A rapid and sensitive procedure for determination of 5-N-acetyl neuraminicacid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H-influenzae strains

In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathog enesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is descri bed and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange ch romatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N -acetyl neuraminic acid residues released by neuraminidase treatment of O-d eacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by H-1 NMR and electrospray ionisation-mass spectrometry (ESI-MS). The ana lysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid wa s found to be a common constituent of LPS in NTHi. Only one strain (NTHi 43 2) did not show any sialylation. Molar ratios (LPS /5-N-acetyl neuraminic a cid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl n euraminic acid could be determined by other methods including 1H NMR and ES I-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD pro cedure. The method was applied to determine levels of terminal 5-N-acetyl n euraminic acid in LPS from NTHi strains grown under different conditions an d mutant strains containing inactive LPS biosynthetic genes. (C) 2001 Elsev ier Science Ltd. All rights reserved.