scholarly journals Modified Lipooligosaccharide Structure Protects Nontypeable Haemophilus influenzae from IgM-Mediated Complement Killing in Experimental Otitis Media

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Jeroen D. Langereis ◽  
Kim Stol ◽  
Elke K. Schweda ◽  
Brigitte Twelkmeyer ◽  
Hester J. Bootsma ◽  
...  
Keyword(s):  
Mouse Model ◽  
Haemophilus Influenzae ◽  
Molecular Mechanism ◽  
Otitis Media ◽  
Middle Ear ◽  
Crucial Role ◽  
Influenza A ◽  
Gram Negative ◽  
Content Type ◽  
Complement Resistance

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a Gram-negative, human-restricted pathogen. Although this bacterium typically colonizes the nasopharynx in the absence of clinical symptoms, it is also one of the major pathogens causing otitis media (OM) in children. Complement represents an important aspect of the host defense against NTHi. In general, NTHi is efficiently killed by complement-mediated killing; however, various resistance mechanisms have also evolved. We measured the complement resistance of NTHi isolates isolated from the nasopharynx and the middle ear fluids of OM patients. Furthermore, we determined the molecular mechanism of NTHi complement resistance. Complement resistance was strongly increased in isolates from the middle ear, which correlated with decreased binding of IgM. We identified a crucial role for the R2866_0112 gene in complement resistance. Deletion of this gene altered the lipooligosaccharide (LOS) composition of the bacterium, which increased IgM binding and complement-mediated lysis. In a novel mouse model of coinfection with influenza virus, we demonstrate decreased virulence for the R2866_0112 deletion mutant. These findings identify a mechanism by which NTHi modifies its LOS structure to prevent recognition by IgM and activation of complement. Importantly, this mechanism plays a crucial role in the ability of NTHi to cause OM.IMPORTANCENontypeableHaemophilus influenzae(NTHi) colonizes the nasopharynx of especially young children without any obvious symptoms. However, NTHi is also a major pathogen in otitis media (OM), one of the most common childhood infections. Although this pathogen is often associated with OM, the mechanism by which this bacterium is able to cause OM is largely unknown. Our study addresses a key biological question that is highly relevant for child health: what is the molecular mechanism that enables NTHi to cause OM? We show that isolates collected from the middle ear fluid exhibit increased complement resistance and that the lipooligosaccharide (LOS) structure determines IgM binding and complement activation. Modification of the LOS structure decreased NTHi virulence in a novel NTHi-influenza A virus coinfection OM mouse model. Our findings may also have important implications for other Gram-negative pathogens harboring LOS, such asNeisseria meningitidis,Moraxella catarrhalis, andBordetella pertussis.

scholarly journals Influenza-Induced Inflammation Drives Pneumococcal Otitis Media

2013 ◽  
Vol 81 (3) ◽  
pp. 645-652 ◽  
Author(s):  
Kirsty R. Short ◽  
Patrick C. Reading ◽  
Lorena E. Brown ◽  
John Pedersen ◽  
Brad Gilbertson ◽  
...  
Keyword(s):  
Otitis Media ◽  
Middle Ear ◽  
Influenza A ◽  
Pneumococcal Disease ◽  
Influenza Viruses ◽  
Dependent Manner ◽  
Proinflammatory Response ◽  
Content Type ◽  
Infant Mouse ◽  
Human Middle Ear

ABSTRACTInfluenza A virus (IAV) predisposes individuals to secondary infections with the bacteriumStreptococcus pneumoniae(the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.

scholarly journals Antibodies Mediate Formation of Neutrophil Extracellular Traps in the Middle Ear and Facilitate Secondary Pneumococcal Otitis Media

2013 ◽  
Vol 82 (1) ◽  
pp. 364-370 ◽  
Author(s):  
Kirsty R. Short ◽  
Maren von Köckritz-Blickwede ◽  
Jeroen D. Langereis ◽  
Keng Yih Chew ◽  
Emma R. Job ◽  
...  
Keyword(s):  
Otitis Media ◽  
Middle Ear ◽  
Influenza A ◽  
Bacterial Load ◽  
Neutrophil Extracellular Trap ◽  
Content Type ◽  
Middle Ear Infection ◽  
Host Inflammatory Response ◽  
Insight Into ◽  
Bacterial Outgrowth

ABSTRACTOtitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) andStreptococcus pneumoniae(the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth ofS. pneumoniaein the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

scholarly journals Correlation between Presence of Viable Bacteria and Presence of Endotoxin in Middle-Ear Effusions

1998 ◽  
Vol 36 (11) ◽  
pp. 3417-3419 ◽  
Author(s):  
Jeffrey R. Dingman ◽  
Mark G. Rayner ◽  
Suman Mishra ◽  
Yingze Zhang ◽  
Miles D. Ehrlich ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Moraxella Catarrhalis ◽  
Pediatric Patients ◽  
Chronic Otitis Media ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Viable Bacteria

The presence of endotoxin (detected by the Limulusamebocyte lysate assay) was compared to the presence of viableHaemophilus influenzae and Moraxella catarrhalis (detected by PCR) in 106 middle-ear effusions from pediatric patients with chronic otitis media. Endotoxin was found in 81 of the 106 specimens. Of these 81 specimens, 66 (81.5%) also tested positive for one or both of the gram-negative bacteria H. influenzae and M. catarrhalis. The data suggest that viable gram-negative bacteria, detectable by PCR but often undetectable by culture, may be the source of endotoxin in middle-ear effusions.

scholarly journals Efficacy of Solithromycin (CEM-101) for Experimental Otitis Media Caused by Nontypeable Haemophilus influenzae and Streptococcus pneumoniae

2016 ◽  
Vol 60 (9) ◽  
pp. 5533-5538 ◽  
Author(s):  
M. Figueira ◽  
P. Fernandes ◽  
S. I. Pelton
Keyword(s):  
Streptococcus Pneumoniae ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Fourth Generation ◽  
Minimal Bactericidal Concentration ◽  
Nontypeable Haemophilus Influenzae ◽  
Time Curve ◽  
Content Type ◽  
Middle Ear Fluid

ABSTRACTSolithromycin (CEM-101) is a “fourth-generation” macrolide, as it has three binding site and is acid stable. The three binding sites confer activity against bacteria resistant to the older macrolides and ketolides, including multidrug-resistantStreptococcus pneumoniaeand nontypeableHaemophilus influenzae(NTHi). The objective of this study was to evaluate solithromycin pharmacokinetics (PK), middle ear fluid (MEF) concentrations, and microbiologic efficacy in a chinchilla model of experimental otitis media (EOM) due to strains ofS. pneumoniaeor NTHi. Plasma PK (maximum concentration of drug in serum [Cmax] and area under the concentration-time curve from 0 to 24 h [AUC0–24]) and middle ear fluid (MEF) concentrations were determined. Isolates with specified antimicrobial susceptibility patterns were inoculated directly into the middle ear (ME). Plasma and MEF were collected for PK and MEF cultures performed to determine efficacy. Solithromycin administered at 150 mg/kg of body weight/day resulted inCmaxand AUC0–24values of 2.2 μg/ml and 27.4 μg · h/ml in plasma and 1.7 μg/ml and 28.2 μg · h/ml in extracellular MEF on day 1. By day 3,Cmaxand AUC0–24values had increased to 4.5 μg/ml and 54 μg · h/ml in plasma and 4.8 μg/ml and 98.6 μg · h/ml in extracellular MEF. For NTHi EOM, three isolates with MIC/minimal bactericidal concentration (MBC) ratios of 0.5/1 μg/ml (isolate BCH1), 2/2 μg/ml (isolate BMC1247C), and 4/4 μg/ml (isolate BMC1213C) were selected. The MEF of >85% of animals infected with BCH1 and BMC1247C was sterilized. For NTHi BMC1213, >85% of MEF cultures remained positive. ForS. pneumoniaeEOM, 3 isolates with MIC/MBC ratios of 0.06/0.125 μg/ml (S. pneumoniae331), 0.125/1 μg/ml (S. pneumoniaeCP-645 [MLSBphenotype]), and 0.5/2 μg/ml (CP-712 [mefAsubclassmefAresistance]) were selected. Solithromycin sterilized MEF in 100% of animals infected withS. pneumoniae331 andS. pneumoniaeCP-645. ME infection persisted in 60% of animals infected with CP-712. In a model of EOM, solithromycin sterilized MEF in >85% of animals challenged with NTHi with an MIC of ≤2 μg/ml and 100% of ME infected withS. pneumoniaewith an MIC of ≤0.125 μg/ml.

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 Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Rachael L. Hardison ◽  
Derek R. Heimlich ◽  
Alistair Harrison ◽  
Wandy L. Beatty ◽  
Sarah Rains ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Bacterial Communities ◽  
Heme Iron ◽  
Content Type ◽  
Iron Restriction ◽  
Intracellular Bacterial Communities ◽  
Intracellular Fate ◽  
Recurrent Otitis Media

ABSTRACTNutrient limitation restricts bacterial growth in privileged sites such as the middle ear. Transient heme-iron restriction of nontypeableHaemophilus influenzae(NTHI), the major causative agent of chronic and recurrent otitis media (OM), promotes new and diverse phenotypes that can influence planktonic, biofilm, and intracellular lifestyles of NTHI. However, the bacterial responses to nutrient restriction that impact intracellular fate and survival of NTHI are unknown. In this work, we provide evidence for the role of transient heme-iron restriction in promoting the formation of intracellular bacterial communities (IBCs) of NTHI bothin vitroandin vivoin a preclinical model of OM. We show that transient heme-iron restriction of NTHI results in significantly increased invasion and intracellular populations that escape or evade the endolysosomal pathway for increased intracellular survival. In contrast, NTHI continuously exposed to heme-iron traffics through the endolysosomal pathway for degradation. The use of pharmacological inhibitors revealed that prior heme-iron status does not appear to influence NTHI internalization through endocytic pathways. However, inhibition of macropinocytosis altered the intracellular fate of transiently restricted NTHI for degradation in the endolysosomal pathway. Furthermore, prevention of macropinocytosis significantly reduced the number of IBCs in cultured middle ear epithelial cells, providing evidence for the feasibility of this approach to reduce OM persistence. These results reveal that microenvironmental cues can influence the intracellular fate of NTHI, leading to new mechanisms for survival during disease progression.IMPORTANCEOtitis media is the most common bacterial infection in childhood. Current therapies are limited in the prevention of chronic or recurrent otitis media which leads to increased antibiotic exposure and represents a significant socioeconomic burden. In this study, we delineate the effect of nutritional limitation on the intracellular trafficking pathways used by nontypeableHaemophilus influenzae(NTHI). Moreover, transient limitation of heme-iron led to the development of intracellular bacterial communities that are known to contribute to persistence and recurrence in other diseases. New approaches for therapeutic interventions that reduce the production of intracellular bacterial communities and promote trafficking through the endolysosomal pathway were revealed through the use of pharmacological inhibition of macropinocytosis. This work demonstrates the importance of an intracellular niche for NTHI and provides new approaches for intervention for acute, chronic, and recurring episodes of otitis media.

The Immune Response to Acute Otitis Media in Children. II. Serum and Middle Ear Fluid Antibody in Otitis Media Due to Haemophilus influenzae

1975 ◽  
Vol 132 (6) ◽  
pp. 685-688 ◽  
Author(s):  
J. L. Sloyer ◽  
C. C. Cate ◽  
V. M. Howie ◽  
J. H. Ploussard ◽  
R. B. Johnston
Keyword(s):  
Immune Response ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Acute Otitis Media ◽  
Middle Ear Fluid

scholarly journals Phylogenetic relatedness and diversity of non-typable Haemophilus influenzae in the nasopharynx and middle ear fluid of children with acute otitis media

2011 ◽  
Vol 60 (12) ◽  
pp. 1841-1848 ◽  
Author(s):  
Ravinder Kaur ◽  
Arthur Chang ◽  
Qingfu Xu ◽  
Janet R. Casey ◽  
Michael E. Pichichero
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Acute Otitis Media ◽  
Phylogenetic Relatedness ◽  
Middle Ear Fluid
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