title: Multiple consecutive lavage samplings reveal greater burden of disease and provide direct access to the nontypeable Haemophilus influenzae biofilm in experimental otitis media.
abstract: 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.
authors.affiliation: Section of Molecular Genetics, Division of Pediatric Infectious Diseases, The Maxwell Finland Laboratory for Infectious Diseases, Boston University School of Medicine, Boston Medical Center, 774 Albany Street, Boston, MA 02118, USA.
authors.forename: Magali
authors.initials: M
authors.lastname: Leroy
authors.forename: Howard
authors.initials: H
authors.lastname: Cabral
authors.forename: Marisol
authors.initials: M
authors.lastname: Figueira
authors.forename: Valérie
authors.initials: V
authors.lastname: Bouchet
authors.forename: Heather
authors.initials: H
authors.lastname: Huot
authors.forename: Sanjay
authors.initials: S
authors.lastname: Ram
authors.forename: Stephen I
authors.initials: SI
authors.lastname: Pelton
authors.forename: Richard
authors.initials: R
authors.lastname: Goldstein
mesh_terms: D000818:Animals; D018441:Biofilms; D001826:Body Fluids / Q000382:microbiology*; D002682:Chinchilla; D015169:Colony Count, Microbial; D004195:Disease Models, Animal; D004432:Ear, Middle / Q000382:microbiology; D020869:Gene Expression Profiling; D006192:Haemophilus Infections / Q000382:microbiology*; D006193:Haemophilus influenzae / Q000302:isolation & purification* / Q000502:physiology; D008856:Microscopy, Fluorescence; D010033:Otitis Media / Q000382:microbiology*; D007507:Therapeutic Irrigation