Table 4).


View this table:
[in this window]
[in a new window]

  		Table 3.. Time–kill results of colistin against carbapenem-resistant A. baumannii strains

 


View this table:
[in this window]
[in a new window]

  		Table 4.. Time–kill results of tigecycline against carbapenem-resistant A. baumannii strains

 
According to the time–kill synergy results, the combination of imipenem and sulbactam, both at a concentration of 1x MIC, appeared to be synergistic against most of the carbapenem-resistant strains after 4 h, excluding the second strain which failed to show synergism (Figure 1). Moreover, a bactericidal effect of imipenem/sulbactam was shown after 8 h. There was sustained synergistic inhibitory activity lasting for more than 24 h; bacterial colony counts were reduced by ≥2 log10 cfu/mL when compared with imipenem alone. Likewise, the combination of colistin and rifampicin was synergistic and bactericidal with a mean decrease of 4.29 ± 1.47 log10 cfu/mL after 8 h (Figure 2).


Figure 1
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1.. Time–kill curves showing synergistic effect of imipenem/sulbactam combination compared with imipenem alone against carbapenem-resistant A. baumannii strains; viable bacterial counts were reduced by at least ≥2 log10 cfu/mL compared with imipenem alone.

 


Figure 2
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2.. Time–kill curves showing synergistic effect of colistin/rifampicin combination compared with colistin alone against carbapenem-resistant A. baumannii strains; viable bacterial counts were reduced by at least ≥2 log10 cfu/mL compared with colistin alone.

 
According to the PCR results for carbapenemases, the blaOXA-51 ß-lactamase gene was detected in all eight strains, but PCR results for other OXA ß-lactamase (OXA-23, -24 and -58) and Ambler class B carbapenemase (blaIMP-1, blaIMP-2, blaVIM-1 and blaVIM-2) genes were all negative.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Transparency declarations
 References
 
Nowadays, the rate of carbapenem resistance among nosocomial A. baumannii isolates is high, particularly in ICUs. Increasing rates of carbapenem resistance have led to widespread use of colistin for the treatment of diverse infectious disease due to A. baumannii. However, colistin-resistant A. baumannii has been reported recently, and heterogeneous colistin resistance among multidrug-resistant isolates has been reported.14 Moreover, according to the results of this study, colistin showed a bactericidal effect only at a high concentration, above 4x MIC, which is clinically important considering the dose-dependent nephrotoxicity of colistin.15 Although some studies have reported clinical efficacy of colistin, the effects of combined antimicrobial agents or antimicrobial susceptibility were not considered in those studies.16,17 In contrast, Motaouakkil et al.18 reported a favourable clinical response to a colistin- and rifampicin-combined regimen against multidrug-resistant A. baumannii. Colistin is a concentration-dependent antimicrobial agent, and it is known to show only a modest post-antibiotic effect against A. baumannii at high concentrations.15 In this study, we could see the synergistic and bactericidal effects of the colistin- and rifampicin-combined regimen just at the MIC level, which was sustained for ≥24 h.

Synergistic activity of imipenem and sulbactam against A. baumannii has been reported previously, and included two imipenem-resistant strains.5 In the present study, we again showed the synergistic and bactericidal effects of this regimen even against carbapenem-resistant strains. This synergistic activity might be related to the concentration of bacterial penicillin-binding protein 2.5 However, there is a chance that those effects can differ according to the resistance mechanisms; serine-ß-lactamase, metallo-ß-lactamase (IMP and VIM type), efflux pumps and outer membrane protein change.19 Especially, VIM-2 ß-lactamase in A. baumannii from Korea showed significantly high level resistance to carbapenem. Actually, the imipenem/sulbactam combination failed to show synergism against one of the carbapenem-resistant strains in this study, which had a higher MIC of imipenem (≥64 mg/L) when compared with the strains. Likewise, colistin did not show a bactericidal effect in some strains (strains 1, 3, 5, 7 and 8) at 4x and 8x MIC concentrations after 4 h from bacterial inoculation. Although we could not find a difference in carbapenemase production among study strains, the therapeutic effectiveness of antibiotic regimens might differ according to the diverse mechanisms of antimicrobial resistance; further studies are required.

Regarding tigecycline, it is very active against Acinetobacter spp. (generally >90% susceptibility, with MIC <8 mg/L).12 Previously, Pachon-Ibanez et al.9 reported that tigecycline has good in vitro bacteriostatic activity against A. baumannii, including one imipenem-intermediate and one imipenem-resistant strain. Likewise in this study, tigecycline showed favourable bacteriostatic activity against all carbapenem-resistant strains even at the MIC level, which was sustained for more than 24 h. Although tigecycline has not been registered in many countries yet, it is thought to be clinically useful, having good tissue penetration. Tigecycline-based combination regimens are expected to be investigated and applied clinically.

In conclusion, imipenem/sulbactam combination, colistin and tigecycline showed good in vitro activities against carbapenem-resistant A. baumannii isolates. Even though colistin is bactericidal against carbapenem-resistant Acinetobacter spp., the colistin/rifampicin combination is more warranted in order to be certain.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
References
 
None to declare.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
1 . Ayats J, Corbella X, Ardanuy C, et al. Epidemiological significance of cutaneous, pharyngeal, and digestive tract colonization by multiresistant Acinetobacter baumannii in ICU patients. J Hosp Infect (1997) 37:287–95.[CrossRef][Web of Science][Medline]

2 . Jawad A, Heritage J, Snelling AM, et al. Influence of relative humidity and suspending menstrua on survival of Acinetobacter spp. on dry surfaces. J Clin Microbiol (1996) 34:2881–7.[Abstract]

3 . Bou G, Cervero G, Dominguez MA, et al. Characterization of a nosocomial outbreak caused by a multiresistant Acinetobacter baumannii strain with a carbapenem-hydrolyzing enzyme: high-level carbapenem resistance in A. baumannii is not due solely to the presence of beta-lactamases. J Clin Microbiol (2000) 38:3299–305.[Abstract/Free Full Text]

4 . Cisneros JM, Reyes MJ, Pachon J, et al. Bacteremia due to Acinetobacter baumannii: epidemiology, clinical findings, and prognostic features. Clin Infect Dis (1996) 22:1026–32.[Web of Science][Medline]

5 . Choi JY, Park YS, Cho CH, et al. Synergic in-vitro activity of imipenem and sulbactam against Acinetobacter baumannii. Clin Microbiol Infect (2004) 10:1098–101.[CrossRef][Web of Science][Medline]

6 . Kiffer CR, Sampaio JL, Sinto S, et al. In vitro synergy test of meropenem and sulbactam against clinical isolates of Acinetobacter baumannii. Diagn Microbiol Infect Dis (2005) 52:317–22.[CrossRef][Web of Science][Medline]

7 . Falagas ME, Kasiakou SK. Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis (2005) 40:1333–41.[CrossRef][Web of Science][Medline]

8 . Petrosillo N, Chinello P, Proietti MF, et al. Combined colistin and rifampicin therapy for carbapenem-resistant Acinetobacter baumannii infections: clinical outcome and adverse events. Clin Microbiol Infect (2005) 11:682–3.[CrossRef][Web of Science][Medline]

9 . Pachon-Ibanez ME, Jimenez-Mejias ME, Pichardo C, et al. Activity of tigecycline (GAR-936) against Acinetobacter baumannii strains, including those resistant to imipenem. Antimicrob Agents Chemother (2004) 48:4479–81.[Abstract/Free Full Text]

10 . National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition: Approved Standard M7-A6 (2003) Wayne, PA, USA: NCCLS.

11 . Giamarellos-Bourboulis EJ, Xirouchaki E, Giamarellou H. Interactions of colistin and rifampin on multidrug-resistant Acinetobacter baumannii. Diagn Microbiol Infect Dis (2001) 40:117–20.[CrossRef][Web of Science][Medline]

12 . Pankey GA. Tigecycline. J Antimicrob Chemother (2005) 56:470–80.[Abstract/Free Full Text]

13 . Montero A, Ariza J, Corbella X, et al. Antibiotic combinations for serious infections caused by carbapenem-resistant Acinetobacter baumannii in a mouse pneumonia model. J Antimicrob Chemother (2004) 54:1085–91.[Abstract/Free Full Text]

14 . Li J, Rayner CR, Nation RL, et al. Heteroresistance to colistin in multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother (2006) 50:2946–50.[Abstract/Free Full Text]

15 . Li J, Nation RL, Turnidge JD, et al. Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. Lancet Infect Dis (2006) 6:589–601.[CrossRef][Web of Science][Medline]

16 . Falagas ME, Kasiakou SK, Kofteridis DP, et al. Effectiveness and nephrotoxicity of intravenous colistin for treatment of patients with infections due to polymyxin-only-susceptible (POS) gram-negative bacteria. Eur J Clin Microbiol Infect Dis (2006) 25:596–9.[CrossRef][Web of Science][Medline]

17 . Reina R, Estenssoro E, Saenz G, et al. Safety and efficacy of colistin in Acinetobacter and Pseudomonas infections: a prospective cohort study. Intensive Care Med (2005) 31:1058–65.[CrossRef][Web of Science][Medline]

18 . Motaouakkil S, Charra B, Hachimi A, et al. Colistin and rifampicin in the treatment of nosocomial infections from multiresistant Acinetobacter baumannii. J Infect (2006) 53:274–8.[CrossRef][Web of Science][Medline]

19 . Bonomo RA, Szabo D. Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa. Clin Infect Dis (2006) 43(Suppl_2):49–56.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Clin. Microbiol. Rev.Home page
D. Landman, C. Georgescu, D. A. Martin, and J. Quale
Polymyxins Revisited
Clin. Microbiol. Rev., July 1, 2008; 21(3): 449 - 465.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Microbiol. Rev.Home page
A. Y. Peleg, H. Seifert, and D. L. Paterson
Acinetobacter baumannii: Emergence of a Successful Pathogen
Clin. Microbiol. Rev., July 1, 2008; 21(3): 538 - 582.
[Abstract] [Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
D. E. Karageorgopoulos, T. Kelesidis, I. Kelesidis, and M. E. Falagas
Tigecycline for the treatment of multidrug-resistant (including carbapenem-resistant) Acinetobacter infections: a review of the scientific evidence
J. Antimicrob. Chemother., July 1, 2008; 62(1): 45 - 55.
[Abstract] [Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
H. A. Lopardo
Acinetobacter spp. and time-kill studies
J. Antimicrob. Chemother., February 1, 2008; 61(2): 464 - 464.
[Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
J. Y. Song and H. J. Cheong
Comment on: Acinetobacter spp. and time-kill studies
J. Antimicrob. Chemother., February 1, 2008; 61(2): 465 - 466.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
60/2/317    most recent
dkm136v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Song, J. Y.
Right arrow Articles by Cheong, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Song, J. Y.
Right arrow Articles by Cheong, H. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?