Commensal non-pathogenic Neisseria spp. live within the human host alongside the pathogenic Neisseria meningitidis and Neisseria gonorrhoeae and due to natural competence, horizontal gene transfer within the genus is possible and has been observed. Four distinct Neisseria spp. isolates taken from the throats of two human volunteers have been assessed here using a combination of microbiological and bioinformatics techniques. Three of the isolates have been identified as Neisseria subflava biovar perflava and one as Neisseria cinerea . Specific gene clusters have been identified within these commensal isolate genome sequences that are believed to encode a Type VI Secretion System, a newly identified CRISPR system, a Type IV Secretion System unlike that in other Neisseria spp., a hemin transporter, and a haem acquisition and utilization system. This investigation is the first to investigate these systems in either the non-pathogenic or pathogenic Neisseria spp. In addition, the N. subflava biovar perflava possess previously unreported capsule loci and sequences have been identified in all four isolates that are similar to genes seen within the pathogens that are associated with virulence. These data from the four commensal isolates provide further evidence for a Neisseria spp. gene pool and highlight the presence of systems within the commensals with functions still to be explored.

Bacterial culture is the crucial step in diagnosingNeisseria gonorrhoeaeinfections and is the gold standard for determining their antimicrobial resistance profile. However, culture ofNeisseriaspp. can be challenging in resource poor areas, relying on specialist incubators supplying 5% CO2for growth of the bacteria. Even when such incubators are available, the CO2to run them may be scarce; there were CO2shortages during the COVID-19 pandemic, for example. Although culture jars with gas packs or candles can be used, these are inefficient in terms of use of incubator space and researcher time. To achieve simplicity in culturing ofN. gonorrhoeae, the standard Oxoid GC base medium, made with the Kellogg’s glucose and iron supplements was improved with the addition of 0.75 g/L sodium bicarbonate (NaHCO3), which is inexpensive and really available. This improved media was able to sustain gonococcal growth as well as standard GC media in 5% CO2. Chocolate agar and Thayer-Martin agar with sodium bicarbonate was also developed, with all showing good growth ofN. gonorrhoeaewithout the need for atmospheric CO2.