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Infection and Immunity, August 2007, p. 4158-4172, Vol. 75, No. 8
0019-9567/07/$08.00+0     doi:10.1128/IAI.00318-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Multiple Consecutive Lavage Samplings Reveal Greater Burden of Disease and Provide Direct Access to the Nontypeable Haemophilus influenzae Biofilm in Experimental Otitis Media

Section of Molecular Genetics, Division of Pediatric Infectious Diseases,¹ Division of Pediatric Infectious Diseases, The Maxwell Finland Laboratory for Infectious Diseases, Boston University School of Medicine, Boston Medical Center, Boston, Massachusetts 02118,² Department of Biostatistics, Boston University School of Public Health, Boston Medical Center, Boston, Massachusetts 02118,³ Division of Infectious Diseases and Immunology, University of Massachusetts Memorial Medical Center, Worcester, Massachusetts 01605⁴

Received 26 February 2007/ Returned for modification 30 March 2007/ Accepted 11 May 2007

The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.

Published ahead of print on 21 May 2007.

Editor: D. L. Burns

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