Abstract
Development of a vaccine to limit the impact of antibiotic resistant Neisseria gonorrhoeae is now a global priority. Serum bactericidal antibody (SBA) is a possible indicator of protective immunity to N. gonorrhoeae, but conventional assays measure colony forming units (CFU), which is time-consuming. A luminescent assay that quantifies ATP as a surrogate measure of bacterial viability was tested on N. gonorrhoeae strains FA1090, MS11 and P9-17 and compared to CFU-based readouts. There was a linear relationship between CFU and ATP levels for all three strains (r > 0.9). Normal human serum (NHS) is a common source of complement for SBA assays, but needs to be screened for non-specific bactericidal activity. NHS from 10 individuals were used for serum sensitivity assays—sensitivity values were significantly reduced with the ATP method for FA1090 (5/10, p < 0.05) and MS11 (10/10, p < 0.05), whereas P9-17 data were comparable for all donors. Our results suggest that measuring ATP underestimates serum sensitivity of N. gonorrhoeae and that the CFU method is a better approach. However, mouse anti-P9-17 outer membrane vesicles (OMV) SBA titres to P9-17 were comparable with both methods (r = 0.97), suggesting this assay can be used to rapidly screen sera for bactericidal antibodies to gonococci.
Highlights
Gonorrhoea is a sexually transmitted disease estimated to cause at least 78 million new cases worldwide per year [1]
Fresh cultures of bacteria were prepared from frozen stocks by streaking onto gonococcal agar (GCA) consisting of gonococcal (GC) agar base supplemented with 5 g/L bovine dried haemoglobin and 1% v/v IsoVitaleX (BD Biosciences, Franklin Lakes, NJ, USA)
To determine whether BacTiter-Glo is a viable choice to quantify N. gonorrhoeae survival, initial experiments compared ATP readings from three N. gonorrhoeae isolates across a broad range of colony forming units (CFU) (~200–107 CFU/well)
Summary
Gonorrhoea is a sexually transmitted disease estimated to cause at least 78 million new cases worldwide per year [1]. The causal organism, Neisseria gonorrhoeae, is listed as a high priority pathogen for research into novel treatments by the WHO [2] due to its ability to rapidly develop resistance to antibiotics [3]. Transmitted infections including N. gonorrhoeae have been linked with a significantly greater risk of contracting HIV [8]. Infection with N. gonorrhoeae results in an initial neutrophilic inflammatory response at the site of infection [9,10] and limited, short-lived humoral responses [11]. Experimental infection of human subjects [12] and longitudinal studies of high risk individuals [13,14] show that acquisition and subsequent clearance of an infection does not protect against further infections with N. gonorrhoeae
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