Development and characterization of a pooled Haemophilus influenzae genomic library for the evaluation of gene expression changes associated with mucosal biofilm formation in otitis media

2003 ◽  
Vol 67 (7) ◽  
pp. 749-755 ◽  
Author(s):  
Geza Erdos ◽  
Sameera Sayeed ◽  
Patricia Antalis ◽  
Fen Ze Hu ◽  
Jay Hayes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Biofilm Formation ◽  
Genomic Library

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

2003 ◽  
Vol 71 (6) ◽  
pp. 3454-3462 ◽  
Author(s):  
Kevin M. Mason ◽  
Robert S. Munson ◽  
Lauren O. Bakaletz
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Fluorescent Protein ◽  
Otitis Media With Effusion ◽  
Acute Otitis Media ◽  
Bacterial Gene ◽  
Nthi Strain

ABSTRACT 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.

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

2007 ◽  
Vol 75 (8) ◽  
pp. 4158-4172 ◽  
Author(s):  
Magali Leroy ◽  
Howard Cabral ◽  
Marisol Figueira ◽  
Valérie Bouchet ◽  
Heather Huot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Vaccine Development ◽  
Ex Vivo ◽  
Direct Analysis ◽  
Direct Access ◽  
Nontypeable Haemophilus Influenzae ◽  
Nasopharyngeal Colonization

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.

scholarly journals Haemophilus influenzae biofilm formation in chronic otitis media with effusion

2016 ◽  
Vol 273 (11) ◽  
pp. 3553-3560 ◽  
Author(s):  
Helen Van Hoecke ◽  
Ann-Sophie De Paepe ◽  
Edward Lambert ◽  
Jonas D Van Belleghem ◽  
Piet Cools ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Biofilm Formation ◽  
Otitis Media With Effusion ◽  
Chronic Otitis Media

scholarly journals Epigenetic Regulation Alters Biofilm Architecture and Composition in Multiple Clinical Isolates of Nontypeable Haemophilus influenzae

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Kenneth L. Brockman ◽  
Patrick N. Azzari ◽  
M. Taylor Branstool ◽  
John M. Atack ◽  
Benjamin L. Schulz ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Biofilm Formation ◽  
Human Pathogens ◽  
Content Type ◽  
Alkaline Conditions ◽  
Tract Infections ◽  
Biofilm Structure

ABSTRACT Biofilms play a critical role in the colonization, persistence, and pathogenesis of many human pathogens. Multiple mucosa-associated pathogens have evolved a mechanism of rapid adaptation, termed the phasevarion, which facilitates a coordinated regulation of numerous genes throughout the bacterial genome. This epigenetic regulation occurs via phase variation of a DNA methyltransferase, Mod. The phasevarion of nontypeable Haemophilus influenzae (NTHI) significantly affects the severity of experimental otitis media and regulates several disease-related processes. However, the role of the NTHI phasevarion in biofilm formation is unclear. The present study shows that the phasevarions of multiple NTHI clinical isolates regulate in vitro biofilm formation under disease-specific microenvironmental conditions. The impact of phasevarion regulation was greatest under alkaline conditions that mimic those known to occur in the middle ear during disease. Under alkaline conditions, NTHI strains that express the ModA2 methyltransferase formed biofilms with significantly greater biomass and less distinct architecture than those formed by a ModA2-deficient population. The biofilms formed by NTHI strains that express ModA2 also contained less extracellular DNA (eDNA) and significantly less extracellular HU, a DNABII DNA-binding protein critical for biofilm structural stability. Stable biofilm structure is critical for bacterial pathogenesis and persistence in multiple experimental models of disease. These results identify a role for the phasevarion in regulation of biofilm formation, a process integral to the chronic nature of many infections. Understanding the role of the phasevarion in biofilm formation is critical to the development of prevention and treatment strategies for these chronic diseases. IMPORTANCE Upper respiratory tract infections are the number one reason for a child to visit the emergency department, and otitis media (middle ear infection) ranks third overall. Biofilms contribute significantly to the chronic nature of bacterial respiratory tract infections, including otitis media, and make these diseases particularly difficult to treat. Several mucosa-associated human pathogens utilize a mechanism of rapid adaptation termed the phasevarion, or phase variable regulon, to resist environmental and host immune pressures. In this study, we assessed the role of the phasevarion in regulation of biofilm formation by nontypeable Haemophilus influenzae (NTHI), which causes numerous respiratory tract diseases. We found that the NTHI phasevarion regulates biofilm structure and critical biofilm matrix components under disease-specific conditions. The findings of this work could be significant in the design of improved strategies against NTHI infections, as well as diseases due to other pathogens that utilize a phasevarion.

Detection and characterization of pediatric serum antibody to the OMP P5-homologous adhesin of nontypeable Haemophilus influenzae during acute otitis media

Vaccine ◽  
2002 ◽  
Vol 20 (29-30) ◽  
pp. 3590-3597 ◽  
Author(s):  
Laura A Novotny ◽  
Michael E Pichichero ◽  
Philippe A Denoël ◽  
Cecil Neyt ◽  
Sylvie Vanderschrick ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Serum Antibody ◽  
Acute Otitis Media ◽  
Nontypeable Haemophilus Influenzae

scholarly journals Incorporation of Phosphorylcholine into the Lipooligosaccharide of Nontypeable Haemophilus influenzae Does Not Correlate with the Level of Biofilm FormationIn Vitro

2014 ◽  
Vol 82 (4) ◽  
pp. 1591-1599 ◽  
Author(s):  
Carmen Puig ◽  
Sara Marti ◽  
Peter W. M. Hermans ◽  
Marien I. de Jonge ◽  
Carmen Ardanuy ◽  
...  
Keyword(s):  
Chronic Obstructive Pulmonary Disease ◽  
Haemophilus Influenzae ◽  
Pulmonary Disease ◽  
Otitis Media ◽  
Middle Ear ◽  
Biofilm Formation ◽  
Community Acquired Pneumonia ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease ◽  
Middle Ear Fluid

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is an opportunistic pathogen that causes otitis media in children and community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium. The objective of this report was to determine the relationship between the presence of phosphorylcholine in the lipooligosaccharide of NTHi and the level of biofilm formation. The study was performed on 111 NTHi clinical isolates collected from oropharyngeal samples of healthy children, middle ear fluid of children with otitis media, and sputum samples of patients with chronic obstructive pulmonary disease or community-acquired pneumonia. NTHi clinical isolates presented a large variation in the level of biofilm formation in a static assay and phosphorylcholine content. Isolates collected from the oropharynx and middle ear fluid of children tended to have more phosphorylcholine and made denser biofilms than isolates collected from sputum samples of patients with chronic obstructive pulmonary disease or community-acquired pneumonia. No correlation was observed between biofilm formation and the presence of phosphorylcholine in the lipooligosaccharide for either planktonic or biofilm growth. This lack of correlation was confirmed by abrogating phosphorylcholine incorporation into lipooligosaccharide throughlicAgene deletion, which had strain-specific effects on biofilm formation. Altogether, we present strong evidence to conclude that there is no correlation between biofilm formation in a static assay and the presence of phosphorylcholine in lipooligosaccharide in a large collection of clinical NTHi isolates collected from different groups of patients.

scholarly journals Increased Biofilm Formation by Nontypeable Haemophilus influenzae Isolates from Patients with Invasive Disease or Otitis Media versus Strains Recovered from Cases of Respiratory Infections

2014 ◽  
Vol 80 (22) ◽  
pp. 7088-7095 ◽  
Author(s):  
Carmen Puig ◽  
Arnau Domenech ◽  
Junkal Garmendia ◽  
Jeroen D. Langereis ◽  
Pascal Mayer ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Respiratory Disease ◽  
Otitis Media ◽  
Crystal Violet ◽  
Solid Surface ◽  
Biofilm Formation ◽  
Invasive Disease ◽  
Crystal Violet Staining ◽  
Content Type ◽  
Initial Adhesion

ABSTRACTBiofilm formation by nontypeable (NT)Haemophilus influenzaeremains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NTH. influenzaeby comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NTH. influenzaeisolation, with significantly increased biofilm formation for NTH. influenzaeisolates collected from patients with invasive disease and OM compared with NTH. influenzaeisolates from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.

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