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Infection and Immunity, June 2003, p. 3454-3462, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3454-3462.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Nontypeable Haemophilus influenzae Gene Expression Induced In Vivo in a Chinchilla Model of Otitis Media

Kevin M. Mason, Robert S. Munson Jr., and Lauren O. Bakaletz*

Columbus Children's Research Institute, Department of Pediatrics, The Ohio State University College of Medicine and Public Health, Columbus, Ohio

Received 13 January 2003/ Returned for modification 19 February 2003/ Accepted 13 March 2003

The gram-negative bacterium nontypeable Haemophilus influenzae (NTHI) is the predominant pathogen in chronic otitis media with effusion and, with Streptococcus pneumoniae and Moraxella catarrhalis, is a causative agent of acute otitis media. To identify potential virulence determinants, bacterial gene expression was monitored by differential fluorescence induction during early disease progression in one specific anatomical niche of a chinchilla model of NTHI-induced otitis media. Genomic DNA fragments from NTHI strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene. NTHI strain 86-028NP served as the host for the promoter trap library. Pools of 2,000 transformants were inoculated into the left and right middle ear cavities of chinchillas. Middle ear effusions were recovered by epitympanic tap at 24 and 48 h, and clones containing promoter elements that were induced in vivo and producing green fluorescent protein were isolated by two-color fluorescence-activated cell sorting. Insert DNA was sequenced and compared to the complete genome sequence of H. influenzae strain Rd. In a screen of 16,000 clones, we have isolated 44 clones that contain unique gene fragments encoding biosynthetic enzymes, metabolic and regulatory proteins, and hypothetical proteins of unknown function. An additional eight clones contain gene fragments unique to our NTHI isolate. Using quantitative reverse transcription-PCR, we have confirmed that 26 clones demonstrated increased gene expression in vivo relative to expression in vitro. These data provide insight into the response of NTHI bacteria as they sense and respond to the middle ear microenvironment during early events of otitis media.

* Corresponding author. Mailing address: Department of Pediatrics, The Ohio State University College of Medicine and Public Health, Columbus Children's Research Institute, 700 Children's Dr., Rm. W302, Columbus, OH 43205-2696. Phone: (614) 722-2915. Fax: (614) 722-2007. E-mail: BakaletL{at}pediatrics.ohio-state.edu.

Editor: D. L. Burns

Infection and Immunity, June 2003, p. 3454-3462, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3454-3462.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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