Methods: A. naeslundii strains (n=107) isolated from human clinical and oral samples (caries-active and caries-free subjects) including type and reference strains of A. naeslundii genospecies 1, 2 and WVA963 and A. viscosus, were cultivated on fastidious anaerobes agar. All selected isolates were sialidase positive. A fragment size of 1077 nucleotides was obtained from all isolates, which covered the active site and the five bacterial neuraminidase repeats (BNRs) found in the gene. The sequence data were aligned in-frame, and recombination events between and within genospecies were investigated.
Results: Data analyses indicated that the gene is under stabilizing selective pressure (mean value for dN/dS < 1), also confirmed by the non-significant D value in the Tajima's test (D = -1.67906, P > 0.05). The Phi test in SplitsTree4 revealed statistically significant evidence for recombination (P < 10-10). The Recombination Detection Program (RDPv3.24) also identified extensive recombination events within and between genospecies 1 and 2, with the various methods (Max Chi, Chimaera, and SiScan). Overall, between 5.6% (for BNR1) and 39.3% (for BNR4) of isolates exhibited amino acid polymorphism within the BNRs. The active site regions exhibited limited variation. Conclusion: The nanH gene of A. naeslundii genospecies 1, 2 and WVA963 exhibited the conserved structure of sialidase genes. There is clear evidence of recombination between genospecies 1 and genospecies 2.
Funded in part by the Welcome Trust.