COLUMBIA BLOOD AGAR BASE

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-pH 7.3 ± 0.2 @ 25°C -Cool to 50°C and add 5% sterile defibrinated blood.

Protocol tips
-pH 7.3 ± 0.2 @ 25°C -Cool to 50°C and add 5% sterile defibrinated blood.

Publication protocol

The method was performed on commercially available 96-well broth microdilution plates for monitoring resistance of anaerobic and Gram-positive bacteria: the Sensititre Anaerobe plate ANO2B format and the Sensititre Gram-Positive plate GPALL1F format (Trek Diagnostic Systems). Thirty antimicrobial agents were tested, including ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, cefotetan, cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, daptomycin, erythromycin, gentamicin, imipenem, levofloxacin, linezolid, meropenem, metronidazole, mezlocillin, moxifloxacin, nitrofurantoin, oxacillin, penicillin, piperacillin, piperacillin/tazobactam, quinupristin/dalfopristin, rifampicin, streptomycin, tetracycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin.

The colonies were selected from 48 h anaerobic culture on 5 % sheep blood agar (Columbia blood agar base; Oxoid). The suspensions and inoculation of microdilution plates were carried out in an aerobic atmosphere, but the organisms were not exposed to air for more than 30 min.

The procedure was carried out using reduced cation-adjusted Mueller–Hinton broth for initial suspension and supplemented Brucella broth (Trek Diagnostic Systems) to make an inoculum containing 1×10 5–1×10 6 c.f.u. ml −1 in final suspension, which was transferred to a broth microdilution plate following the manufacturer’s instructions. The plates were incubated at 35 °C for 48 h in anaerobic conditions (GENbox anaerobic jar and GENbox Anaer generators; bioMérieux).

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