In recent years, Acinetobacter strains have emerged as one of the most important nosocomial pathogens, especially in patients admitted to an intensive care unit (ICU). The progressively increasing antibiotic resistance against A. baumannii is now a major problem in our country as it is throughout the world. This resistance against A.baumannii has increased and led clinicians to find alternative antibiotics or antibiotic combinations. In the present study, it is aimed to evaluate the interaction between colistinrifampicin, colistin-imipenem, tigecycline-rifampicin and tigecycline-imipenem antibiotic combinations using microdilution checkerboard and E-test methods against ten multidrug resistant A. baumannii strains. Since A. baumannii strains have become frequently observed as an infection factor and since antimicrobial resistance rates have increased, there should be newly developed drugs for better treatments. In this study, 50 A. baumannii strains were isolated from various clinical specimens between June 2005 and September 2009 in Celal Bayar University, Faculty of Medicine, Department of Microbiology and Clinical Microbiology, and in Bacteriology Laboratory. Isolation and identification procedures were performed by using conventional biochemical tests as well as by BBL Crystal GN; N/F ID (Becton Dickinson, USA) or Phoenix 100 BD systems (Becton Dickinson, USA). The antibiotic susceptibilities of strains were investigated by using the disk diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute. According to our in vitro study results, the checkerboard method, which was used to examine the synergy between colistin-rifampicin and colistin-imipenem, showed 80% synergistic activity. Tigecycline-imipenem combination had lowest synergetic (10%) efficiency and highest antagonistic effect (30%). The consistency between checkerboard and E-test methods was 52,5% (range 10-70%). Further comparison studies of the E-test synergy technique with the checkerboard and time-kill methods are warranted.
Acinetobacter has been detected as one of the most common nosocomial infections particularly in intensive care unit (ICU) [1,2]. Recently, there has been an increasing resistance against antimicrobial agents that are commonly used for infections caused by Acinetobacter baumannii strains and this resistance has given rise to an important health issue in Turkey as it is the case throughout the world. New options of drugs should be searched and established since A. baumannii infections have become more and more diverse and the antimicrobial agent resistance has increased [1-3].
There have been new studies which try to develop different treatment protocols because of the increment in the resistance rates of microorganisms. Combination therapies and the production of new antibiotics are some of the focuses of these studies. Antimicrobial drug combinations are used in order to obtain a broad spectrum, prevent the development of resistant strains, minimize the toxicity, and obtain a synergetic effect between two drugs [4,5].
In this study, the aims are to examine the in vitro effect of different antibiotic combinations on A.baumannii isolates using synergistic tests such as checkerboard and E-test methods and to assess the compatibility between these two methods.
Materials and Methods
This study was approved by Celal Bayar University, Dean of the Faculty of Medicine, and Ethics Committee with Decision No. 390 in the scientific meeting which was held on 06.18.2009.
This study was conducted in Celal Bayar University, School of Medicine, Department of Medical Microbiology, and Laboratory of Bacteriology. 50 A.baumannii strains were isolated from various clinical samples between 2005 and 2009. Only one clinical isolate from each patient was included in the study. In order to evaluate these tests, we isolated 10 A. baumannii strains, which were isolated from different patients, which had different sensitivity profiles, and which were resistant against at least three antibiotic groups.
Bacteria were identified at the species level by using BBL Crystal Enteric/Nonfermenter ID Kit (Becton Dickinson, USA) or Phoenix 100 BD system (Becton Dickinson, USA). The sensitivities of A.baumannii strains were determined using disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) criteria.
The efficiency of colistin-rifampicin, colistin-imipenem, rifampicin-tigecycline, tigecycline-imipenem combinations were examined in 10 A. baumannii strains which had multiple drug resistance determined using checkerboard and E-test techniques. The consistency between these two techniques was compared. Evaluations were performed with reference to checkerboard method 6.
Interaction between antibiotic combinations was examined using checkerboard method for each antibiotic and strain whose minimum inhibitory concentration (MIC) values were determined using broth microdilution method. In this study, colistin-imipenem, colistin-rifampicin, imipenem-tigecycline and tigecycline-rifampin combinations were examined.
Fractional inhibitory concentration (FIC) values of antibiotics were detected using the first horizontal row (A row) for the first antibiotic in the combination and the first vertical row (number 1 column) for the second antibiotic in the combination. The first antibiotic was diluted for four times and each dilution was put in the microdilution plate as starting from the number 1 column till the 8th column. In case of four times dilutions of the second antibiotic, each dilution was put in first 8 rows in the micro dilution plate.
These combinations were also examined using E-test method. FIC index was used in both methods in order to determine the effectiveness of combinations and interactions between antibiotics were recorded as synergy, indifference and antagonism [7]. FIC index was calculated as:
FIC A=MIC value of A in the presence of B/MIC value of A alone
FIC B=MIC value of B in the presence of A/MIC value of B alone
S FIC index=FIC A+ FIC B
Evaluation:
S FIC index = 0.5 : synergy (4 times decrease)
S FIC index = 1 and 4 = : indifference (no interaction)
S FIC index >4 : antagonism
Statistical analyses
Sommer' D, Chi-square, Mann-Whitney-U, Student's t-test and Logistic Regression analyses were performed by using SPSS 13.0 software. ?p? values less than 0.05 (p<0.05) were accepted as statistically significant.
Statistical analyses of data obtained from the results of in vitro interactions of colistin-imipenem, colistin-rifampicin, imipenem-tigecycline, tigecycline-rifampin combinations by using Fisher?s chi-square test with the help of SPSS (SPSS Incorporated, Chicago, USA) program.
Results
In this study, out of 50 Acinetobacter strains, 27 (54%) of them were isolated from anesthesia ICU samples, 8 (16%) of them were isolated from pulmonary unit samples, 7 (14%) of them were isolated from neurology unit samples, 4 (8%) of them were isolated from general surgery unit samples, and 4 (8%) of them were isolated from brain surgery unit samples.
Acinetobacter was isolated from blood [39 (78%)], respiratory samples [6 (12%)] or various clinical samples [5 (10%)]. According to the antibiogram results of A. baumannii strains that were obtained with the help of disc diffusion method, the maximum resistance was observed against ceftriaxone whereas the minimum resistance was observed against netilmycin. The rate of the resistance against imipenem was 70%.
The efficiency of antibiotic combinations was examined for 10 A. baumannii strains which had multi drug resistance. When the microdilution of strains was primarily examined, all strains were found to be colistin and tigecycline sensitive and their MIC50 values were detected as 0.07 µg/ml and 0.7 µg/ml; respectively. Out of 10 strains, 8 were found to be imipenem resistant.
When efficiencies of drug combinations were assessed using checkerboard method, it was detected that the highest synergy (80%) was found for combinations of colistin with imipenem and rifampicin (Figure 1). Tigecycline-imipenem pair was detected as a combination which had lowest synergetic (10%) efficiency and highest antagonistic effect (30%) (Table 1).
When the consistencies of the techniques (checkerboard and Etest) were examined, the highest sensitivity rate (70%) was detected for tigecycline combinations. Furthermore, in cases of colistin-imipenem combination, 8 of the strains had synergistic interaction with these antibiotics according to the results of checkerboard test whereas they were shown as indifference according to E-test results. The consistency was calculated as 10% (Figure 2, Table 2).
Table 2: The consistency rates between checkerboard and E-test methods (n=10)*. *The interpretation of the K values specified as: (-0.3) (+0.3): inconsistent +0.3) (+0.5) : weak positive, +0.5 (+0.7): moderate positive, >0.7: strong positive. **Kappa values could not be calculated since the sub efficiency categories of diagnostic tests were not paired.
The percentages of liquid values were failed to be compared among combinations due to the fact that the assumption on the expected values of chi-square test did not hold. Therefore, the analysis was performed by colistin and tigecycline combinations. When the efficiencies of the colistin and tigecycline combinations (with imipenem and rifampicin), the combination efficiency of colistin was found two times synergetic compared to tigecycline (p = 0.05) (Table 3).
Discussion
A. baumannii strains have been isolated frequently from nosocomial pathogens for the last 15 years [8]. These strains lead to infections with high mortality and their high resistance against various antibiotics causes a severe issue [9]. Increment in the isolation of multiple drug resistant strains throughout the world as well as in Turkey and increasing resistance against antibiotics decrease the treatment options of clinicians in inpatients who were suspected of suffering with A. baumannii [10,11]. It is suggested to use antibiotic combinations in order to both ensure the success and prevent the resistance development in the treatment of the infections due to the multidrug resistant A. baumanni strains [12].
When colistin was in combination with rifampicin, 8 of 10 stains (80%) had synergetic interaction with this combination of antibiotics. Hogg et al.[13] performed a study in which they used checkerboard method with their isolates, they detected synergetic association between antibiotics in 11 of the 13 strains. Timurkaynak et al. [14] showed that all strains (in 25 of the strains, 100%) had synergetic interaction with antibiotics. According to the study of Giamarellos et al. [15] in which they used time-kill technique, they indicated in 2001 that 39 strains showed (66.7%) synergy in the 24th hours for colistinrifampicin combinations. Tripodi et al. [16] also used time-kill technique and they showed the 100% synergy of 9 isolates with colistin-rifampicin combinations in 2007. In Turkey, there was a 60% synergy using E-test for colistin-rifampicin combinations in A.baumannii strains isolated from 25 different patients [17]. As a result, our results are consistent with all of these findings, and it has been decided that colistin-rifampicin combinations are efficient and convenient for the treatment of A.baumannii infection.
There was a synergy in 8 strains (80%) for colistin-imipenem combinations when we used checkerboard method. According to the results of study performed by Pongpech et al. [18] in 2010 in Thailand, they determined that colistin-imipenem combinations were 100% synergistic when they applied the same technique to 30 isolates. In a study which was conducted by Haddad et al. [5] between 1999 and 2003 in New York with 10 A.baumannii isolates that were resistant against all routinely used antibiotics, they used E-test method and detected a 50% synergy for colistin-imipenem combinations. High synergy rates detected in bacterial strains including imipenem resistant strains in all of these studies and particularly in our study let us think that colistin-imipenem combinations are efficient enough for the treatment.
There was a synergy in 6 strains (60%) for the tigecyclinerifampicin combination when we used checkerboard technique. Petersen et al. [19] detected a synergy only in one strain out of 9 strains for tigecycline-rifampicin combination in 2005 using checkerboard method. Dizbay et al. [17] specified that they detected 12% synergy for tigecycline-rifampicin combination using E-test method. In our study, we detected higher synergy rates in this combination compared to other studies.
There was a 10% synergy in one strain for tigecyclineimipenem combinations. Principe et al. [20] performed a study with 22 isolates using checkerboard technique, and they showed 8.3% synergy for tigecycline-imipenem combination. To sum up, tigecycline-imipenem combinations are not effective option for the treatment.
The rate of synergistic interaction is greater in the colistin combinations compared to the tigesiklin combinations; this result was statistically significant (P<0.05).
E-test method was also used in addition to checkerboard method in order to examine the antibiotic combinations and the consistency between these methods.
When the consistency of efficiencies which were detected by using E-test method was also examined using checkerboard method, the mean consistency was found to be 52.5% (10%-70%). Bonapace et al. [21] compared the consistency between E-test and checkerboard methods in 10 A. baumanii isolates for trovafloxacin or tobramycin-cefepime or piperacillin. They found the consistency between time kill method with E-test and checkerboard methods as 72% and 51%, respectively.
Conclusively, multi-drug resistance (MDR) A.baumanii strains are important problems in hospitals and treatment options are limited. Due to high synergy rates that were determined for colistin-imipenem, colistin-rifampicin and tigecyclinerifampicin combinations, it has been thought that these combinations can be efficiently used in the treatment of MDR A.baumanii infections. However, it should be noted that some antimicrobial agents in combination with colistin lead to decrease in the activity and create the antagonistic effect. Further studies should be performed since in vitro experiences are limited for colistin and tigecycline. Since A. baumannii strains are resistant against majority of antibiotics, surveillance results should be considered when we select empirical antibiotic therapy that will be applied to patients in critical condition.
Acknowledgement
This study was supported by Celal Bayar University, Scientific Research Projects (Project no: 2009-069).
References
Bergogne-BerezinE, Towner KJ. Acinetobacter spp. as nosocomial pathogens: Microbiological, clinical and epidemiological features. ClinMicrobiol Rev 1996; 9: 148-165.
Roberts SA, Findlay R, Lang SDR. Investigation of an outbreak of multi-drug resistant Acinetobacterbaumannii in an intensive care burns unit. Journal of Hospital Infection 2001; 48: 228-32.
Chastre J. Infections due to Acinetobacterbaumannii in the ICU. Seminars in Respiratory and Critical Care Medicine. 2003; 24: 69-77.
Moody J. Susceptibility tests to evaluate synergism. In: Isenberg HD, editor. Clinical microbiology procedures handbook. Washington, DC: ASM Press; 2004. pp. 5.12.1-23.
Haddad FA, Van Horn K, Carbonaro C, Aguero-Rosenfeld M, Wormser GP. Evaluation antibiotic combinations against multidrug- resistant Acinetobacterbaumannii using E-test. Eur J ClinMicrobiol Infect Dis 2005; 24: 577-9.
Clinical and Laboratory Standards Institute. 2008Â Practice Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard M2-A9 ve M7-A7.
Gulay Z. In-Vitro Activity Meter Tests of the Antibiotic Combination Standardization Meeting of Antimicrobial Susceptibility Testing Congress book 1997: 85-100.
Saltoglu N. Acinetobacterbaumannii infections and treatment XIII. Turkish Congress of Clinical Microbiology and Infectious Diseases 14-18 March 2007, Congress book 2007: 204-7.
Pachon Ibanez ME, Jimenez Mejias ME, Pichardo C, Llanos AC, Pachon J. Activity of tigecyline (GAR-936) against Acinetobacterbaumannii strains, including those resistant to imipenem. Antimicrob Agent Chemother 2004; 48: 4479-81.
Yavuz MT, Sahin I, Behcet M, Ozturk E, Kaya D: Antibiotic Susceptibility of Acinetobacterbaumannii Strains Isolated from Various Clinical Samples, Journal of Ankem2006; 20: 107-10.
11.Cetin ES, Kaya S, Tetik T, Aridogan BC. Distribution and Antibiotic Susceptibility of Acinetobacterbaumannii Strains Isolated from Clinical Samples Journal of Ankem 2006; 20: 202-5.
Marques MB, Brookings ES, Moser SA, Sonke PM, Waites KB. Comparative in vitro antimicrobial susceptibilities of nosocomial isolates of Acinetobacterbaumannii and synergistic activities of nine antimicrobial combinations.Antimicrob Agents Chemother 1997; 41: 881-5.
Hogg GM, Barr JG, Webb CH. In-vitroactivity of the combination of colistin and rifampicin against multidrug-resistant strains of Acinetobacterbaumannii. J AntimicrobChemother 1998; 41:494-5.
Timurkaynak F, Can F, Azap OK, Demirbilek M, Arslan H, Karaman SO. In vitro activities of non-traditional antimicrobial saloneor in combination against multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacterbaumannii isolated from intensive care units. Int J Antimicrob Agents. 2006; 27(3): 224-8.
Giamarellos-Bourboulis EJ, Xirouchaki E, GiamarellouH.Interactions of colistin and rifampin on multidrug-resistant Acinetobacterbaumannii. DiagnMicrobiol Infect Dis. 2001; 40(3): 117-20.
Tripodi MF, Durante-Mangoni E, Fortunato R, Utili R, Zarrilli R. Comparative activities of colistin, rifampicin, imipenem and sulbactam/ampicillin alone or in combination against epidemic multidrug-resistant Acinetobacterbaumannii isolates producing OXA-58 carbapenemases. Int J AntimicrobAgents. 2007; 30(6): 537-40.
Dizbay M, Tozlu DK, Ozdemir K, Arman D. "Extensive Drug - Resistant (XDR)" Acinetobacterbaumannii isolates Tigecycline and Colistin Combination of In- Vitro Synergistic Activity Journal of Ankem 2009; 23(Add 1): 1.
PongpechP,Amornnopparattanakul S, Panapakdee S, et al. Antibacterial Activity of Carbapenem-Based Combinations Againts Multidrug-Resistant Acinetobacterbaumannii. J MedAssocThai 2010; 93 (2): 161-71.
Petersen PJ, Labthavikul P, Jones CH, Patricia A Invitro antibacterial activities of tigecycline in combination with other antimicrobial agents determined by chequerboard and time-kill kinetic analysis. Bradford Journal of AntimicrobialChemotherapy. 2006; 57(3): 573?6.
Principe L, D'Arezzo S, Capone A, Nicola Petrosillo and PaoloViscaInvitro activity of tigecycline in combination with various antimicrobials against multi drug resistant Acinetobacterbaumannii Annals of Clinical Microbiology and Antimicrobial. 2009; 8:18.
Bonapece CR, White RL, Friedrich LV, Bosso JA. Evaluation of antibiotic synergy against Acinetobacterbaumannii: A comparison with Etest, time-kill and checkerboard methods. DiagnMicrobiol Infect Dis 2000; 38: 43-50.
