Bru SIC: a novel selective medium for the primary isolation of Brucella in veterinary samples

Abstract

Farrell’s medium (FM) and CITA medium (CM), recommended for Brucella isolation in animal samples, are inhibitory for certain strains. A reformulated Brucella selective medium (BSM), containing half the CM vancomycin and colistin concentrations, improved the isolation of B. melitensis , but not Brucella abortus bv1. Brucellosis, a re-emerging zoonotic infection, threatens animal welfare and public health with serious economic consequences. A definitive diagnosis requires Brucella isolation by culturing field specimens in specific media. This study aimed to (i) assess the effectivity of recommended Farrell’s médium (FM) and CITA medium (CM) for the isolation of four Brucella melitensis strains (16M, Rev1, and the 16MΔ wzm and Rev1Δ wzm in-frame deletion mutants) with variable susceptibility to polymyxins; (ii) develop a Brucella selective medium (BSM) suitable for these strains; (iii) test BSM, FM, and CM with other Brucella species; and (iv) develop an improved selective culture medium ( Bru SIC) for all brucellae, including B. abortus bv1. The four B. melitensis strains were strongly inhibited in FM and (except Rev1) CM. Since Rev1Δ wzm ’s CM inhibition was due to a synergistic effect of colistin and vancomycin, we formulated BSM with half the concentrations of both antibiotics, achieving a similar growth of B. melitensis to blood agar base (BAB) and an inhibition of contaminant microorganisms comparable to CM; CM performance was surpassed by BSM for the primary isolation of B. melitensis when tested in 1,789 real sheep samples. For other brucellae, BSM and CM were more inhibitory than FM for B. abortus bv1 when using plates immediately after preparation but not after ≥4 weeks of storage. To address this, we developed the improved solid medium Bru SIC by replacing the calf serum in BSM with activated charcoal. Bru SIC yielded faster colony growth than BSM and CM and similar CFU numbers than BAB (including for B. ovis in BAB-Serum) and inhibited accompanying microorganisms in sheep and cow samples as effectively as BSM. IMPORTANCE Farrell’s medium (FM) and CITA medium (CM), recommended for Brucella isolation in animal samples, are inhibitory for certain strains. A reformulated Brucella selective medium (BSM), containing half the CM vancomycin and colistin concentrations, improved the isolation of B. melitensis , but not Brucella abortus bv1. A novel Brucella selective culture medium ( Bru SIC), in which calf serum is replaced by activated charcoal, retains the selectivity and improves the productivity of BSM and CM. Bru SIC allows the growth of all brucellae faster than in CM or BSM, and at CFU number equivalent to BAB supplemented by calf serum, including B. abortus bv1 and the serum-dependent Brucella ovis . Due to its performance and reduced cost, Bru SIC represents an added-value alternative to the existing selective culture media for these bacteria.