Comparison of E Test to Microdilution for Determining In Vitro Activities of Antibiotics against Brucella melitensis

The standard procedures for in vitro testing have not been determined for brucellae. We compared two susceptibility testing methods for this organism. Isolates were from blood or bone marrow cultures of patients admitted to Hacettepe University Hospital. MICs were determined by microdilution testing of 22 strains for the following antibiotics: azithromycin (Pfizer), doxycycline (FAKO), ofloxacin (Hoechst Marion Roussel), rifampin (Sigma), and streptomycin (Sigma). Antibiotics were diluted twofold in Mueller-Hinton broth (Oxoid), supplemented with 1% PoliViteX (BioMerieux). Bacterial inoculum was prepared by adjusting the turbidity of the 48-h broth culture to that of a 0.5 McFarland turbidity standard to give a final inoculum of 10⁵ to 10⁶ CFU/well. The microplate trays were incubated at 35°C in ambient air, and the results were read after 48 h. The lowest antibiotic concentration that completely inhibited visible growth was considered to be the MIC (4).

E test (AB Biodisk) was performed in parallel with the broth microdilution tests, by using the same bacterial inoculum. Bacterial suspension was spread on Mueller-Hinton agar plates supplemented with 5% sheep blood, and E-test strips were applied. The plates were incubated at 35°C in ambient air, and the results were read after 48 h.

When results of the two methods were compared, agreement rates of MIC end points within fourfold dilutions by the two tests were 82% for ofloxacin and streptomycin, 91% for rifampin, 95% for azithromycin, and 100% for doxycycline (Table 1).

TABLE 1.

MIC end points with E test compared to microbroth dilution

Fold variation in MIC end pointsa	No. of strains (%)
	Azithro-mycin	Doxy-cycline	Oflox-acin	Rifampin	Strepto-mycin
None	6 (27)	12 (55)	8 (36)	13 (59)	7 (32)
−2	6 (27)	3 (14)	3 (14)	4 (18)	7 (32)
+2	3 (14)	4 (18)	1 (5)	3 (14)	2 (10)
−4	4 (18)	2 (10)	6 (27)	0	2 (10)
+4	2 (10)	1 (5)	0	1 (5)	1 (5)
−8	1 (5)	0	4 (18)	1 (5)	2 (10)
+8	0	0	0	0	0
−16	0	0	0	0	1 (5)

^aCompared to microbroth dilution. 

In vitro susceptibility tests are not critical in brucellosis mainly because resistance to antibiotics is not well correlated with relapse associated with these organisms (1). The guidelines of the National Committee for Clinical Laboratory Standards which are followed for a wide range of organisms are not applicable for this group of bacteria. It has been shown that factors such as pH, inoculum size, and medium have a significant effect on the results (2, 3). Our results indicated that E test compared with broth microdilution test is in an acceptable range of difference (±4-fold) for 82 to 100% of isolates with various antibiotics. For two strains, MICs of streptomycin were 256 μg/ml with the broth microdilution test; the MICs were 256 and 128 μg/ml with the E test. We suggest that E test can be applied for antibiotic susceptibility testing of Brucella spp. as it is less labor-intensive, less time-consuming, and more practical than the broth microdilution method.