|Year : 2016 | Volume
| Issue : 3 | Page : 342-345
Antimicrobial susceptibility testing of Brucella melitensis isolated from patients with acute brucellosis in a centre of Iran
R Razzaghi1, R Rastegar1, M Momen-Heravi1, M Erami2, M Nazeri3
1 Department of Infectious Disease, Medical Faculty, Kashan University of Medical Sciences, Kashan, Iran
2 Department of Clinical Laboratory, Beheshti Hospital, Kashan University of Medical Sciences, Kashan, Iran
3 Department of Medical Parasitology and Mycology, Medical Faculty, Kashan University of Medical Sciences, Kashan, Iran
|Date of Submission||03-Aug-2015|
|Date of Acceptance||30-Jun-2016|
|Date of Web Publication||12-Aug-2016|
Department of Medical Parasitology and Mycology, Medical Faculty, Kashan University of Medical Sciences, Kashan
Source of Support: None, Conflict of Interest: None
Structured to Purpose: Human brucellosis is one of the most common zoonotic infections worldwide, which remains one of the major problems for public health. Despite the World Health Organization's recommendation for human brucellosis treatment, sporadic cases of relapse have been reported. The aim of this study was to assess the susceptibility of Brucella isolates to common antibiotics that are prescribed by the physician for the treatment of brucellosis and also to determine the minimum inhibitory concentration 50% (MIC 50 ) and MIC 90 for these antibiotics. Materials and Methods: Forty-eight Brucella strains were collected from patients with acute brucellosis. Species identification was made based on the conventional methods. MIC of rifampin, doxycycline, ciprofloxacin, trimethoprim- sulfamethoxazole, streptomycin, azithromycin and ceftriaxone was determined by E-test. Results: All the 48 Brucella isolates (47 blood samples and one synovial fluid) were identified as Brucella melitensis. No antimicrobial-resistant strains were recognised. Trimethoprim-sulfamethoxazole had the lowest MIC 50 (0.016 μg/ml) and MIC 90 (0.064 μg/ml), whereas MIC 50 and MIC 90 of streptomycin and azithromycin had the highest level at 0.625, 1.5 µg/ml and 0.25, 1 µg/ml, respectively. All the isolates were susceptible to rifampin, and only one of the isolates had a reduced sensitivity to rifampin (1 μg/ml). Conclusions: Although all the Brucella isolates were susceptible, antimicrobial susceptibility test should be recommended in patients with recurrent brucellosis or life-threatening organ involvement.
Keywords: Antimicrobial susceptibility, Brucella melitensis, Iran
|How to cite this article:|
Razzaghi R, Rastegar R, Momen-Heravi M, Erami M, Nazeri M. Antimicrobial susceptibility testing of Brucella melitensis isolated from patients with acute brucellosis in a centre of Iran. Indian J Med Microbiol 2016;34:342-5
|How to cite this URL:|
Razzaghi R, Rastegar R, Momen-Heravi M, Erami M, Nazeri M. Antimicrobial susceptibility testing of Brucella melitensis isolated from patients with acute brucellosis in a centre of Iran. Indian J Med Microbiol [serial online] 2016 [cited 2020 Jan 20];34:342-5. Available from: http://www.ijmm.org/text.asp?2016/34/3/342/188336
| ~ Introduction|| |
Human brucellosis, with more than 500,000 new cases, is one of the most common zoonotic infections worldwide, which is caused by Gram-negative bacteria, Brucella spp.  In endemic areas, infection is mainly transmitted to humans by the consumption of unpasteurised dairy products, whereas other routes of transmission such as direct contact with infected animals or inhalation of aerosols are less common in these areas. 
In Iran, approximately, 238.6 cases/million population of human brucellosis (overwhelmingly due to Brucella melitensis) are reported annually.  Isfahan province, in the centre of Iran, has been described as one the most important endemic areas of brucellosis for many years. 
Since Brucella species are intracellular pathogens, it is obvious that combined antibiotic therapy with confirmed intracellular activity is the best therapeutic regimen in brucellosis. Thus, effective antibiotics are limited to tetracyclines, rifampin, trimethoprim-sulfamethoxazole, streptomycin, fluoroquinolones and some members of new class of antibiotics such as tigecycline. 
According to the World Health Organization's (WHO) recommendation for human brucellosis, doxycycline combined with rifampin or streptomycin for 6 weeks is effective.  Despite the use of this regimen, sporadic cases of relapse have been reported near to 30%.  In vitro antimicrobial susceptibility testing of Brucella spp. is not routine for all isolated strains from patients due to risk of laboratory-acquired infection and the necessity of biological safety level 3 precautions.  The WHO suggests only a limited number of antibiotics with good intracellular access and confirmed clinical effectiveness for the treatment of brucellosis. Usually, Brucella species are susceptible to the WHO-recommended antibiotics, though sporadic cases of antimicrobial resistance happen. However, in vitro susceptibility testing of Brucella spp. can differ across geographical regions. 
The purpose of this study was to evaluate the minimum inhibitory concentration 50% (MIC 50 ) and MIC 90 values of various antibiotics against B. melitensis isolated from patients with Brucellosis using the E-test.
| ~ Materials and Methods|| |
Specimens and bacterial isolation
In this experimental study, 48 Brucella isolates were collected prospectively from patients with acute brucellosis who were hospitalised at the Infectious Disease Department in Sahid Beheshti Hospital in Kashan, Iran, between 2011 and 2013. Forty-seven isolates were obtained from blood cultures except one sample which was obtained from synovial fluid. All the samples were cultured using an automated blood culture system (OrganonTecnicaBacT/Alert, Biomerioux; , France).
All patients had prolonged fever, night sweating and the musculoskeletal disorders including myalgia, and arthralgia was the most common complaint (96%), while two patients (5%) had gastrointestinal symptoms (nausea and vomiting) and neurologic symptoms. These patients were diagnosed as brucellosis with serological titre ≥1/160 and culture-positive.
Species identification was made on the basis of conventional standard procedure: CO 2 requirement for growth, H 2 S and urease production, sensitivity to the dyes (basic fuchsin and thionine) and species identification confirmed by susceptibility to Tbilisi phage.
Isolated strains were stored in brain heart infusion broth with 5% glycerol at −70°C and they were sub-cultured twice before the antimicrobial susceptibility testing.
Antimicrobial susceptibility test
MIC of rifampin, doxycycline, ciprofloxacin, trimethoprim-sulfamethoxazole, streptomycin, azithromycin and ceftriaxone was evaluated by E-test (Liofilchem S.R.L., Italy) method according to the Clinical Laboratory Standards Institute (CLSI) guidelines for slow-growing bacteria such as Haemophilus spp. The bacterial suspension equal to 0.5 McFarland turbidity was inoculated on Mueller-Hinton agar plates, supplemented with 5% sheep blood, and E-test strips of selected antibacterial were applied. The plates were incubated in ambient air at 35°C, and the results were read after 48 h. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as controls for antimicrobial susceptibility testing. 
| ~ Results|| |
All the 48 Brucella isolates (47 blood samples and one synovial fluid) were identified as B. melitensis by using conventional methods for Brucella identification.
The MIC 50 and MIC 90 values of seven antibiotics which are able to penetrate the macrophage are shown in [Table 1].
|Table 1: MIC ranges, MIC50 and MIC90 values of seven antibiotics against 48 Brucella melitensis isolates |
Click here to view
According to the CLSI criteria for slow-growing bacteria, all the isolates were susceptible. Trimethoprim-sulfamethoxazole had the lowest MIC 50 and MIC 90 values (0.016 and 0.064 μg/ml) and followed by doxycycline which had MIC 50 and MIC 90 of 0.047 and 0.1 μg/ml, respectively. Whereas MIC 50 and MIC 90 of streptomycin and azithromycin, which had the highest level, were 0.625, 1.5 μg/ml and 0.25, 1 μg/ml, respectively.
All the isolates were susceptible to rifampin except one isolate that showed reduced susceptibility to rifampin (1 μg/ml).
| ~ Discussion|| |
Human brucellosis is one of the major zoonotic infections in the world, especially in the Middle East.  Hence, Iran is one of the endemic countries for brucellosis. The infection is treated effectively with combined regimens that are recommended by the WHO.  However, there are some problems such as long-term treatment, emergence of resistant strains during treatment and change in antibiotic susceptibility patterns depending on the geographic region. 
This study showed that trimethoprim-sulfamethoxazole had the lowest MIC that was in agreement with other studies reported in Italy, Peru and Egypt. ,, Nevertheless, trimethoprim-sulfamethoxazole resistance has been reported in several studies in other countries between 2% and 62%. , These high rates of resistance could be related to susceptibility testing method such as Kirby-Bauer disc diffusion technique, which is not generally recommended.  High level of sensitivity to trimethoprim-sulfamethoxazole is important for several reasons such as oral form, potential to be prescribed in pregnant women and children and lower cost compared to other drugs in combination regimen that this latter one can be of importance in the developing countries. 
Despite the fact that streptomycin is the most effective antibiotic for brucellosis treatment, there are several side effects for the prolonged use of streptomycin including nephrotoxicity, ototoxicity and parenteral administration.  In the present study, streptomycin with MIC 90 (1.5 μg/ml) had the highest MIC, which was in agreement with Kaya et al.'s study.  MIC 90 level for streptomycin has been evaluated at a difference to be between 0.5 and 8 μg/ml. However, according to the CLSI guidelines, all the isolates were susceptible (≤8 μg/ml) that was in agreement with the previous studies. ,,,
Doxycycline, which is one of the principal antibacterial component in therapeutic regimens against brucellosis, appeared to have a MIC 90 (0.1 μg/ml) that was higher than that in the previous reports. ,, Although higher MIC has been reported in Egypt. 
Ceftriaxone has been proven to be effective in pyogenic infections of the central nervous system, so it could be more effective for neurobrucellosis treatment.  The MIC range values for ceftriaxone were between 0.125 and 1 μg/ml, which was in agreement with Baykam et al.'s study  Nevertheless, another study reported higher MIC range. 
Azithromycin is a new macrolide with good intracellular penetration and in vitro activity against Brucella. However, in acidic pH environment, at phagolysosome conditions, this activity is decreased. In this study, MIC 90 for azithromycin was 1 μg/ml that had good activity and was concordant with the previous reports in different regions of Turkey,  which is in the neighbourhood of Iran. Although Yamazhan et al. reported high MIC (32 μg/ml) values for azithromycin with the agar dilution method,  this difference may be due to the geographical variations and different methods.
Ciprofloxacin is a fluoroquinolone that has features such as broad spectrum of antibacterial activity, oral consumption, high tissue accumulation and intracellular penetration potency. 
These features define these antimicrobial agents as candidates for the treatment of intracellular bacterial infections such as brucellosis. However, several problems have been reported for ciprofloxacin including a lack of bactericidal activity against Brucella spp., development of resistance in B. melitensis during ciprofloxacin therapy, cross-resistance to other fluoroquinolones and high incidence of relapse when it is used for treatment alone. 
In this study, MIC range value for ciprofloxacin was between 0.094 and 0.5 μg/ml, with MIC 90 (0.38 μg/ml) value, so all the isolates were susceptible, which was in agreement with other studies. ,, In two studies, ciprofloxacin at MIC 90 value of 2 μg/ml was higher than the CLSI breakpoints. As was mentioned earlier, this high MIC 90 value could be due to different evaluation methods. 
Rifampin has good intracellular penetration with good activity against Brucella spp., especially in an acidic pH environment.  However, there are no defined criteria for rifampin against Brucella species in CLSI guidelines. Hence, in this situation, MIC values for rifampin were evaluated according to criteria for slow-growing bacteria. In this criteria, MIC 90 values of ≤1 μg/ml, 2-3 μg/ml and ≥4 μg/ml were considered susceptible, intermediate (reduced susceptibility) and resistant, respectively. 
All the Brucella isolates with MIC range between 0.006 and 1 μg/ml and MIC 90 of 0.75 μg/ml for rifampin were susceptible, which was in agreement with other studies. , The highest rate of reduced susceptibility (45%) and resistance (19%) to rifampin among Brucella isolates has been reported from Egypt,  although a reduced susceptibility to rifampin was reported in others studies from Turkey, , Kuwait,  and Mexico.  This sensitivity to rifampin could be important for several reasons. The first one is that rifampin is the basic drug in the treatment of Mycobacterium tuberculosis. Hence, any increase in rifampin resistance in brucellosis may lead to the emergence of resistance to M. tuberculosis species.  Moreover, experimental studies showed that the mycobacterial resistance to rifampin may result in the development of resistance to other antibiotics.  This should not be neglected that some regions in Iran are endemic for tuberculosis. 
Our results showed that there is no resistance to these drugs. Therefore, reported treatment failure could be due to others factors such as inappropriate dose, short period prescription, insufficient drug potency, poor patient compliance and host immune status. Therefore, antimicrobial susceptibility test should be recommended for patients with recurrent brucellosis or life-threatening organ involvement.
This is the first study to evaluate antimicrobial susceptibility by E-test method for the Brucella isolates in Iran. Although no antimicrobial resistance in our strains was identified, continual examination for the emergence of antimicrobial resistance in brucellosis is necessary.
This article is based on the thesis of an M.D. degree supported by the Kashan University of Medical Sciences, Research Fund (Grant #9181).
We are also grateful to Dr. Hossein Hooshyar and Dr. Rezvan Moniri for their valuable opinions and advice throughout the study.
Financial support and sponsorship
This study was supported by the Kashan University of Medical Sciences, Research Fund (Grant #9181).
Conflicts of interest
There are no conflicts of interest.
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