scholarly journals Comparative Profile of Heme Acquisition Genes in Disease-Causing and Colonizing Nontypeable Haemophilus influenzae and Haemophilus haemolyticus

2015 ◽  
Vol 53 (7) ◽  
pp. 2132-2137 ◽  
Author(s):  
Nurul I. Hariadi ◽  
Lixin Zhang ◽  
Mayuri Patel ◽  
Sara A. Sandstedt ◽  
Gregg S. Davis ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Middle Ear ◽  
Acute Otitis Media ◽  
Healthy Children ◽  
Nontypeable Haemophilus Influenzae ◽  
Closely Related Species ◽  
Content Type ◽  
Nutritional Immunity ◽  
Heme Acquisition ◽  
Tract Infections

NontypeableHaemophilus influenzae(NTHI) are Gram-negative bacteria that colonize the human pharynx and can cause respiratory tract infections, such as acute otitis media (AOM). Since NTHI require iron from their hosts for aerobic growth, the heme acquisition genes may play a significant role in avoiding host nutritional immunity and determining virulence. Therefore, we employed a hybridization-based technique to compare the prevalence of five heme acquisition genes (hxuA,hxuB,hxuC,hemR, andhup) between 514 middle ear strains from children with AOM and 235 throat strains from healthy children. We also investigated their prevalences in 148Haemophilus haemolyticusstrains, a closely related species that colonizes the human pharynx and is considered to be nonpathogenic. Four out of five genes (hxuA,hxuB,hxuC, andhemR) were significantly more prevalent in the middle ear strains (96%, 100%, 100%, and 97%, respectively) than in throat strains (80%, 92%, 93%, and 85%, respectively) of NTHI, suggesting that strains possessing these genes have a virulence advantage over those lacking them. All five genes were dramatically more prevalent in NTHI strains than inH. haemolyticus, with 91% versus 9%hxuA, 98% versus 11%hxuB, 98% versus 11%hxuC, 93% versus 20%hemR, and 97% versus 34%hup, supporting their potential role in virulence and highlighting their possibility to serve as biomarkers to distinguishH. influenzaefromH. haemolyticus. In summary, this study demonstrates that heme acquisition genes are more prevalent in disease-causing NTHI strains isolated from the middle ear than in colonizing NTHI strains andH. haemolyticusisolated from the pharynx.

scholarly journals Role of Sialic Acid and Complex Carbohydrate Biosynthesis in Biofilm Formation by Nontypeable Haemophilus influenzae in the Chinchilla Middle Ear

2005 ◽  
Vol 73 (6) ◽  
pp. 3210-3218 ◽  
Author(s):  
Joseph Jurcisek ◽  
Laura Greiner ◽  
Hiroshi Watanabe ◽  
Anthony Zaleski ◽  
Michael A. Apicella ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Middle Ear ◽  
Biofilm Formation ◽  
Mammalian Host ◽  
Nontypeable Haemophilus Influenzae ◽  
Biofilm Production ◽  
Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an important pathogen in respiratory tract infections, including otitis media (OM). NTHI forms biofilms in vitro as well as in the chinchilla middle ear, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of OM. We've previously shown that SiaA, SiaB, and WecA are involved in biofilm production by NTHI in vitro. To investigate whether these gene products were also involved in biofilm production in vivo, NTHI strain 2019 and five isogenic mutants with deletions in genes involved in carbohydrate biosynthesis were inoculated into the middle ears of chinchillas. The wild-type strain formed a large, well-organized, and viable biofilm; however, the wecA, lsgB, siaA, pgm, and siaB mutants were either unable to form biofilms or formed biofilms of markedly reduced mass, organization, and viability. Despite their compromised ability to form a biofilm in vivo, wecA, lsgB, and siaA mutants survived in the chinchilla, inducing culture-positive middle ear effusions, whereas pgm and siaB mutants were extremely sensitive to the bactericidal activity of chinchilla serum and thus did not survive. Lectin analysis indicated that sialic acid was an important component of the NTHI 2019 biofilm produced in vivo. Our data suggested that genes involved in carbohydrate biosynthesis and assembly play an important role in the ability of NTHI to form a biofilm in vivo. Collectively, we found that when modeled in a mammalian host, whereas biofilm formation was not essential for survivability of NTHI in vivo, lipooligosaccharide sialylation was indispensable.

scholarly journals Design and Characterization of Protein E-PilA, a Candidate Fusion Antigen for Nontypeable Haemophilus influenzae Vaccine

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Normand Blais ◽  
Don Somers ◽  
Denis Faubert ◽  
Steve Labbé ◽  
Cindy Castado ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Peptide Mapping ◽  
3D Structure ◽  
Acute Otitis Media ◽  
Vaccine Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chronic Obstructive ◽  
Nontypeable Haemophilus Influenzae ◽  
Tract Infections ◽  
Adhesive Proteins

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a pathogen known for being a frequent cause of acute otitis media in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In the present study, a vaccine antigen based on the fusion of two known NTHi adhesive proteins, protein E (PE) and a pilin subunit (PilA), was developed. The quality of the combined antigen was investigated through functional, biophysical, and structural analyses. It was shown that the PE and PilA individual structures are not modified in the PE-PilA fusion and that PE-PilA assembles as a dimer in solution, reflecting PE dimerization. PE-PilA was found to bind vitronectin by enzyme-linked immunosorbent assay, as isolated PE does. Disulfide bridges were conserved and homogeneous, which was determined by peptide mapping and top-down analysis of PE, PilA, and PE-PilA molecules. Finally, the PE-PilA crystal showed a PE entity with a three-dimensional (3D) structure similar to that of the recently published isolated PE, while the structure of the PilA entity was similar to that of a 3D model elaborated from two other type 4 pilin subunits. Taken together, our observations suggest that the two tethered proteins behave independently within the chimeric molecule and display structures similar to those of the respective isolated antigens, which are important characteristics for eliciting optimal antibody-mediated immunity. PE and PilA can thus be further developed as a single fusion protein in a vaccine perspective, in the knowledge that tethering the two antigens does not perceptibly compromise the structural attributes offered by the individual antigens.

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 N-Acetyl-l-Cysteine and Cysteamine as New Strategies against Mixed Biofilms of Nonencapsulated Streptococcus pneumoniae and Nontypeable Haemophilus influenzae

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Mirian Domenech ◽  
Ernesto García
Keyword(s):  
Streptococcus Pneumoniae ◽  
Haemophilus Influenzae ◽  
Laser Scanning ◽  
Acute Otitis Media ◽  
Public Health Problem ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type

ABSTRACT Acute otitis media, a polymicrobial disease of the middle ear cavity of children, is a significant public health problem worldwide. It is most frequently caused by encapsulated Streptococcus pneumoniae and nontypeable Haemophilus influenzae, although the widespread use of pneumococcal conjugate vaccines is apparently producing an increase in the carriage of nonencapsulated S. pneumoniae. Frequently, pneumococci and H. influenzae live together in the human nasopharynx, forming a self-produced biofilm. Biofilms present a global medical challenge since the inherent antibiotic resistance of their producers demands the use of large doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence. Here, we describe the development of an in vitro nonencapsulated S. pneumoniae-nontypeable H. influenzae biofilm system with polystyrene or glass-bottom plates. Confocal laser scanning microscopy and specific fluorescent labeling of pneumococcal cells with Helix pomatia agglutinin revealed an even distribution of both species within the biofilm. This simple and robust protocol of mixed biofilms was used to test the antimicrobial properties of two well-known antioxidants that are widely used in the clinical setting, i.e., N-acetyl-l-cysteine and cysteamine. This repurposing approach showed the high potency of N-acetyl-l-cysteine and cysteamine against mixed biofilms of nonencapsulated S. pneumoniae and nontypeable H. influenzae. Decades of clinical use mean that these compounds are safe to use, which may accelerate their evaluation in humans.

scholarly journals Experimental Acute Otitis Media Due to Nontypeable Haemophilus influenzae: Comparison of High and Low Azithromycin Doses with Placebo

2002 ◽  
Vol 46 (7) ◽  
pp. 2194-2199 ◽  
Author(s):  
Franz E. Babl ◽  
Stephen I. Pelton ◽  
Zhong Li
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Clinical Success ◽  
Acute Otitis Media ◽  
Published Data ◽  
Nontypeable Haemophilus Influenzae ◽  
Ear Infection ◽  
Dependent Effect ◽  
Middle Ear Infection

ABSTRACT Treatment of acute otitis media (AOM) with azithromycin results in apparent clinical success, but tympanocentesis performed 4 to 6 days after initiation of therapy in children with nontypeable Haemophilus influenzae (NTHI) recovered from initial middle ear cultures demonstrates persistence of infection in more than 50% of episodes. We sought to determine the effect of azithromycin at different doses on the density of middle ear infection due to NTHI to provide additional understanding of this dichotomy between clinical and microbiologic outcome measures in AOM. In a chinchilla model of experimental otitis media (EOM), animals treated with placebo were compared to animals receiving a single daily dose 30 or 120 mg of azithromycin per kg of body weight per day for 5 days. Microbiologic outcome was assessed by obtaining quantitative cultures from the middle ear during a 5-day course and for 1 week following therapy. Azithromycin concentrations were measured to ascertain whether a concentration-dependent effect was present. Azithromycin at 30 and 120 mg/kg/day demonstrated a dose-dependent effect on the quantitative assessment of middle ear infection due to NTHI. A 30-mg/kg dose of azithromycin daily resulted in levels in serum and areas under the serum concentration-time curve at 24 h comparable to published data obtained with children given azithromycin at 5 to 10 mg/kg in multiday regimens. Increased doses of azithromycin (120 mg/kg) achieved 2.5- to 4-fold-higher levels in serum and 3- to 6-fold-higher total levels and levels in extracellular middle ear fluid as well as more rapid reduction in bacterial density and a greater proportion of middle ears with complete sterilization than either placebo or the 30-mg/kg/day regimen.

scholarly journals Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications

2016 ◽  
Vol 84 (3) ◽  
pp. 765-774 ◽  
Author(s):  
Deborah M. B. Post ◽  
Margaret R. Ketterer ◽  
Jeremy E. Coffin ◽  
Lorri M. Reinders ◽  
Robert S. Munson ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Mass Spectrometric ◽  
Lower Airway ◽  
Titration Calorimetry ◽  
Acid Uptake ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type ◽  
Comparative Analyses ◽  
Tract Infections

Haemophilus haemolyticusand nontypeableHaemophilus influenzae(NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, whileH. haemolyticusrarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent thanH. haemolyticusLOS did. Genomic analyses of 10 strains demonstrated thatH. haemolyticuslacked the sialyltransferase geneslic3Aandlic3B(9/10) andsiaA(10/10), but all strains contained the sialic acid uptake genessiaPandsiaT(10/10). However, isothermal titration calorimetry analyses of SiaP from twoH. haemolyticusstrains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS fromH. haemolyticuscontained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures fromH. haemolyticusand NTHi may explain some of the differences in their propensities to cause disease.

scholarly journals Identification of the Lipooligosaccharide Biosynthesis Gene lic2B as a Putative Virulence Factor in Strains of Nontypeable Haemophilus influenzae That Cause Otitis Media

2002 ◽  
Vol 70 (7) ◽  
pp. 3551-3556 ◽  
Author(s):  
M. M. Pettigrew ◽  
B. Foxman ◽  
C. F. Marrs ◽  
J. R. Gilsdorf
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Day Care ◽  
Amino Acid Level ◽  
Acute Otitis Media ◽  
Putative Virulence Factor ◽  
Type B ◽  
Nontypeable Haemophilus Influenzae ◽  
Biosynthesis Gene

ABSTRACT Nontypeable (NT) strains of Haemophilus influenzae are an important cause of acute otitis media (OM). The pathogenic process by which NT H. influenzae strains cause OM is poorly understood. In order to identify specific virulence factors important for OM pathogenesis, genomic subtraction of the NT H. influenzae middle ear isolate G622 against H. influenzae strain Rd was conducted and the resulting subtraction products were used to screen a panel of H. influenzae isolates. Subtraction identified 36 PCR fragments unique to strain G622, which were used in a preliminary screen of 48 middle ear isolates and 46 nasopharyngeal and throat isolates to identify genes found more frequently among middle ear isolates. These experiments identified a PCR fragment with high homology to the lipooligosaccharide biosynthesis gene lic2B (originally identified in an H. influenzae type b strain) among 52% of the middle ear isolates and 9% of nasopharyngeal and throat isolates. The lic2B gene cloned from NT H. influenzae strain G622 was 99% identical at the amino acid level to that of the H. influenzae type b strain RM7004. The lic2B gene was used to screen a larger panel of H. influenzae isolates including the original 48 middle ear isolates, 40 invasive type b isolates, 90 NT H. influenzae throat isolates from children attending day care, and 32 NT H. influenzae nasopharyngeal clinical isolates. The lic2B gene was found 3.7 times more frequently among middle ear isolates than in throat isolates from children attending day care. These data suggest that a specific NT H. influenzae gene is associated with OM.

scholarly journals The Effect of the 10-Valent Pneumococcal NontypeableHaemophilus influenzaeProtein D Conjugate Vaccine onH. influenzaein Healthy Carriers and Middle Ear Infections in Iceland

2019 ◽  
Vol 57 (7) ◽  
Author(s):  
Hildigunnur Sveinsdóttir ◽  
Jana Birta Björnsdóttir ◽  
Helga Erlendsdóttir ◽  
Martha Á. Hjálmarsdóttir ◽  
Birgir Hrafnkelsson ◽  
...  
Keyword(s):  
Otitis Media ◽  
Middle Ear ◽  
Acute Otitis Media ◽  
Annual Number ◽  
Healthy Children ◽  
Carriage Rate ◽  
Content Type ◽  
Ear Infections ◽  
Culture Positive ◽  
Healthy Carriers

ABSTRACTVaccinations with the 10-valent pneumococcal conjugated vaccine (PHiD-CV) started in Iceland in 2011. Protein D (PD) fromH. influenzae, which is coded for by thehpdgene, is used as a conjugate in the vaccine and may provide protection against PD-positiveH. influenzae. We aimed to evaluate the effect of PHiD-CV vaccination onH. influenzaein children, both in carriage and in acute otitis media (AOM).H. influenzaewas isolated from nasopharyngeal swabs collected from healthy children attending 15 day care centers in 2009 and from 2012 to 2017 and from middle ear (ME) samples from children with AOM collected from 2012 to 2017. All isolates were identified using PCR for thehpdandfucKgenes. Of the 3,600 samples collected from healthy children, 2,465 were culture positive forH. influenzae(68.5% carriage rate); of these, 151 (6.1%) containedhpd-negative isolates. Of the 2,847 ME samples collected, 889 (31.2%) were culture positive forH. influenzae; of these, 71 (8.0%) werehpdnegative. Despite the same practice throughout the study, the annual number of ME samples reduced from 660 in 2012 to 330 in 2017. The proportions ofhpd-negative isolates in unvaccinated versus vaccinated children were 5.6% and 7.0%, respectively, in healthy carriers, and 5.4% and 7.8%, respectively, in ME samples. The proportion ofhpd-negative isolates increased with time in ME samples but not in healthy carriers. The number of ME samples from children with AOM decreased. The PHiD-CV had no effect on the proportion of thehpdgene inH. influenzaefrom carriage, but there was an increase inhpd-negativeH. influenzaein otitis media. The proportions ofhpd-negative isolates remained similar in vaccinated and unvaccinated children.

scholarly journals Histidine Auxotrophy in Commensal and Disease-Causing Nontypeable Haemophilus influenzae

2007 ◽  
Vol 189 (14) ◽  
pp. 4994-5001 ◽  
Author(s):  
Patricia C. Juliao ◽  
Carl F. Marrs ◽  
Jingping Xie ◽  
Janet R. Gilsdorf
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Bacterial Species ◽  
Blot Hybridization ◽  
Acute Otitis Media ◽  
Prevalence Ratio ◽  
Nontypeable Haemophilus Influenzae ◽  
Dot Blot ◽  
Growth And Survival

ABSTRACT Histidine biosynthesis is one of the best studied metabolic pathways in bacteria. Although this pathway is thought to be highly conserved within and between bacterial species, a previous study identified a genetic region within the histidine operon (his) of nontypeable strains of Haemophilus influenzae (NTHI) that was more prevalent among otitis media strains than among throat commensal NTHI strains. In the present study, we further characterized this region and showed that genes in the complete his operon (hisG, -D, -C, -NB, -H, -A, -F, and -IE) are >99% conserved among four fully sequenced NTHI strains, are present in the same location in these four genomes, and are situated in the same gene order. Using PCR and dot blot hybridization, we determined that the his operon was significantly more prevalent in otitis media NTHI strains (106/121; 87.7%) than in throat strains (74/137; 54%) (prevalence ratio, 1.62; P < 0.0001), suggesting a possible role in middle ear survival and/or acute otitis media. NTHI strains lacking the his operon showed attenuated growth in histidine-restricted media, confirming them as his-negative auxotrophs. Our results suggest that the ability to make histidine is an important factor in bacterial growth and survival in the middle ear, where nutrients such as histidine may be found in limited amounts. Those isolates lacking the histidine pathway were still able to survive well in the throat, which suggests that histidine is readily available in the throat environment.

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