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In vitro antimicrobial susceptibility of Brucella species
International Journal of Antimicrobial Agents 23 (2004) 405–407
In vitro antimicrobial susceptibility of Brucella species Nurcan Baykam∗ , Harika Esener, Önder Ergönül, Sebnem ¸ Eren, Aysel K. Çel˙ikbas, Ba¸sak Dokuzoˇguz Ankara Numune Research and Education Hospital, First Infectious Diseases and Clinical Microbiology Department, Ankara, Turkey Received 9 July 2003; accepted 22 September 2003
Abstract MIC50 and MIC90 values of doxycycline, rifampicin, ciprofloxacin, trimethoprim-sulphamethoxazole and ceftriaxone for 42 blood isolates of Brucella species were determined using the Etest. Thirty-seven isolates were identified as B. melitensis and five as B. abortus. Doxycycline had the lowest and rifampicin the highest MIC50 values. Four strains were non-susceptible to rifampicin, and one strain was resistant to trimethoprim-sulphamethoxazole. There is no significantly important resistance problem for antibiotics targeted against Brucella species in Turkey. However, since rifampicin is commonly used for prevalent diseases such as tuberculosis, the regional susceptibility pattern of rifampicin should be assessed periodically. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Brucella spp.; MIC; Susceptibility; Etest
1. Introduction Human Brucellosis is a multisystemic disease that may present with a broad spectrum of clinical manifestations [1]. As Brucella species are intracellular pathogens, the treatment requires not only combined regimens but agents that may efficiently penetrate macrophages as well [2,3]. The type of antibiotics used for treatment of brucellosis influences the recurrence rate and it is clear that a synergistic combination with marked intracellular activity is the best therapeutic regimen in Brucellosis [4]. The agents that are used in these combinations are tetracyclines, rifampicin, fluoroquinolones, trimethoprim-sulphamethoxazole (TMP-SMX), third generation cephalosporins, streptomycin and other aminoglycosides [1,5]. Published guidelines that have originated from WHO recommendations, have suggested rifampicin plus doxycycline management for human brucellosis for more than a decade [6]. However, in vitro susceptibilities of these antibiotics may change over time and from one geographical region to another. Moreover, in vitro susceptibility tests are not standardised for Brucella species and they are not routinely ∗
Corresponding author. E-mail address: [email protected] (N. Baykam).
performed [7]. The aim of this study is to evaluate using the Etest, the MIC 50 and MIC 90 values of various antibiotics against clinical isolates of Brucella spp. from a brucellosis endemic region.
2. Materials and methods The isolates were collected prospectively between 2000 and 2003 from patients hospitalised in the First Infectious Diseases and Clinical Microbiology Department of the Numune Education and Research Hospital, which is the largest tertiary state hospital in Ankara. All isolates were obtained from blood cultures. Samples of blood were cultured using an automated culture system (Organon Tecnica BacT/Alert, Biomerioux® , France). The biotyping of the bacteria used the following tests: CO2 requirement, H2 S production, urease positivity, dye sensitivity (20–40 g/ml basic fuchsin) and growth in thionine. All the isolates were stored in skimmed milk powder at −20 ◦ C. MIC50 and MIC90 values of doxycycline, rifampicin, ciprofloxacin, TMP-SMX and ceftriaxone for the isolates were determined by the Etest. Bacterial suspension was spread on Mueller Hinton agar (Oxoid® ) plates supplemented with 5% sheep blood agar and Etest strips (AB
0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2003.09.024
406
N. Baykam et al. / International Journal of Antimicrobial Agents 23 (2004) 405–407
Biodisk® ) were applied. The plates were incubated at 35 ◦ C in ambient air, and the results were evaluated after 48 h. The procedure was performed according to The National Committee for Clinical Laboratory Standards (NCCLS)-M 100-S [8]. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 26213 and Brucella abortus S 19 served as controls. The results were evaluated according to Etest manufacturer’s instructions. The susceptibilities of Brucella species were presented as MIC50 and MIC90, and these findings were also evaluated using NCCLS susceptibility criteria for slow growing bacteria.
3. Results Forty-two Brucella species isolated from blood cultures were studied. Thirty-seven of these were identified as B. melitensis and five as B. abortus. The MIC50 and MIC90 values of the species are shown in Table 1. Doxycycline had the lowest and rifampicin the highest MIC50 values; TMP-SMX had the highest MIC90 value (Table 1). According to evaluation based on NCCLS slow growing bacteria standards, all isolates were sensitive to all antibiotics except one which was resistant to TMP-SMX and four which were non-susceptible to rifampicin (Table 2). The range of MIC and MIC50 values of B. melitensis and B. abortus are shown in Table 3. MIC50 values of TMP-SMX and rifampicin against B. abortus were lower than B. melitensis, whereas MIC50 values of ceftriaxone and ciprofloxacin for B. melitensis were lower than B. abortus (Table 3). The subtype of TMP-SMX resistant strain was Brucella melitensis biovar 3. The subtypes of rifampicin non-susceptible strains were Brucella melitensis biovar 1 and biovar 3 (Table 2).
Table 1 MIC50 and MIC90 values of Brucella species under study
Doxycycline Co-trimoxazole Ceftriaxone Rifampicin Ciprofloxacin
Range (mg/l)
MIC50
MIC90
<0.016–0.094 0.047–3.0 0.125–1 0.19–1.5 0.064–0.50
0.032 0.50 0.38 0.75 0.094
0.064 1.5 0.50 1.0 0.19
Table 3 MIC results of Brucella melitensis and Brucella abortus
Doxycycline Co-trimoxazole Ceftriaxone Rifampicin Ciprofloxacin
Brucella melitensis (MIC50 (min, max))
Brucella abortus (MIC50 (min, max))
0.032 (0.016, 0.094) 0.50 (0.047, 3.0) 0.25 (0.125, 1.0) 0.75 (0.019, 1.5) 0.094 (0.064, 0.50)
0.032 (0.023, 0.064) 0.38 (0.25, 1.0) 0.50 (0.25, 0.50) 0.50 (0.50, 0.75) 0.125 (0.094, 0.125)
4. Discussion There are three principal components of the treatment for brucellosis. Adequate intracellular concentration of antibiotic should be achieved, the antibiotic combination should be chosen because of its synergistic effect and thirdly, in vitro susceptibilities of the antibiotics should be evaluated. In this study we focused on in vitro susceptibilities of antibiotics commonly used against Brucella species. The Etest was used to determine the antibiotic susceptibilities of Brucella spp. as it has been reported to be less labour-intensive, less time consuming and more practical than the broth microdilution method for antibiotic susceptibility testing of Brucella spp. [9]. Since susceptibility testing of brucellae is not routinely used in clinical practice, there are few studies on in vitro susceptibilities of Brucella organisms in the literature and most have been poorly standardised [4,10]. Doxycycline, which is a major component of most therapeutic regimens in brucella infections, was found to have the lowest MIC50 and MIC90 values (Table 1). Previously, the average MIC of all tetracyclines has been reported as <1 mg/l [11]. In another study, the in vitro resistance rate was reported as 0.6% of 143 isolates for tetracycline [12]. The minimum bactericidal concentration of rifampicin for Brucella spp. is between 0.06 and 1 mg/l [13] and MIC value of four of our isolates of the total 42 (9.5%) were found to be 1.5 mg/l (Table 1). These isolates were considered as non-susceptible to rifampicin. On the other hand, according to the NCCLS, slow growing bacteria are intermediately susceptible against rifampicin at an MIC value of 2 mg/l [8]. By this definition, our four non-susceptible isolates might be considered as intermediate susceptible (Table 2). Memish et al. reported an in vitro resistance rate of 3.5% for rifampicin [12]. Although no rifampicin resistance was
Table 2 Distribution of antibiotic susceptibilities of brucella subtypes based on NCCLS slow growing bacteria values Doxycycline
M1 M3 A3
Co-trimoxazole
Ceftriaxone
Rifampicin
Ciprofloxacin
S
IM
R
S
IM
R
S
IM
R
S
IM
R
S
IM
R
8 29 5
– – –
– – –
8 28 5
– – –
– 1 –
8 29 5
– – –
– – –
6 27 5
2 2 –
– – –
8 29 5
– – –
– – –
M1: Brucella melitensis biovar 1, M3: Brucella melitensis biovar 3, A3: Brucella abortus biovar 3, S: susceptible, IM: intermediate susceptible, R: resistant.
N. Baykam et al. / International Journal of Antimicrobial Agents 23 (2004) 405–407
detected against Brucella species, the resistance rate of rifampicin against M. tuberculosis was reported as 15–58% in Turkey [14]. Our findings should alert us to the potential emergence of rifampicin resistance of Brucella in the region. TMP-SMX resistance was detected only in one strain (2%), which was much lower than previous reports (Table 2). In one study, the in vitro resistance rate was 29% [12]. Kinsara et al. reported 38% of 37 brucella isolates sensitive to TMP-SMX. They also reported all but one isolate sensitive to rifampicin, tetracycline, gentamicin and ciprofloxacin [15]. All the species were sensitive to ceftriaxone with MIC values of between 0.125 and 1.0 mg/l (Table 1). Palenque et al. reported the in vitro susceptibility of B. melitensis to ceftriaxone and cefotaxime, with MICs ranging from 0.25 to 2.0 mg/l [16]. In conclusion, there is no significantly important resistance problem for antibiotics targeted to Brucella species in Turkey. Based on these findings we do not recommend antibiotic susceptibility tests for every brucella isolate. However, antibiotic susceptibility tests should be considered where there is recurrent brucellosis. Therefore, we suggest, regional periodic assessment of susceptibility of strains to rifampicin,. References [1] Young EJ. Brucella species. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and practice of infectious diseases. New York: Churchill Livingstone; 2000. p. 2386–93. [2] Trujillano-Martin I, Garcia Sanchez E, Martinez IM, et al. In vitro activities of six new fluoroquinolones against Brucella melitensis. Antimicrob Agents Chemother 1999;43(1):194–5. [3] Garcia-Rodriguez JA, Garcia Sanchez E, Trujillano I. Lack of effective bactericidal activity of new quinolones against Brucella spp. Antimicrob Agents Chemother 1991;35(4):756–9.
407
[4] Arıza J, Bosch J, Gudiol F, et al. Relevance of in vitro antimicrobial susceptibility of Brucella melitensis to relapse rate in human brucellosis. Antimicrob Agents Chemother 1986;30(6):958– 60. [5] Gotuzzo E, Cellillo C. Brucella. In: Gorbach SL, Bartlett JG, Blacklow NR, editors. Infectious diseases. London: Saunders; 1992. p. 1513–21. [6] Joint Food and Agriculture Organization/World Health Organization. FAO–WHO Expert Committee on Brucellosis (sixth report). WHO Technical Report Series No. 740. Geneva: World Health Organisation; 1986. p. 56–7. [7] Gür D, Kocagöz S, Akova M. Comparison of E-test to microdilution for determining in vitro activities of antibiotics against Brucella melitensis. Antimicrob Agents Chemother 1999;43(9):2337. [8] National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Eighth informational supplement. NCCLS document M 100-S8, vol. 18. Wayne, PA: National Committee for Clinical Laboratory Standards; 1998. [9] Kocagöz S, Akova M, Ünal S. Comparison of E test to microdilution for determining in vitro activities of antibiotics against Brucella melitensis. Antimicrob Agents Chemother 1999;43:2337. [10] Shapiro SD, Wong JD. Brucella. In: Murray P, editor. Manuel of clinical microbiology, vol. 1. 7th ed. ASM Press; 2000. p. 625–31. [11] Farrell ID, Hinchcliffe PM, Robertson L. Sensitivity of Brucella spp. to tetracyline and its analogues. J Clin Pathol 1976;29:1097– 100. [12] Memish Z, Mah MW, Al Mahmoud S, Al Shaalan M, Khan MY. Brucella bacteraemia: clinical and laboratory observations in 160 patients. J Infect 2000;40(1):59–63. [13] Solera J, Martinez-Alfaro E, Espinosa A. Recognition and optimum treatment of brucellosis. Drugs 1997;53(2):246–56. [14] Kocaba¸s A. Akci˘ger Tüberkülozu. In: Topçu Willke A, Söyletir ˙ G, Do˘ganay M, editors. Infeksiyon Hastalıkları ve Mikrobiyolo˙ jisi.Istanbul: Nobel Tıp Kitabevleri, 2002; p. 38–591. [15] Kinsara A, Al-Mowallad A, Osoba AO. Increasing resistance of brucellae to co-trimoxazole. Antimicrob Agents Chemother 1999;43(6):1531. [16] Palenque E, Otero JR, Noriega AR. In vitro susceptibility of Brucella melitensis to new cephalosporins crossing the blood–brain barrier. Antimicrob Agents Chemother 1986;12:182–3.
In vitro antimicrobial susceptibility of Brucella species Nurcan Baykam∗ , Harika Esener, Önder Ergönül, Sebnem ¸ Eren, Aysel K. Çel˙ikbas, Ba¸sak Dokuzoˇguz Ankara Numune Research and Education Hospital, First Infectious Diseases and Clinical Microbiology Department, Ankara, Turkey Received 9 July 2003; accepted 22 September 2003
Abstract MIC50 and MIC90 values of doxycycline, rifampicin, ciprofloxacin, trimethoprim-sulphamethoxazole and ceftriaxone for 42 blood isolates of Brucella species were determined using the Etest. Thirty-seven isolates were identified as B. melitensis and five as B. abortus. Doxycycline had the lowest and rifampicin the highest MIC50 values. Four strains were non-susceptible to rifampicin, and one strain was resistant to trimethoprim-sulphamethoxazole. There is no significantly important resistance problem for antibiotics targeted against Brucella species in Turkey. However, since rifampicin is commonly used for prevalent diseases such as tuberculosis, the regional susceptibility pattern of rifampicin should be assessed periodically. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Brucella spp.; MIC; Susceptibility; Etest
1. Introduction Human Brucellosis is a multisystemic disease that may present with a broad spectrum of clinical manifestations [1]. As Brucella species are intracellular pathogens, the treatment requires not only combined regimens but agents that may efficiently penetrate macrophages as well [2,3]. The type of antibiotics used for treatment of brucellosis influences the recurrence rate and it is clear that a synergistic combination with marked intracellular activity is the best therapeutic regimen in Brucellosis [4]. The agents that are used in these combinations are tetracyclines, rifampicin, fluoroquinolones, trimethoprim-sulphamethoxazole (TMP-SMX), third generation cephalosporins, streptomycin and other aminoglycosides [1,5]. Published guidelines that have originated from WHO recommendations, have suggested rifampicin plus doxycycline management for human brucellosis for more than a decade [6]. However, in vitro susceptibilities of these antibiotics may change over time and from one geographical region to another. Moreover, in vitro susceptibility tests are not standardised for Brucella species and they are not routinely ∗
Corresponding author. E-mail address: [email protected] (N. Baykam).
performed [7]. The aim of this study is to evaluate using the Etest, the MIC 50 and MIC 90 values of various antibiotics against clinical isolates of Brucella spp. from a brucellosis endemic region.
2. Materials and methods The isolates were collected prospectively between 2000 and 2003 from patients hospitalised in the First Infectious Diseases and Clinical Microbiology Department of the Numune Education and Research Hospital, which is the largest tertiary state hospital in Ankara. All isolates were obtained from blood cultures. Samples of blood were cultured using an automated culture system (Organon Tecnica BacT/Alert, Biomerioux® , France). The biotyping of the bacteria used the following tests: CO2 requirement, H2 S production, urease positivity, dye sensitivity (20–40 g/ml basic fuchsin) and growth in thionine. All the isolates were stored in skimmed milk powder at −20 ◦ C. MIC50 and MIC90 values of doxycycline, rifampicin, ciprofloxacin, TMP-SMX and ceftriaxone for the isolates were determined by the Etest. Bacterial suspension was spread on Mueller Hinton agar (Oxoid® ) plates supplemented with 5% sheep blood agar and Etest strips (AB
0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2003.09.024
406
N. Baykam et al. / International Journal of Antimicrobial Agents 23 (2004) 405–407
Biodisk® ) were applied. The plates were incubated at 35 ◦ C in ambient air, and the results were evaluated after 48 h. The procedure was performed according to The National Committee for Clinical Laboratory Standards (NCCLS)-M 100-S [8]. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 26213 and Brucella abortus S 19 served as controls. The results were evaluated according to Etest manufacturer’s instructions. The susceptibilities of Brucella species were presented as MIC50 and MIC90, and these findings were also evaluated using NCCLS susceptibility criteria for slow growing bacteria.
3. Results Forty-two Brucella species isolated from blood cultures were studied. Thirty-seven of these were identified as B. melitensis and five as B. abortus. The MIC50 and MIC90 values of the species are shown in Table 1. Doxycycline had the lowest and rifampicin the highest MIC50 values; TMP-SMX had the highest MIC90 value (Table 1). According to evaluation based on NCCLS slow growing bacteria standards, all isolates were sensitive to all antibiotics except one which was resistant to TMP-SMX and four which were non-susceptible to rifampicin (Table 2). The range of MIC and MIC50 values of B. melitensis and B. abortus are shown in Table 3. MIC50 values of TMP-SMX and rifampicin against B. abortus were lower than B. melitensis, whereas MIC50 values of ceftriaxone and ciprofloxacin for B. melitensis were lower than B. abortus (Table 3). The subtype of TMP-SMX resistant strain was Brucella melitensis biovar 3. The subtypes of rifampicin non-susceptible strains were Brucella melitensis biovar 1 and biovar 3 (Table 2).
Table 1 MIC50 and MIC90 values of Brucella species under study
Doxycycline Co-trimoxazole Ceftriaxone Rifampicin Ciprofloxacin
Range (mg/l)
MIC50
MIC90
<0.016–0.094 0.047–3.0 0.125–1 0.19–1.5 0.064–0.50
0.032 0.50 0.38 0.75 0.094
0.064 1.5 0.50 1.0 0.19
Table 3 MIC results of Brucella melitensis and Brucella abortus
Doxycycline Co-trimoxazole Ceftriaxone Rifampicin Ciprofloxacin
Brucella melitensis (MIC50 (min, max))
Brucella abortus (MIC50 (min, max))
0.032 (0.016, 0.094) 0.50 (0.047, 3.0) 0.25 (0.125, 1.0) 0.75 (0.019, 1.5) 0.094 (0.064, 0.50)
0.032 (0.023, 0.064) 0.38 (0.25, 1.0) 0.50 (0.25, 0.50) 0.50 (0.50, 0.75) 0.125 (0.094, 0.125)
4. Discussion There are three principal components of the treatment for brucellosis. Adequate intracellular concentration of antibiotic should be achieved, the antibiotic combination should be chosen because of its synergistic effect and thirdly, in vitro susceptibilities of the antibiotics should be evaluated. In this study we focused on in vitro susceptibilities of antibiotics commonly used against Brucella species. The Etest was used to determine the antibiotic susceptibilities of Brucella spp. as it has been reported to be less labour-intensive, less time consuming and more practical than the broth microdilution method for antibiotic susceptibility testing of Brucella spp. [9]. Since susceptibility testing of brucellae is not routinely used in clinical practice, there are few studies on in vitro susceptibilities of Brucella organisms in the literature and most have been poorly standardised [4,10]. Doxycycline, which is a major component of most therapeutic regimens in brucella infections, was found to have the lowest MIC50 and MIC90 values (Table 1). Previously, the average MIC of all tetracyclines has been reported as <1 mg/l [11]. In another study, the in vitro resistance rate was reported as 0.6% of 143 isolates for tetracycline [12]. The minimum bactericidal concentration of rifampicin for Brucella spp. is between 0.06 and 1 mg/l [13] and MIC value of four of our isolates of the total 42 (9.5%) were found to be 1.5 mg/l (Table 1). These isolates were considered as non-susceptible to rifampicin. On the other hand, according to the NCCLS, slow growing bacteria are intermediately susceptible against rifampicin at an MIC value of 2 mg/l [8]. By this definition, our four non-susceptible isolates might be considered as intermediate susceptible (Table 2). Memish et al. reported an in vitro resistance rate of 3.5% for rifampicin [12]. Although no rifampicin resistance was
Table 2 Distribution of antibiotic susceptibilities of brucella subtypes based on NCCLS slow growing bacteria values Doxycycline
M1 M3 A3
Co-trimoxazole
Ceftriaxone
Rifampicin
Ciprofloxacin
S
IM
R
S
IM
R
S
IM
R
S
IM
R
S
IM
R
8 29 5
– – –
– – –
8 28 5
– – –
– 1 –
8 29 5
– – –
– – –
6 27 5
2 2 –
– – –
8 29 5
– – –
– – –
M1: Brucella melitensis biovar 1, M3: Brucella melitensis biovar 3, A3: Brucella abortus biovar 3, S: susceptible, IM: intermediate susceptible, R: resistant.
N. Baykam et al. / International Journal of Antimicrobial Agents 23 (2004) 405–407
detected against Brucella species, the resistance rate of rifampicin against M. tuberculosis was reported as 15–58% in Turkey [14]. Our findings should alert us to the potential emergence of rifampicin resistance of Brucella in the region. TMP-SMX resistance was detected only in one strain (2%), which was much lower than previous reports (Table 2). In one study, the in vitro resistance rate was 29% [12]. Kinsara et al. reported 38% of 37 brucella isolates sensitive to TMP-SMX. They also reported all but one isolate sensitive to rifampicin, tetracycline, gentamicin and ciprofloxacin [15]. All the species were sensitive to ceftriaxone with MIC values of between 0.125 and 1.0 mg/l (Table 1). Palenque et al. reported the in vitro susceptibility of B. melitensis to ceftriaxone and cefotaxime, with MICs ranging from 0.25 to 2.0 mg/l [16]. In conclusion, there is no significantly important resistance problem for antibiotics targeted to Brucella species in Turkey. Based on these findings we do not recommend antibiotic susceptibility tests for every brucella isolate. However, antibiotic susceptibility tests should be considered where there is recurrent brucellosis. Therefore, we suggest, regional periodic assessment of susceptibility of strains to rifampicin,. References [1] Young EJ. Brucella species. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and practice of infectious diseases. New York: Churchill Livingstone; 2000. p. 2386–93. [2] Trujillano-Martin I, Garcia Sanchez E, Martinez IM, et al. In vitro activities of six new fluoroquinolones against Brucella melitensis. Antimicrob Agents Chemother 1999;43(1):194–5. [3] Garcia-Rodriguez JA, Garcia Sanchez E, Trujillano I. Lack of effective bactericidal activity of new quinolones against Brucella spp. Antimicrob Agents Chemother 1991;35(4):756–9.
407
[4] Arıza J, Bosch J, Gudiol F, et al. Relevance of in vitro antimicrobial susceptibility of Brucella melitensis to relapse rate in human brucellosis. Antimicrob Agents Chemother 1986;30(6):958– 60. [5] Gotuzzo E, Cellillo C. Brucella. In: Gorbach SL, Bartlett JG, Blacklow NR, editors. Infectious diseases. London: Saunders; 1992. p. 1513–21. [6] Joint Food and Agriculture Organization/World Health Organization. FAO–WHO Expert Committee on Brucellosis (sixth report). WHO Technical Report Series No. 740. Geneva: World Health Organisation; 1986. p. 56–7. [7] Gür D, Kocagöz S, Akova M. Comparison of E-test to microdilution for determining in vitro activities of antibiotics against Brucella melitensis. Antimicrob Agents Chemother 1999;43(9):2337. [8] National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Eighth informational supplement. NCCLS document M 100-S8, vol. 18. Wayne, PA: National Committee for Clinical Laboratory Standards; 1998. [9] Kocagöz S, Akova M, Ünal S. Comparison of E test to microdilution for determining in vitro activities of antibiotics against Brucella melitensis. Antimicrob Agents Chemother 1999;43:2337. [10] Shapiro SD, Wong JD. Brucella. In: Murray P, editor. Manuel of clinical microbiology, vol. 1. 7th ed. ASM Press; 2000. p. 625–31. [11] Farrell ID, Hinchcliffe PM, Robertson L. Sensitivity of Brucella spp. to tetracyline and its analogues. J Clin Pathol 1976;29:1097– 100. [12] Memish Z, Mah MW, Al Mahmoud S, Al Shaalan M, Khan MY. Brucella bacteraemia: clinical and laboratory observations in 160 patients. J Infect 2000;40(1):59–63. [13] Solera J, Martinez-Alfaro E, Espinosa A. Recognition and optimum treatment of brucellosis. Drugs 1997;53(2):246–56. [14] Kocaba¸s A. Akci˘ger Tüberkülozu. In: Topçu Willke A, Söyletir ˙ G, Do˘ganay M, editors. Infeksiyon Hastalıkları ve Mikrobiyolo˙ jisi.Istanbul: Nobel Tıp Kitabevleri, 2002; p. 38–591. [15] Kinsara A, Al-Mowallad A, Osoba AO. Increasing resistance of brucellae to co-trimoxazole. Antimicrob Agents Chemother 1999;43(6):1531. [16] Palenque E, Otero JR, Noriega AR. In vitro susceptibility of Brucella melitensis to new cephalosporins crossing the blood–brain barrier. Antimicrob Agents Chemother 1986;12:182–3.