K-083. Anaerobic Growth of Neisseria gonorrhoeae in Liquid Medium

M. A. Graver, J. J. Wade;
King's Coll. Hosp., London, UNITED KINGDOM.

Background: Evidence accrues that Neisseria gonorrhoeae (NG) grows anaerobically in vivo: NG may be recovered with obligate anaerobes from clinical material and NG proteins induced anaerobically include a nitrite reductase, antibodies to which are present in sera of infected patients. To date, growth of NG under anaerobic conditions in vitro has required nitrite as a terminal electron acceptor. We sought to adapt our published liquid medium (GW medium) to permit growth of NG anaerobically. Methods: The changes to GW medium were: sodium acetate and lactate omitted and glucose reduced to 2.0g/L; 1mL ‘Oxyrase® for Broth’ (Oxyrase Inc, Ohio) per 50mL medium was added. The final evaluation used 21 strains of NG (two distinguishable strains each of 10 auxotypes, plus NCTC 8375) and Fusobacterium nucleatum ATCC 25586 (FN). For each bacterium, 25mL medium was inoculated with 300μL of a 10-3 dilution of a 0.5 McFarland suspension in phosphate-buffered saline (PBS); from this, three 7mL aliquots in 7mL bijoux were prepared and time 0 counts performed on chocolate agar (CA) or fastidious anaerobe agar (FAA) using the remnant. Uninoculated controls included 7mL aliquots with methylene blue and resazurin sodium at 0.002g/L, each with and without Oxyrase. Bijoux lids were tightly closed and all incubated at 37oC. Anaerobiosis indicators were examined and counts done at 20h, 25h and 30h on serial 10-2 dilutions in PBS on CA or FAA using a spiral plater. Experiments were done at least in triplicate. Results: Indicators confirmed that anaerobiosis was maintained to 30h. Twelve of the 21 NG grew well in triplicate initially; 9 were repeated on one or more occasions to achieve optimum growth thrice; for one NG this required a higher inoculum. Counts (median log10 cfu/mL) for FN at times 0, 20, 25 and 30h were 3.03, 7.98, 8.36 and 8.27, respectively. Despite the NG strains showing variable growth rates, median counts for the 21 NG at these time intervals were 3.16, 6.27, 6.76 and 6.86, respectively. Conclusion: This medium provides a novel way to study anaerobic growth of NG and may provide insights in to a nitrite-independent mechanism, possibly of physiological relevance.