Antimicrobial susceptibility testing of Bilophila wadsworthia by using triphenyltetrazolium chloride to facilitate endpoint determination

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Abstract
Initial susceptibility studies of Bilophila wadsworthia indicated significant resistance to several beta-lactam antibiotics, including imipenem and cefoxitin. NO beta-lactamase production was detected. However, some B. wadsworthia strains may grow as a heavy "haze" at up to the highest concentration of an antibiotic on standard antimicrobial agent-containing plates, and it is often difficult to determine the point at which conventional growth stops and haze growth begins. We investigated the nature of the haze growth of B. wadsworthia by using triphenyltetrazolium chloride (TTC) as an indicator of viability during antimicrobial susceptibility testing, by determining viability counts on antimicrobial agent-containing plates at various times, and by microscopically inspecting stained preparations of the growth on the control plate and the haze area. TTC MICs were determined by applying a TTC solution over the growth on plates inside the anaerobic chamber or within 5 min after exposure to air (aerobic TTC MICs). The haze growth reduced TTC in the chamber but not under aerobic conditions, whereas TTC was reduced by the conventional growth in both atmospheres. The aerobic TTC MICs correlated with the viability counts. Separated proteins resolved by polyacrylamide gel electrophoresis and isoelectric focusing showed TTC-reactive bands only when stained under anaerobic conditions, further demonstrating the sensitivity of TTC reduction to aerobic conditions. Microscopic examination of the haze growth indicated spheroplast formation. A new antibiogram for B. wadsworthia has been established by use of aerobic TTC endpoints; we believe that the lower MICs obtained with the TTC method are likely the ones that are clinically relevant and should be used in tests of B. wadsworthia. Also, we found that when the organisms were grown on pyruvate-containing medium, 87% of 56 strains tested were Beta-lactamase positive.