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 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 LuxS Promotes Biofilm Maturation and Persistence of Nontypeable Haemophilus influenzae In Vivo via Modulation of Lipooligosaccharides on the Bacterial Surface

2009 ◽  
Vol 77 (9) ◽  
pp. 4081-4091 ◽  
Author(s):  
Chelsie E. Armbruster ◽  
Wenzhou Hong ◽  
Bing Pang ◽  
Kristin E. Dew ◽  
Richard A. Juneau ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Opportunistic Infections ◽  
Bacterial Species ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nontypeable Haemophilus Influenzae ◽  
Bacterial Persistence ◽  
Soluble Mediator

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

scholarly journals Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media

2006 ◽  
Vol 75 (2) ◽  
pp. 958-965 ◽  
Author(s):  
Wenzhou Hong ◽  
Kevin Mason ◽  
Joseph Jurcisek ◽  
Laura Novotny ◽  
Lauren O. Bakaletz ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Fluorescent Antibody ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Bacterial Surface ◽  
Culture Cells ◽  
Antibody Staining

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a leading causative agent of otitis media. Much of the inflammation occurring during NTHi disease is initiated by lipooligosaccharides (LOS) on the bacterial surface. Phosphorylcholine (PCho) is added to some LOS forms in a phase-variable manner, and these PCho+ variants predominate in vivo. Thus, we asked whether this modification confers some advantage during infection. Virulence of an otitis media isolate (NTHi strain 86-028NP) was compared with that of an isogenic PCho transferase (licD) mutant using a chinchilla (Chinchilla lanigera) model of otitis media. Animals infected with NTHi 86-028NP licD demonstrated increased early inflammation and a delayed increase in bacterial counts compared to animals infected with NTHi 86-028NP. LOS purified from chinchilla-passed NTHi 86-028NP had increased PCho content compared to LOS purified from the inoculum. Both strains were recovered from middle ear fluids as long as 14 days postinfection. Biofilms were macroscopically visible in the middle ears of euthanized animals infected with NTHi 86-028NP 7 days and 14 days postchallenge. Conversely, less dense biofilms were observed in animals infected with NTHi 86-028NP licD 7 days postinfection, and none of the animals infected with NTHi 86-028NP licD had a visible biofilm by 14 days. Fluorescent antibody staining revealed PCho+ variants within biofilms, similar to our prior results with tissue culture cells in vitro (S. L. West-Barnette, A. Rockel, and W. E. Swords, Infect. Immun. 74:1828-1836, 2006). Animals coinfected with equal proportions of both strains had equal persistence of each strain and somewhat greater severity of disease. We thus conclude that PCho promotes NTHi infection and persistence by reducing the host inflammatory response and by promoting formation of stable biofilm communities.

scholarly journals Nasal Delivery of a Commensal Pasteurellaceae Species Inhibits Nontypeable Haemophilus influenzae Colonization and Delays Onset of Otitis Media in Mice

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Caitlyn M. Granland ◽  
Naomi M. Scott ◽  
Jean-Francois Lauzon-Joset ◽  
Jeroen D. Langereis ◽  
Camilla de Gier ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Influenza A ◽  
Clinical Score ◽  
Nasal Delivery ◽  
Tissue Homogenate ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type

ABSTRACT Nasopharyngeal colonization with nontypeable Haemophilus influenzae (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi colonization may prevent disease development. We previously demonstrated that Haemophilus haemolyticus, a closely related human commensal, can inhibit NTHi colonization and infection of human respiratory epithelium in vitro. We have now assessed whether Muribacter muris (a rodent commensal from the same family) can prevent NTHi colonization and disease in vivo using a murine NTHi otitis media model. Otitis media was modeled in BALB/c mice using coinfection with 1 × 104.5 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 107 CFU of NTHi R2866 Specr. Mice were pretreated or not with an intranasal inoculation of 5 × 107 CFU M. muris 24 h before coinfection. NTHi and M. muris viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1β [IL-1β], IL-6, keratinocyte chemoattractant [KC], and IL-10) were measured in nasal washes and middle ear tissue homogenate. M. muris pretreatment decreased the median colonization density of NTHi from 6 × 105 CFU/ml to 9 × 103 CFU/ml (P = 0.0004). Only 1/12 M. muris-pretreated mice developed otitis media on day 5 compared to 8/15 mice with no pretreatment (8% versus 53%, P = 0.0192). Inflammation, clinical score, and weight loss were also lower in M. muris-pretreated mice. We have demonstrated that a single dose of a closely related commensal can delay onset of NTHi otitis media in vivo. Human challenge studies investigating prevention of NTHi colonization are warranted to reduce the global burden of otitis media and other NTHi diseases.

scholarly journals Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine

BMC Medicine ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Rebecca Webster ◽  
Silvana Sekuloski ◽  
Anand Odedra ◽  
Stephen Woolley ◽  
Helen Jennings ◽  
...  
Keyword(s):  
Gene Expression ◽  
Malaria Vaccine ◽  
Vaccine Development ◽  
Ex Vivo ◽  
Clinical Investigation ◽  
Blood Stage ◽  
Trial Registration ◽  
Influenza B ◽  
Gene Encoding

Abstract Background There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. Methods The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). Results None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. Conclusions This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. Trial registration Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369; date: 06 June 2017).

scholarly journals Nasopharyngeal Colonization With Nontypeable Haemophilus Influenzae And Recurrent Otitis Media

1994 ◽  
Vol 170 (4) ◽  
pp. 862-866 ◽  
Author(s):  
Y. Harabuchi ◽  
H. Faden ◽  
N. Yamanaka ◽  
L. Duffy ◽  
J. Wolf ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Nontypeable Haemophilus Influenzae ◽  
Nasopharyngeal Colonization ◽  
Recurrent Otitis Media

scholarly journals Nasopharyngeal Colonization with Nontypeable Haemophilus influenzae in Chinchillas

1998 ◽  
Vol 66 (5) ◽  
pp. 1973-1980 ◽  
Author(s):  
Yan-ping Yang ◽  
Sheena M. Loosmore ◽  
Brian J. Underdown ◽  
Michel H. Klein
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Candidate Vaccine ◽  
Nontypeable Haemophilus Influenzae ◽  
Nasopharyngeal Colonization ◽  
Ear Infection ◽  
Middle Ear Infection ◽  
Significant Difference ◽  
Vaccine Antigens

ABSTRACT Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.

Epidemiology of Nasopharyngeal Colonization with Nontypeable Haemophilus influenzae in the First 2 Years of Life

1995 ◽  
Vol 172 (1) ◽  
pp. 132-135 ◽  
Author(s):  
H. Faden ◽  
L. Duffy ◽  
A. Williams ◽  
D. A. Krystofik ◽  
J. Wolf ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Nontypeable Haemophilus Influenzae ◽  
Nasopharyngeal Colonization

scholarly journals Healthy versus inflamed lung environments differentially effect MSCs

2021 ◽  
pp. 2004149
Author(s):  
Sara Rolandsson Enes ◽  
Thomas H. Hampton ◽  
Jayita Barua ◽  
David H. McKenna ◽  
Claudia C. dos Santos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Healthy Volunteers ◽  
Cell Injury ◽  
Ex Vivo ◽  
Lavage Fluid ◽  
Cell Therapies ◽  
Inflammatory Microenvironment ◽  
Self Recognition ◽  
Clinical Investigations

BackgroundDespite increased interest in MSC-based cell therapies for the acute respiratory distress syndrome (ARDS), clinical investigations have not yet been successful and understanding of the potential in vivo mechanisms of MSC actions in ARDS remain limited. ARDS is driven by an acute severe innate immune dysregulation, often characterised by inflammation, coagulation, and cell injury. How this inflammatory microenvironment influences MSC functions remains to be determined.AimTo comparatively assess how the inflammatory environment present in ARDS lungs versus the lung environment present in healthy volunteers alters MSC behaviors.MethodsClinical grade human bone marrow-derived MSCs (hMSCs) were exposed to bronchoalveolar lavage fluid (BALF) samples obtained from ARDS patients or from healthy volunteers. Following exposure, hMSCs and their conditioned media were evaluated for a broad panel of relevant properties including viability, levels of expression of inflammatory cytokines, gene expression, cell surface HLA expression, and activation of coagulation and complement pathways.ResultsPro-inflammatory, pro-coagulant, and major histocompatibility complex (self recognition) related gene expression was markedly up-regulated in hMSCs exposed ex vivo to BALF obtained from healthy volunteers. In contrast, these changes were less apparent and often opposite in hMSCs exposed to ARDS BALF samples.ConclusionThese data provide new insights into how hMSCs behave in healthy versus inflamed lung environments strongly suggesting that the inflamed environment in ARDS induces hMSC responses potentially benefical for cell survival and actions. This further highlights the need to understand how different disease environments affect hMSC functions.

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