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Vol. 59, no 1, 2010, 55-60

Evaluation of in vitro Activities of Tigecycline and Various Antibiotics BETIL OZHAK-BAYSAN1, GOZDE ONGUT1, DILARA OGUNC1, FILIZ GUNSEREN2*, NEVGUN SEPIN-OZEN1, FERYAL OZTURK3, ORHAN CEM AKTEPE4 and MERAL GULTEKIN1 1 Akdeniz University Faculty of Medicine, Department of Medical Microbiology, Antalya, Turkey 2 Akdeniz University Faculty of Medicine, Department of Clinical Microbiology 4 Afyon Kocatepe University Faculty of Medicine, Department of Medical Microbiology, Afyon, Turkey Received 2 July 2009, revised 14 December 2009, accepted 20 December 2009 Brucellosis is a zoonosis with a worldwide distribution and remains a significant public health problem mainly in the developing world.
In this study we evaluated the in vitro activities and synergistic effects of antibiotic combinations against blood culture isolates of Brucella spp. In vitro susceptibilities of 76 blood culture isolates of Brucella melitensis and one blood culture isolate of Brucella abortus to doxycycline, streptomycin, gentamicin, trimethoprim-sulfamethoxazole, moxifloxacin, rifampin, ciprofloxacin, and tigecycline were exam- ined by Etest method. For 37 patients with Brucella spp. isolates (36 B. melitensis, 1 B. abortus), antibiotic combinations used for treatment were identified with those tested in vitro for synergy using Etest method. Trimethoprim-sulfamethoxazole and tigecycline were the most active of the compounds tested with MIC value of 0.094 mg/l. Among antibiotic combinations only streptomycin-rifampin combination was synergistic for one Brucella spp. isolate. The other antibiotic combinations revealed antagonistic or indifferent activity. Complete clinical response was achieved in all patients. Further studies are required to determine the correlation between the antimicrobial susceptibility and synergy test results with the clinical course of patients. Brucellosis can be adequately treated with existing regimens in our region.
K e y w o r d s: brucellosis, Etest for susceptibility, synergy testing, susceptibility, tigecycline a six-week regimen containing doxycycline plus rifam- pin or doxycycline plus streptomycin for the treatment Human brucellosis is a multisystem disease that of brucellosis (World Health Organization, 1986).
may involve any organ or system. Successful treat- Most cases respond to standard regimens, with fewer ment of brucellosis requires combined antimicrobials than 10% manifesting relapses, usually in the first with synergy and good intracellular penetration and year after treatment. Treatment failures are of concern should be prolonged (Young, 2005). Recently, Cen- especially in patients with serious or complicated ters for Disease Control (CDC) listed Brucella as brucellosis such as spondylitis, neurobrucellosis and a potential biological weapon and the European Union endocarditis (Bossi et al., 2004; Pappas et al., 2006b).
(EU) released Bichat guidelines for clinical and bio- Clinical Brucella isolates are generally susceptible terrorism related events of brucellosis (Bossi et al., to the antibiotic combinations recommended by WHO, 2004; Yagupsky and Baron, 2005). Moreover, Clinical however the results of in vitro susceptibility tests do and Laboratory Standards Institute (CLSI, formerly not always predict the clinical outcome. A number of the National Committee for Clinical Laboratory Stan- methods used for detection of in vitro synergy between dards [NCCLS]) released the first MIC interpretive antibiotics have been described (White et al., 1996).
standards for Brucella spp. in January 2006 (Clinical The Etest method has been used both for in vitro sus- and Laboratory Standards Institute, 2006). In 1986, ceptibility and synergy testing of Brucella spp. as it is the World Health Organization (WHO) recommended less time consuming and less labor intensive. However * Corresponding author: F. Gunseren, Akdeniz University, Faculty of Medicine, Department of Clinical Microbiology and Infectious Diseases, Antalya, Turkey; phone (+90) 2422496762; fax. (+90) 2422272535; e-mail: filizg@akdeniz.edu.tr synergy testing methods are not yet standardized (Gur The plates were read after 48 h incubation at 35°C under aerobic conditions according to manufacturer’s In this study, we evaluated the in vitro activities recommendations. MIC values of streptomycin (STR), and synergistic effects of antibiotic combinations doxycycline (DOX), rifampin (RIF), gentamicin (GEN), against Brucella spp. using Etest method and compared trimethoprim-sulfamethoxazole (SXT), ciprofloxacin (CIP), moxifloxacin (MXF), and tigecycline (TIG) Synergy testing. Rifampin-doxycycline, strepto- mycin-doxycycline, streptomycin-rifampin, trimetho- prim-sulfamethoxazole-rifampin, and gentamicin- doxycycline combinations were tested against 28 (27 B. melitensis, 1 B. abortus), two, one, five and A total of 77 Brucella spp. strains isolated at one Brucella spp. isolates, respectively.
Akdeniz University Hospital and Afyon Kocatepe Synergy tests were performed on Mueller-Hinton University Hospital Central Laboratories between agar supplemented with 5% sheep blood by Etest.
September 2001 and June 2006 were tested. The col- First, strip A was placed on the inoculated agar sur- lection included 76 strains of Brucella melitensis and face and left for one hour. The strip’s position was one strain of B. abortus. All isolates were obtained marked on the back of the plate. Strip A was removed from blood using BACTEC 9240 (Becton Dickinson, and strip B was placed on the imprint of A, vertically U.K.) blood culture system. The isolates were collected transposed so MICA and MICB overlap at the same at the individual study sites and were sent to Akdeniz position. Strip B was left on the agar plate and incu- University Hospital Central Laboratory for identifi- bated for 48 h. To evaluate the effect of the combina- cation and susceptibility testing and were stored in tions, the fractional inhibitory concentration (FIC) stock cultures at –20°C until used. The isolates were was calculated for each antibiotic in each combina- identified to the species-level using standard classifi- tion. The following formulas were used to calculate cation tests, including Gram stain, growth character- the FIC index: FIC of drug A = MIC of drug A in istics, oxidase activity, urease activity, H2S production, combination/ MIC of drug A alone. FIC of drug dye sensitivity such as basic fuchsin and thionin and B = MIC of drug B in combination/MIC of drug B seroagglutination. Identification was confirmed using alone, and FIC index = FIC of drug A+FIC of drug B.
a semi-nested real-time PCR assay targeting a 223-bp Synergy was defined as an FIC index of ≤0.5. Indif- fragment of gene encoding the cell surface protein ference was defined as an FIC index of >0.5 but of (BCSP31), specific for the Brucella genus. A class II ≤4. Antagonism was defined as an FIC index of >4.
biological safety cabinet was used to perform the tests.
Quality Control. Quality control isolates, Escheri- In the first part of our study, we investigated the chia coli ATCC 25922 and Streptococcus pneumoniae minimum inhibitory concentration (MIC) values of ATCC 49619 were included in all runs.
antibiotics for all isolates from the two centers by Etest method. In the second part, the patient charts of 37 subjects with Brucella spp. isolates (36 B. meliten- sis, 1 B. abortus) of the 46 patients from Akdeniz University Hospital, were taken for further assessment.
MIC ranges, MIC50 and MIC90 values of the anti- Demographic data, treatment protocol, as well as biotics for the isolates are shown in Table I. Accord- clinical outcome were evaluated, retrospectively.
ing to their MIC90 values, SXT and TIG were the most Each patient’s antibiotic combinations used for treat- active compounds against all Brucella spp. isolates.
ment were identified with those tested in vitro for Among quinolones, MXF demonstrated a lower MIC synergy using E-test method. The results were com- (MIC50: 0.125 mg/l, MIC90: 0.25 mg/l) than that of CIP (MIC50: 0.19 mg/l, MIC90: 0.38 mg/l). MIC range The study was approved by Akdeniz University of RIF was between 0.064–3 mg/l and a MIC90 value Medical Faculty Ethical Committee with B.30.2.AKD.
of 1.5 mg/l for RIF was found. DOX, STR and GEN 0.01.00.00/Etik/483 protocol number.
had good activities against all isolates with a MIC90 Antimicrobial susceptibility tests. The Etest value below standard CLSI breakpoints. A complete method was performed according to manufacturer’s clinical response was achieved in all patients and no instructions. An inoculum equal to a 1 McFarland relapses were recorded in the patients’ charts.
turbidity standard was prepared from each Brucella Synergy test results of antibiotic combinations isolate and 10 µl of the suspension was inoculated against Brucella spp. isolates (36 B. melitensis, onto Mueller-Hinton agar plates with 5% sheep blood 1 B. abortus) from 37 patients are shown in Table II.
and Etest strips were applied to the inoculated surface.
In our study synergy tests were performed using Etest Activity of antibiotics against Brucella sp.
MIC ranges, MIC and MIC values of antibiotics against Brucella spp. isolates (n = 77) * MIC values and CLSI breakpoints are expressed in mg/l.
Synergy test results of antibiotic combinations against Brucella spp. isolates * Twenty-four isolates of B. melitensis and one isolate of B. abortus RIF, rifampin; GEN, gentamicin; SXT, trimethoprim-sulfamethoxazole; STR, streptomycin; DOX, doxycycline.
method. The combination of RIF-DOX yielded anta- MIC90 values of previous studies ranging from 2 to gonism against 89.3% and indifference against 10.7% 4 mg/l (Lopez-Merino et al., 2004; Turkmani et al., of the isolates. The STR-DOX combination exhibited 2006; Khan et al., 1989; Rubinstein et al., 1991; Akova antagonism against all of the isolates tested. The STR- et al., 1999). However, DOX was found to have RIF combination was tested in one isolate and was a higher MIC90 value when compared to those previ- found synergistic. For the combination of SXT-RIF ously reported (Baykam et al., 2004; Akova et al., we found antagonism against 60%, and indifference 1999; Bodur et al., 2003). Several other studies found against 40% of the isolates. The GEN-DOX combina- even higher MIC90 values for DOX for Brucella iso- tion exhibited indifference against one tested isolate lates (Trujillano-Martin et al., 1999; Yamazhan et al., (Table II). Demographic data, clinical characteristics of 2005). GEN had a good activity against all isolates patients and synergy test results of individual patient’s with a MIC90 value below standard CLSI breakpoint.
antibiotic combinations are presented in Table III.
This finding agrees with data from previous studies (Qadri and Ueno, 1993; Turkmani et al., 2006). RIF breakpoints have not been established for Brucella spp. by CLSI. MIC of RIF for our isolates ranges be- tween 0.064–3 mg/l. MIC values of RIF ranging from As shown in several earlier studies, Brucella spp.
0.02 to 16 mg/l have already been reported (Bosch are generally susceptible to traditional drugs used for et al., 1986; Qadri and Ueno, 1993; Lopez-Merino the treatment of brucellosis (Bosch et al., 1986; Qadri et al., 2004; Turkmani et al., 2006; Khan et al., 1986; and Ueno, 1993; Baykam et al., 2004). However Bodur et al., 2003; Al-Orainey et al., 1991). MIC strains with decreased susceptibility to RIF and STR values of SXT ranging from 0.032 to >32 mg/l have have been reported (Baykam et al., 2004; Lopez- been reported previously (Bosch et al., 1986; Qadri Merino et al., 2004). In our study all strains were sus- and Ueno, 1993; Baykam et al., 2004; Lopez-Merino ceptible to STR (MIC90: 1 mg/l) and DOX (MIC90: et al., 2004; Turkmani et al., 2006; Khan et al., 1986; 0.125 mg/l) according to CLSI interpretive breakpoints Bodur et al., 2003; Al-Orainey et al., 1991). CLSI of ≤8 mg/l and ≤1 mg/l, respectively. Furthermore, determined interpretive breakpoints for SXT in 2006 MIC90 value of STR in our study is lower than the (Clinical and Laboratory Standards Institute, 2006).
Characteristics of brucellosis patients and synergy test results of antibiotic combinations a F: female, M: male; bA: antagonism, I: indifference, S: synergism.
RIF: rifampin, GEN: gentamicin, SXT: trimethoprim-sulfamethoxazole, STR: streptomycin, DOX: doxycycline.
All isolates in our study were susceptible to SXT with value of 0.38 mg/l for CIP which is consistent with MIC values ranging from 0.016 to 0.125 mg/l.
data from previous reports (Qadri and Ueno, 1993; An alternative approach may be the use of quinolo- Lopez-Merino et al., 2004). Our MIC90 value for MXF nes for the treatment of brucellosis. Ciprofloxacin and (0.25 mg/l) was lower than those previously reported ofloxacin have been used clinically for the treatment (0.5–8 mg/l) (Lopez-Merino et al., 2004; Trujillano- of human brucellosis in various combinations. In vitro Martin et al., 1999; Yamazhan et al., 2005).
susceptibility testing results exist for several other Tigecycline is a broad-spectrum glycylcycline anti- quinolones including gatifloxacin, levofloxacin, nor- microbial agent and seems to have excellent in vitro floxacin and pefloxacin (Falagas and Bliziotis, 2006; activity against many Gram-positive and Gram-nega- Pappas et al., 2006a). In our study, we found a MIC90 tive microorganisms (Nathwani, 2005). Pappas et al.
Activity of antibiotics against Brucella sp.
suggested a shorter duration of treatment with the use for the combinations used could not be evaluated ap- of TIG as a single agent (Pappas et al., 2005). MIC90 propriately because of very low MICs of tetracycline value of TIG was 0.094 mg/l for our isolates. There and DOX for all the isolates. Similarly, all our iso- are conflicting data about the MIC of TIG against lates chosen for synergy testing had very low MICs Brucella in Turkey. In a study of Turan et al. the MIC of DOX, STR, SXT, and GEN according to their CLSI of TIG was found to be lower than those of RIF and breakpoints. In our study, performance of time-kill CIP but greater than that of DOX (Turan et al., 2007).
studies on the isolate for which synergy was observed TIG was found to be superior to DOX and its MIC90 as well as some of the antagonistic organism/drug values were the lowest in the study of Dizbay et al.
combinations might strengthen our findings with (2007). This new compound may have a use in the Etest. Further studies are needed to clarify this issue.
treatment of brucellosis in the future, especially in In endemic regions, conventional antibrucellar anti- complicated or serious forms of disease. However biotics are cheap, easy to use and mostly well toler- it is costly, intravenously administered and its wide ated. We believe that brucellosis can be adequately use may lead to development of resistance for other treated with existing regimens, with protracted admi- serious nosocomial pathogens in countries where bru- nistration of appropriate drug combinations to mini- cellosis is endemic (Pappas et al., 2006b).
mize the percentage of relapses even for the most Time-kill, checkerboard and Etest are most exten- sively used in vitro methods for detecting synergy (White et al., 1996). There are few reports about in vitro combination studies for Brucellae (Orhan et al., This study was supported by Akdeniz University Scientific 2005; Rubinstein et al., 1991; Akova et al., 1999).
Research Project Unit. Etest strips were provided by Wyeth Phar- Akova et al. (1999) tested activities of RIF-DOX and STR-DOX by checkerboard method against 20 B. melitensis isolates. They found that RIF-DOX combination was synergistic for 17 isolates, additive for two isolates and indifferent for one isolate. The combination STR-DOX has showed synergistic effect Akova M., D. Gur, D.M. Livermore, T. Kocagoz and H.E.
for 18 isolates and was indifferent for two isolates.
Akalýn. 1999. In vitro activities of antibiotics alone and in com- bination against Brucella melitensis at neutral and acidic pHs.
Dizbay et al. in their study reported TIG-RIF and Antimicrob. Agents. Chemother. 43: 1298–1300.
DOX-RIF combinations demonstrated synergistic acti- Al-Orainey I.O., E.N.S. Saeed, A.M.M. Kambal and M.E.
vity in all strains. They didn’t detect synergy for DOX- Eltigani. 1991. Sensitivity of Brucella melitensis to chemothera- STR combination in any strain, but found that 18.75% peutic agents. Saudi. Med. J. 12: 119–120.
of their isolates exhibited antagonism for that combi- Ariza J., J. Bosch, F. Gudiol, J. Liñares, P.F. Viladrich and
R. Martín. 1986. Relevance of in vitro antimicrobial susceptibility nation (Dizbay et al., 2007). On the other hand, Orhan of Brucella melitensis to relapse rate in human brucellosis. Anti- et al. found a high rate of synergy for DOX-STR com- microb. Agents. Chemother. 30: 958–960.
bination. They also reported that the results of Etest Baykam N., H. Esener, O. Ergonul, S. Eren, A.K. Celikbas and and checkerboard methods agreed with a rate of 55% B. Dokuzoguz. 2004. In vitro antimicrobial susceptibility of Bru- in 16 B. melitensis isolates (Orhan et al., 2005).
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streptomycin, co-trimoxazole, rifampin and six fluoro- Bosch J., J. Linares, M.J. Lopez de Goicoechea, J. Ariza, quinolones and also studied the possible synergistic M.C. Cisnal and R. Martin. 1986. In vitro activity of ciprofloxacin, effects of several combinations of these antibiotics. ceftriaxone and five other antimicrobial agents against 95 strains of Brucella melitensis. J. Antimicrob. Chemother. 17: 459–461.
They found that no combination exhibited synergy Bossi P., A. Tegnell, A. Baka, F. Van Loock, J. Hendriks, against any of the tested strains by checkerboard test A. Werner, H. Maidhof and G. Gouvras. 2004. Bichat guide- lines for the clinical management of brucellosis and bioterrorism- Differences in agreement among synergy testing related brucellosis. Euro. Surveill. 9: 12.
methods have been reported previously most of which Clinical and Laboratory Standards Institute. 2006. Perfor- mance Standards for Antimicrobial Testing, Document M100-S16.
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