Essential role of Bordetella NadC in a quinolinate salvage pathway for NAD biosynthesis.

Nicotinamide adenine dinucleotide (NAD) is produced via de novo biosynthesis pathways and by salvage or recycling routes. The classical Bordetella bacterial species are known to be auxotrophic for nicotinamide or nicotinic acid. This study confirmed that Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis have the recycling/salvage pathway genes pncA and pncB, for use of nicotinamide or nicotinic acid, respectively, for NAD synthesis. Although these Bordetellae lack the nadA and nadB genes needed for de novo NAD biosynthesis, remarkably, they have one de novo pathway gene, nadC, encoding quinolinate phosphoribosyltransferase. Genomic analyses of taxonomically related Bordetella and Achromobacter species also indicated the presence of an 'orphan' nadC and the absence of nadA and nadB. When supplied as the sole NAD precursor, quinolinate promoted B. bronchiseptica growth, and the ability to use it required nadC. Co-expression of Bordetella nadC with the nadB and nadA genes of Paraburkholderia phytofirmans allowed B. bronchiseptica to grow in the absence of supplied pyridines, indicative of de novo NAD synthesis and functional confirmation of Bordetella NadC activity. Expression of nadC in B. bronchiseptica was influenced by nicotinic acid and by a NadQ family transcriptional repressor, indicating that these organisms prioritize their use of pyridines for NAD biosynthesis.

Inappropriate use of D-values for determining biocidal activity of various antimicrobials.

The objective of this study was to investigate the application of established D-value calculations to survival curves for various bacteria using the following antimicrobials: acidified sodium chlorite, triclosan, octanoic acid, and sodium hydroxide. D-values can be calculated in 3 ways, a linear regression, an endpoint calculation, or an average of multiple endpoint calculations. The assumption made in calculating a D-value is that the rate of kill follows 1st-order kinetics under specified treatment conditions. Each antimicrobial solution was challenged with approximately 108 CFU/mL of Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica subsp. enterica, and Escherichia coli independently and in triplicate. Test systems were sampled at each of the 10 time points over a period of 7 min, neutralized, pour plated then incubated at 35 °C for 48 h (AOAC official method 960.09). Survival curves using the log-transformed data were calculated using regression analysis. Correlations coefficients for all linear regression analyses ranged between 0.291 and 0.982, with 6 of the 16 different treatment systems having an R2 value below 0.7. Methods used for calculating D-values should lead to the same result if the survival curve in a given condition is linear. The calculated D-values were different using endpoint analysis (Stumbo method), linear regression, and average of multiple endpoints. This study demonstrates the nonlinearity of inactivation curves of antimicrobials. D-value estimations cannot be reliably used to illustrate biocidal activity in antimicrobial test systems.