Molecular characterisation of Haemophilus influenzae type a and untypeable strains isolated simultaneously from cerebrospinal fluid and blood: novel use.

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Molecular characterisation of Haemophilus influenzae type a and untypeable strains isolated simultaneously from cerebrospinal fluid and blood: novel use of quantitative real-time PCR based on the cap copy number to determine virulence K. Ohkusu, K.A. Nash, C.B. Inderlied Clinical Microbiology and Infection Volume 11, Issue 8, Pages 637-643 (August 2005) DOI: 10.1111/j.1469-0691.2005.01203.x Copyright © 2005 European Society of Clinical Infectious Diseases Terms and Conditions

Fig. 1 Pulsed-field gel fingerprints of Haemophilus influenzae isolates digested with (a) SmaI, (b) ApaI, (c) SacII, or (d) NaeI. (e) The c. 530-kb SmaI fragment from the cerebrospinal fluid isolate (KO100), and the c. 495-kb SmaI fragment from the blood isolate (KO101) following digestion with KpnI. M, lambda ladder molecular size marker. Arrows indicate a single band shift difference between KO100 and KO101 of c. 35 kb. Clinical Microbiology and Infection 2005 11, 637-643DOI: (10.1111/j.1469-0691.2005.01203.x) Copyright © 2005 European Society of Clinical Infectious Diseases Terms and Conditions

Fig. 2 Southern hybridisation analysis of SmaI or ApaI digests with a 1.1-kb probe for serotype a-specific genes. (a) Chromosomal DNA digested with SmaI or ApaI and separated by field inversion gel electrophoresis. (b) Southern blot of chromosomal DNA from KO100 and KO101 hybridised to the 1.1-kb type a-specific probe. M, lambda ladder molecular size marker. Clinical Microbiology and Infection 2005 11, 637-643DOI: (10.1111/j.1469-0691.2005.01203.x) Copyright © 2005 European Society of Clinical Infectious Diseases Terms and Conditions

Fig. 3 Schematic diagram of the cap locus in KO100 and KO101. (a) The KO100 locus consists of directly repeated duplication of cap genes, with each set containing three DNA segments flanked by copies of IS1016, region I, region II and region III, as indicated. KO101 has one copy of IS1016. (b) Primer sets A, B, C, D, E, F, G and H were used to generate the amplification products indicated. CSF, cerebrospinal fluid. Clinical Microbiology and Infection 2005 11, 637-643DOI: (10.1111/j.1469-0691.2005.01203.x) Copyright © 2005 European Society of Clinical Infectious Diseases Terms and Conditions