The genetic similarity between isolates was calculated with the Jaccard index.
Average pairwise diversity between isolates was calculated from the 3,714-bp 3,714-bpated sequenconcatenatedloci for each isequenceofned in-frame using MEGA4 (27 ).
The genetic distance between individual isolates was calculated using the binary data with all isolates, including the references, PowerMarker software (Liu and Muse 2005), and the neighbor-joining (NJ) method based on the proportion of shared allele distance.
Significance of selection between groups of isolates was calculated with a two-tailed t-test.
High quality SNVs between the shared genome portion of each isolate were calculated as recently described for V. cholerae [ 18] by mapping the trimmed reads against the RIMD 2210633 genome [ 13] and considering only SNVs with depth coverage higher than 0.4 times the average depth of coverage for each strain (values varied from 8 to 16), and a minimum variant frequency of 75%.
Viable counts for each isolate were calculated every second day.
Mutation rate differences between the two isolate groups were calculated from the interaction effects of a standard linear regression.
Genetic distance was calculated on the basis of the number of shared bands between isolates, and similarity matrices were calculated by using the Dice coefficient (14 ).
To demonstrate the degree to which isolates have diverged from year to year, nucleotide sequences were grouped by year and the average pairwise distances between groups were calculated.
Genetic divergence and gene flow (F ST ) were calculated between 58 gonococcal isolates collected in Liverpool (1981 1989), 38 gonococcal isolates collected in Liverpool between 2000 and 2001, 53 gonococcal isolates collected worldwide, 217 Czech carried meningococci and 103 N. lactamica isolates (Table 3).
