The modA10 phasevarion of nontypeable Haemophilus influenzae R2866 regulates multiple virulence-associated traits.

Non-typeable Haemophilus influenzae (NTHi) is a human restricted commensal and pathogen that elicits inflammation by adhering to and invading airway epithelia cells: transcytosis across these cells can result in systemic infection. NTHi strain R2866 was isolated from the blood of a normal 30-month old infant with meningitis, and is unusual for NTHi in that it is able to cause systemic infection. Strain R2866 is able to replicate in normal human serum due to expression of lgtC which mimics human blood group p(k). R2866 contains a phase-variable DNA methyltransferase, modA10 which switches ON and OFF randomly and reversibly due to polymerase slippage over a long tetrameric repeat tract located in its open reading frame. Random gain or loss of repeats during replication can results in expressed (ON), or not expressed (OFF) states, the latter due to a frameshift or transcriptional termination at a premature stop codon. We sought to determine if the unusual virulence of R2866 was modified by modA10 phase-variation. A modA10 knockout mutant was found to have increased adherence to, and invasion of, human ear and airway monolayers in culture, and increased invasion and transcytosis of polarized human bronchial epithelial cells. Intriguingly, the rate of bacteremia was lower in the infant rat model of infection than a wild-type R2866 strain, but the fatality rate was greater. Transcriptional analysis comparing the modA10 knockout to the R2866 wild-type parent strain showed increased expression of genes in the modA10 knockout whose products mediate cellular adherence. We conclude that loss of ModA10 function in strain R2866 enhances colonization and invasion by increasing expression of genes that allow for increased adherence, which can contribute to the increased virulence of this strain.

Bacteremia and malaria in Tanzanian children hospitalized for acute febrile illness.

We recorded the reason for presentation to a rural hospital in an area endemic for malaria in 909 children between January 2006 and March 2009. Blood smears were examined for Plasmodium falciparum parasites, and blood spots dried on filter paper were prepared for 464 children. A PCR assay utilizing the stored blood spots was developed for Streptococcus pneumoniae (lytA) and Haemophilus influenzae (pal). Malaria was present in 299 children whose blood was tested by polymerase chain reaction (PCR); 19 had lytA and 15 had pal. The overall prevalence of lytA was 25 of the 464 children, while that of pal was 18 children. Fever was present in 369 children of whom 19 had lytA DNA while 11 had pal DNA detected. Of the 95 afebrile children, six had lytA and seven pal. We conclude that there are no clinical features that distinguish malaria alone from bacteremia alone or the presence of both infections.

Molecular basis of increased serum resistance among pulmonary isolates of non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies.

Haemophilus influenzae phasevarions have evolved from type III DNA restriction systems into epigenetic regulators of gene expression.

Phase variably expressed (randomly switching) methyltransferases associated with type III restriction-modification (R-M) systems have been identified in a variety of pathogenic bacteria. We have previously shown that a phase variable methyltransferase (Mod) associated with a type III R-M system in Haemophilus influenzae strain Rd coordinates the random switching of expression of multiple genes, and constitutes a phase variable regulon--'phasevarion'. We have now identified the recognition site for the Mod methyltransferase in H. influenzae strain Rd as 5'-CGAAT-3'. This is the same recognition site as the previously described HinfIII system. A survey of 59 H. influenzae strains indicated significant sequence heterogeneity in the central, variable region of the mod gene associated with target site recognition. Intra- and inter-strain transformation experiments using Mod methylated or non-methylated plasmids, and a methylation site assay demonstrated that the sequence heterogeneity seen in the region encoding target site specificity does correlate to distinct target sites. Mutations were identified within the res gene in several strains surveyed indicating that Res is not functional. These data suggest that evolution of this type III R-M system into an epigenetic mechanism for controlling gene expression has, in some strains, resulted in loss of the DNA restriction function.

Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing.

The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.

Nontypeable Haemophilus influenzae: understanding virulence and commensal behavior.

Haemophilus influenzae is genetically diverse and exists as a near-ubiquitous human commensal or as a pathogen. Invasive type b disease has been almost eliminated in developed countries; however, unencapsulated strains - nontypeable H. influenzae (NTHi) - remain important as causes of respiratory infections. Respiratory tract disease occurs when NTHi adhere to or invade respiratory epithelial cells, initiating one or more of several proinflammatory pathways. Biofilm formation explains many of the observations seen in chronic otitis media and chronic bronchitis. However, NTHi biofilms seem to lack a biofilm-specific polysaccharide in the extracellular matrix, a source of controversy regarding their relevance. Successful commensalism requires dampening of the inflammatory response and evasion of host defenses, accomplished in part through phase variation.

Health-related quality of life in children with hepatitis C acquired in the first year of life.

AIMS: The first aim of this study was to determine the health-related quality of life (HRQoL) of children with chronic hepatitis C virus (HCV) infection and compare HRQoL as reported by parents. The second aim was to ascertain parents' perceptions and concerns about current and future life for their child with HCV, and compare these findings with those reported by adolescents. METHODS: The study group comprised children attending a tertiary pediatric HCV-clinic in Melbourne, Australia, who acquired HCV prior to 12 months of age by vertical transmission or blood transfusion. Two validated (parent- and self-reported) questionnaires of HRQoL were completed (CHQ-PF 50 and CHQ-CF 50). Scores for children with HCV were compared with normative data (representative sample of 3119 age-matched Victorian children). A study-designed questionnaire relating to the impact of the diagnosis of HCV on parent and child perceptions of current and future health was administered. RESULTS: In total, 83% (19/23) questionnaires were returned. Physical and psychosocial summary scores were significantly lower in HCV than non-HCV children (45.3 vs 49.6 and 44.0 vs 50.1, respectively). Nine out of 11 scale scores were significantly lower in children with HCV, most notably the General health (49.9 vs 77.1; P < 0.001) and Parent impact-emotional (45.6 vs 80.3; P < 0.001) scales. Children reported reduced physical functioning (82.8% vs 94.4%) but were otherwise less concerned than their parents about their future health. CONCLUSIONS: Despite being "asymptomatic" on routine medical history, children with early acquired HCV have significantly poorer health status than community controls. These findings suggest the need for services currently available for adult HCV patients to support families and children with HCV.

A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

H. influenzae Consortium: integrative study of H. influenzae-human interactions.

Developments in high-throughput analysis tools coupled with integrative computational techniques have enabled biological studies to reach new levels. The ability to correlate large volumes of diverse data types into cohesive models of organism function has spawned a new systematic approach to biological investigation. The creation of a new consortium has been proposed to investigate a single organism utilizing these comprehensive approaches. The Haemophilus influenzae Consortium (HIC) would be comprised of five laboratories, each providing separate and complementary areas of expertise in the study of Haemophilus influenzae (HI). The 5-year study proposes to develop coherent models of HI, both as a stand-alone organism, and more importantly, as a human pathogen. Studies in growth condition specificity followed by genomic, metabolic, and proteomic experimentation will be combined and integrated through computational and experimental analyses to form dynamic and predictive models of HI and its responses. Data from the HIC will allow greater understanding of cellular behavior, pathogen-host interactions, bacterial infection, and provide future scientific endeavors with a template for studies of other pathogens.

In Silico Metabolic Model and Protein Expression of Haemophilus influenzae Strain Rd KW20 in Rich Medium.

The intermediary metabolism of Haemophilus influenzae strain Rd KW20 was studied by a combination of protein expression analysis using a recently developed direct proteomics approach, mutational analysis, and mathematical modeling. Special emphasis was placed on carbon utilization, sugar fermentation, TCA cycle, and electron transport of H. influenzae cells grown microaerobically and anaerobically in a rich medium. The data indicate that several H. influenzae metabolic proteins similar to Escherichia coli proteins, known to be regulated by low concentrations of oxygen, were well expressed in both growth conditions in H. influenzae. An in silico model of the H. influenzae metabolic network was used to study the effects of selective deletion of certain enzymatic steps. This allowed us to define proteins predicted to be essential or non-essential for cell growth and to address numerous unresolved questions about intermediary metabolism of H. influenzae. Comparison of data from in vivo protein expression with the protein list associated with a genome-scale metabolic model showed significant coverage of the known metabolic proteome. This study demonstrates the significance of an integrated approach to the characterization of H. influenzae metabolism.

Identification and characterization of a nontypeable Haemophilus influenzae putative toxin-antitoxin locus.

BACKGROUND: Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NTHi survival inside human cells, virulence-associated protein D (vapDHi, encoded by gene HI0450). Both vapDHi and a flanking gene, HI0451, exhibit the genetic and physical characteristics of a toxin/antitoxin (TA) locus, with VapDHi serving as the toxin moiety and HI0451 as the antitoxin. We propose the name VapXHi for the HI0451 antitoxin protein. Originally identified on plasmids, TA loci have been found on the chromosomes of a number of bacterial pathogens, and have been implicated in the control of translation during stressful conditions. Translation arrest would enhance survival within human cells and facilitate persistent or chronic mucosal infections. RESULTS: Isogenic mutants in vapDHi were attenuated for survival inside human respiratory epithelial cells (NCI-H292) and human brain microvascular endothelial cells (HBMEC), the in vitro models of mucosal infection and the blood-brain barrier, respectively. Transcomplementation with a vapDHi allele restored wild-type NTHi survival within both cell lines. A PCR survey of 59 H. influenzae strains isolated from various anatomical sites determined the presence of a vapDHiallele in 100% of strains. Two isoforms of the gene were identified in this population; one that was 91 residues in length, and another that was truncated to 45 amino acids due to an in-frame deletion. The truncated allele failed to transcomplement the NTHi vapDHi survival defect in HBMEC. Subunits of full-length VapDHi homodimerized, but subunits of the truncated protein did not. However, truncated protein subunits did interact with full-length subunits, and this interaction resulted in a dominant-negative phenotype. Although Escherichia coli does not contain a homologue of either vapDHi or vapXHi, overexpression of the VapDHi toxin in trans resulted in E. coli cell growth arrest. This arrest could be rescued by providing the VapXHi antitoxin on a compatible plasmid. CONCLUSION: We conclude that vapDHi and vapXHi may constitute a H. influenzae TA locus that functions to enhance NTHi survival within human epithelial and endothelial cells.

Murine beta-defensin-3 is an inducible peptide with limited tissue expression and broad-spectrum antimicrobial activity.

Beta-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. Similarities between mammalian beta-defensins may permit the use of murine models to further define the role of these peptides in innate host defense. Murine beta-defensin-3 (mBD-3) is a peptide that exhibits homology at the gene level to human beta-defensin-2 (hBD-2), one of four beta-defensins identified in man. The purpose of this study was to determine the antimicrobial activity of mBD-3, the tissue distribution of mBD-3 expression, and the effect of gram-negative bacterial infection on mBD-3 expression. Based on the sequence deduced from mBD-3 cDNA, a 40-amino acid peptide was assembled using automated [n-(9-fluorenyl)methoxycarbonyl] solid-phase synthesis. The antimicrobial activity of synthetic mBD-3 was evaluated in microdilution broth assays using bacterial and fungal organisms. mBD-3 mRNA expression was assayed by polymerase chain reaction (PCR) using cDNA derived from a panel of tissues. Expression of mBD-3 was also evaluated in tissues obtained from mice 24 h after intraperitoneal infection with Escherichia coli using reverse transcriptase (RT)-PCR. Synthetic mBD-3 inhibited the growth of E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans at concentrations from 25 to 50 microg/mL. Constitutive expression of mBD-3 mRNA was not consistently found in any organ using RT-PCR. In an E. coli peritonitis model, expression of mBD-3 mRNA was upregulated only in the esophagus and tongue. We conclude that mBD-3 is an inducible peptide with limited tissue expression during E. coli peritonitis. Because it exhibits broad-spectrum antimicrobial activity, this peptide may serve as an innate defense against microbial invasion at specific mucosal surfaces in the mouse.

Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis.

STUDY OBJECTIVE: To determine the relationship between the antibiotic susceptibility of Pseudomonas aeruginosa isolated from the sputum of patients with cystic fibrosis (CF) and the patient's response to parenteral antibiotic administration, we performed a retrospective analysis using data from patients in the placebo arm of a phase 3 trial of tobramycin solution for inhalation. All patients were chronically infected with P aeruginosa. Seventy-seven of the 262 patients receiving placebo experienced a pulmonary exacerbation during the trial for which they received therapy with IV tobramycin and ceftazidime. The susceptibility of the P aeruginosa isolates to ceftazidime and tobramycin was determined at trial enrollment by broth microdilution. DESIGN: The clinical response to combination antibiotic therapy was assessed by analyzing differences in spirometry before and after antibiotic administration. The FEV(1) percent predicted at the first visit after the conclusion of antibiotic administration was compared to the FEV(1) percent predicted prior to antibiotic therapy. The results were analyzed both descriptively and by regression analyses. RESULTS: The conditions of 54 patients improved, and those of 9 patients worsened, and in 14 patients there was no change in FEV(1) with antibiotic administration. No correlation was observed between the susceptibility of P aeruginosa to tobramycin or ceftazidime and clinical response. Only the three following variables were observed to significantly correlate with FEV(1) after antibiotic treatment on regression analysis: FEV(1) prior to treatment (p < 0.0001); number of days elapsed between the previous FEV(1) measurement and the initiation of IV antibiotic therapy (p < 0.002); and the number of days elapsed between the determination of the minimum inhibitory concentration and the initiation of IV therapy (p < 0.03). No significant trends were observed between the antibiotic susceptibility of P aeruginosa isolates and treatment outcomes. CONCLUSION: While lack of statistical significance for a trend between bacterial susceptibilities and the response to parenteral antibiotic administration does not mean that no such trend exists, the precision of the confidence intervals allows us to conclude that even if isolate antibiotic susceptibilities affect outcome, the impact would be small and not clinically relevant.

Haemophilus influenzae Rd KW20 has virulence properties.

Haemophilus influenzae is a human-adapted commensal and pathogen that can cause mucosal infections such as sinusitis, otitis media and bronchitis. Certain strains also cause bacteraemia and meningitis. Clinical isolates are genetically heterogeneous and are often recalcitrant to standard genetic manipulation. H. influenzae strain Rd KW20 has traditionally been considered avirulent, since it does not survive in the bloodstream of animals, is readily killed by normal adult human sera and cannot colonize the nasopharynx of infant rats. The purpose of this study was to determine whether Rd KW20 could be used in certain infection models. It is shown here that strain Rd KW20 can invade certain human epithelial cell lines grown either as monolayers or as differentiated epithelium at the air-liquid interface. In addition, Rd KW20 can invade a monolayer of immortalized human brain microvascular endothelial cells. Finally, this strain can replicate and survive in human bronchial xenografts for up to 3 weeks. The complete genomic sequence of Rd KW20 is available and it is readily amenable to genetic manipulation. These properties and the results reported here indicate that this strain is a viable alternative to the use of clinical isolates for the investigation of H. influenzae virulence.

Construction of a nontypeable Haemophilus influenzae-specific ectopic delivery vector.

Complementation of chromosomal mutations in trans can introduce artifacts due to the number of episomal copies of the gene in question. One solution is to study the gene expressed at a single ectopic site in cis. We have designed and constructed a vector that allows homologous recombination into a gene encoding a frame-shifted IS1016-V6 protein in the Haemophilus influenzae Rd KW20 chromosome (HI1018). This site is the location of the > or = 35 kilobase capsule locus in encapsulated type b and d strains. This locus is not present in the nontypeable Rd KW20 strain, thus allowing ectopic expression of genes homologously recombined into HI1018 without polar effects.

Tobramycin Serum Concentrations After Aerosol and Oral Administration in Cystic Fibrosis.

We sought to describe tobramycin absorption after aerosol administration to cystic fibrosis (CF) subjects. Serum tobramycin concentrations were determined by modification of the radioimmuno-assay (RIA) technique, lowering the limit of detection from 1.0 &mgr;g ml(minus sign1) to 0.05 &mgr;g ml(minus sign1). In 37 studies, after aerosol delivery of 666 plus minus 195 mg to the airway of 24 patients, in which 222 samples were assayed, only 1 serum sample contained tobramycin at a concentration greater than 1.0 &mgr;g ml(minus sign1). Twenty-six of the 37 studies permitted estimation of pharmacokinetic parameters of tobramycin. The serum clearance of tobramycin following aerosol adminstration is 39.13 plus minus 0.393 L h(minus sign1) (mean plus minus standard error of the mean), with an elimination half-life of 3.072 plus minus 0.194 h. The half-life was significantly longer than that found after intravenous adminstration. The elimination rate constant (K(e)) was calculated to be 0.234 plus minus 0.002 h(minus sign1). Estimated total-body clearance in which systemic absorption was determined from sputum and urinary recovery of tobramycin was 0.094 plus minus 0.002 1 hr(minus sign1) kg(minus sign1). We also studied tobramycin absorption in six CF subjects after ingestion of a 80-mg m(minus sign2) dose, to gain insight into the tobramycin levels observed after swallowing an aerosol. Four out of the six subjects had measurable serum tobramycin concentration after ingestion. The serum concentration-time curve mirrored what was seen after aerosol administration. We concluded that tobramycin has poor systemic absorption in CF subjects after aerosol administration. Tobramycin in serum after aerosol administration is in part due to the gastrointestinal absorption of swallowed drug, as well as absorption from lower respiratory tract.

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates.

The incidence of invasive infections due to Haemophilus influenzae has decreased significantly in developed countries with high rates of vaccination against H. influenzae serotype b (Hib). This vaccine provides no protection against H. influenzae serotype f (Hif), typically associated with invasive infections in adults with chronic disease and/or immunodeficiency, and rarely in otherwise healthy adults and children. The specific properties of Hif associated with virulence remain largely uncharacterized. A panel of 26 Hif strains consisting of both invasive disease-associated and mucosal surface non-invasive disease-associated isolates was surveyed by DNA fingerprinting, biotyping and PCR detection of hmw1, hmw2, hsf, the hif fimbrial locus and the lipo-oligosaccharide (LOS) biosynthetic island, and assessment of ß-lactamase expression and determination of resistance to the bactericidal activity of normal adult human serum. Repetitive sequence-based PCR fingerprinting differentiated the 26 strains into three clusters, with the majority of isolates (22/26, 84.6 %) clustered into a single indistinguishable group. Most isolates (24/26, 92.3 %) were of biotype I and two isolates produced ß-lactamase with detection of a conjugative plasmid, and the isolates displayed a range of resistances to the bactericidal activity of human serum. All 26 isolates carried the adhesin hsf, 21 carried a partial hif fimbrial operon and 4 had the adhesin genes hmw1/2. A LOS biosynthetic island was detected in 20 isolates consisting of the genes lic2BC. It was concluded that Hif has many recognized virulence properties and comprises a relatively homogeneous group independent of the anatomical source from which it was isolated.

Infant rat infection modifies phenotypic properties of an invasive nontypeable Haemophilus influenzae.

Enhancing the virulence trait of a specific bacterium in an animal model is often performed prior to the use of the strain for ex vivo human studies, such as reactivity with complement and antibody, or with phagocytic cells. For example, in Streptococcus pneumoniae mouse passage is used to enhance capsule production. While investigating an unusual serum-resistant unencapsulated Haemophilus influenzae (R2866), we found that animal passage yielded an isolate (R3392) which had decreased resistance to human serum, but increased virulence in Chang conjunctival cell monolayers, but with less invasion and transcytosis of polar H292 cells. We examined 90 colonies recovered from three infant rats for phase variants of LPS biosynthetic genes. In 88 colonies lgtC was OFF due to tetrameric repeat mediated slipped-strand mispairing at the time of DNA replication, while there was no variation in lic1A, lic2A, lic3A, lexA and oaf A. With lgtC OFF the LPS lacks Galα1-4ßGal, an epitope mimicking the human p(k) blood group, and molecular mimicry is lost. Selection for strain susceptible to NHS in the infant rat was not antibody mediated. We conclude that the passage of pathogens virulent in humans and animals may select for phenotypes only relevant for the animal species used.

Characterization of extended co-culture of non-typeable Haemophilus influenzae with primary human respiratory tissues.

Non-typeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that comprise part of the normal flora of the human upper airway, but are also responsible for a number of mucosal infections such as otitis media and bronchitis. These infections often recur and can become chronic. To characterize the effect of long-term co-culture of NTHi with human tissues, we infected primary respiratory epithelial cells grown at the air-liquid interface with three NTHi strains over a range of 1-10 days. Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded. Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues. Although the tissues elaborated the cytokine profile reported for NTHi-caused otitis media in vivo, there was little change in the dynamics of cytokine secretion over the time points tested. Finally, we report that NTHi strains released outer membrane vesicles (OMVs) during extended co-culture with the tissues, and show that these OMVs directly interact with host cell membranes.