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Characterization of Genetic and Phenotypic Diversity of Invasive Nontypeable Haemophilus influenzae

Alice L. Erwin,^1* Kevin L. Nelson,¹ Tendai Mhlanga-Mutangadura,^2, Paul J. Bonthuis,¹ Jennifer L. Geelhood,¹ Gregory Morlin,² William C. T. Unrath,¹ Jose Campos,³ Derrick W. Crook,⁴ Monica M. Farley,⁵ Frederick W. Henderson,⁶ Richard F. Jacobs,⁷ Kathrin Mühlemann,⁸ Sarah W. Satola,⁵ Loek van Alphen,⁹ Miriam Golomb,² and Arnold L. Smith¹

Bacterial Pathogenesis Program, Seattle Biomedical Research Institute, Seattle, Washington 98109,¹ Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211,² Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera de Pozuelo, 28220 Majadahonda, Madrid, Spain,³ Infectious Diseases and Clinical Microbiology, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford, United Kingdom,⁴ Atlanta Veterans Affairs Medical Center and Department of Medicine, Emory University School of Medicine, Decatur, Georgia 30033,⁵ Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599,⁶ Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, Arkansas 72202,⁷ Institute for Infectious Diseases, University of Bern, Bern, Switzerland,⁸ Netherlands Vaccine Institute (NVI), NL-3720 AL Bilthoven, The Netherlands⁹

Received 7 February 2005/ Returned for modification 30 March 2005/ Accepted 10 May 2005

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.

Editor: J. N. Weiser

Present address: Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211.

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