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Article Reference Phosphorylcholine expression by nontypeable Haemophilus influenzae correlates with maturation of biofilm communities in vitro and in vivo.
Nontypeable Haemophilus influenzae (NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019 licD) or were constitutively locked in the PCho-positive phase (NTHI 2019 lic(ON)). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019 lic(ON). The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.
Article Reference Multiple consecutive lavage samplings reveal greater burden of disease and provide direct access to the nontypeable Haemophilus influenzae biofilm in experimental otitis media.
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.
Article Reference Biofilms formed by nontypeable Haemophilus influenzae in vivo contain both double-stranded DNA and type IV pilin protein.
Nontypeable Haemophilus influenzae (NTHI) strains are members of the normal human nasopharyngeal flora, as well as frequent opportunistic pathogens of both the upper and lower respiratory tracts. Recently, it has been shown that NTHI can form biofilms both in vitro and in vivo. NTHI strains within in vitro-formed biofilms differentially express both epitopes of lipooligosaccharide (LOS) and the outer membrane proteins P2, P5, and P6, whereas those generated either in a 96-well plate assay in vitro or in a mammalian host have been shown to incorporate a specific glycoform of sialylated LOS within the biofilm matrix. While DNA has been identified as a key component of the biofilm matrix formed in vitro by several bacterial pathogens, here we demonstrate for the first time that in addition to sialylated LOS, the biofilm formed by NTHI in vivo contains both type IV pilin protein and a significant amount of double-stranded DNA. The DNA appeared to be arranged in a dense interlaced meshwork of fine strands as well as in individual thicker ``ropes'' that span water channels, suggesting that DNA could be imparting structural stability to the biofilm produced by NTHI in vivo. The presence of type IV pilin protein both appearing as small aggregates within the biofilm matrix and tracking along DNA strands supports our observations which showed that type IV pili are expressed by NTHI during experimental otitis media when these bacteria form a biofilm in the middle ear space.
Article Reference 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.
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.
Article Reference Nontypeable Haemophilus influenzae strains with the capsule-associated insertion element IS1016 may mimic encapsulated strains.
With the elimination of Haemophilus influenzae type b through vaccination, it has been suggested that other types of H. influenzae strains might acquire virulence traits and emerge as important pathogens. The gene sequence IS1016 has been associated with an increased capacity to cause severe infections. It is usually present in encapsulated strains but is sometimes harbored by nontypeable H. influenzae strains. To explore this further, 118 H. influenzae isolates, collected from both patients and healthy carriers, were investigated with PCR with reference to this gene sequence. Isolates positive for the insertion element were bio- and serotyped. The presence of hmw genes for adherence, the genetic profile, and the ability to form biofilm in vitro were investigated. A total of 15 isolates were IS1016-positive, whereof 12 were nontypeable. All 12 nontypeable isolates were obtained from healthy carriers, and 92% of the isolates were biotype I. They cross-reacted to some extent with type-specific antisera or exhibited a restricted genetic diversity like encapsulated strains. Furthermore, they lacked hmw-genes, and their ability to form biofilms was comparable with a capsule-deficient type b strain. Although this subset of strains mimicked traits usually exhibited by encapsulated strains, the isolation frequency did not seem to have been affected by vaccination.
Article Reference Identification of biofilm proteins in non-typeable Haemophilus Influenzae.
Non-typeable Haemophilus influenzae biofilm formation is implicated in a number of chronic infections including otitis media, sinusitis and bronchitis. Biofilm structure includes cells and secreted extracellular matrix that is ``slimy'' and believed to contribute to the antibiotic resistant properties of biofilm bacteria. Components of biofilm extracellular matrix are largely unknown. In order to identify such biofilm proteins an ex-vivo biofilm of a non-typeable Haemophilus influenzae isolate, originally from an otitis media patent, was produced by on-filter growth. Extracellular matrix fraction was subjected to proteomic analysis via LC-MS/MS to identify proteins.
Article Reference Multiple combination antibiotic susceptibility testing of nontypeable Haemophilus influenzae biofilms.
Haemophilus influenzae is a cause of otitis media with effusion (OME). Animal models demonstrate growth of H. influenzae biofilms in OME, which may explain why OME does not respond well to conventional antibiotic therapy. Using a previously developed in vitro model, we performed H. influenzae susceptibility studies to see if H. influenzae biofilm cultures were more resistant to antibiotics than planktonic (broth) cultures, and to determine which antibiotics were most effective against H. influenzae biofilms. H. influenzae isolates were grown as biofilms on polystyrene pins. Biofilm and planktonic minimum inhibitory concentrations (MICs) were measured for 8 antibiotics, and multiple combination testing was performed with 66 groupings of 1, 2, or 3 antibiotics. We found that biofilm cultures were more resistant to antibiotics than planktonic ones. Antibiotic combinations containing rifampin and ciprofloxacin were most effective against biofilms. Biofilm testing reveals differences in effectiveness among antibiotics not apparent from conventional susceptibility testing, and suggests novel antibiotic regimens that could be studied for treatment of OME.
Article Reference Haemophilus influenzae forms biofilms on airway epithelia: implications in cystic fibrosis.
Nontypeable Haemophilus influenzae (NTHi) commonly infects patients with cystic fibrosis (CF), especially early in childhood. Bacteria biofilms are increasingly recognized as contributing to bacterial persistence and disease pathogenesis in CF.
Article Reference Distribution of bacterial proteins in biofilms formed by non-typeable Haemophilus influenzae.
The ability to preserve the fragile ultrastructural organization of bacterial biofilms using cryo-preparation methods for electron microscopy has enabled us to probe sections through non-typeable Haemophilus influenzae (NTHi) biofilms and determine the localization of NTHi-specific lipooligosaccharide (LOS) and proteins within these structures. Some of the proteins we examined are currently being considered as candidates for vaccine development, so it is important that their distribution and accessibility within the biofilms formed by NTHi be determined. We have localized LOS to the extracellular matrix (ECM) of the biofilm and the P6 outer membrane protein to the membrane of what appear to be viable bacteria within the biofilm. The Hap and HWM1/HMW2 adhesive proteins were associated with bacteria within the biofilm and were present in the biofilm ECM. The IgA1 protease is a secreted protein that was also associated with NTHi in the biofilm and was in the ECM, but was more concentrated in the top region of the biofilm, suggesting a role in protecting biofilm bacteria from antibody attack.
Article Reference Biofilm growth increases phosphorylcholine content and decreases potency of nontypeable Haemophilus influenzae endotoxins.
Nontypeable Haemophilus influenzae (NTHI) is a common respiratory commensal and opportunistic pathogen. NTHI is normally contained within the airways by host innate defenses that include recognition of bacterial endotoxins by Toll-like receptor 4 (TLR4). NTHI produces lipooligosaccharide (LOS) endotoxins which lack polymeric O side chains and which may contain host glycolipids. We recently showed that NTHI biofilms contain variants with sialylated LOS glycoforms that are essential to biofilm formation. In this study, we show that NTHI forms biofilms on epithelial cell layers. Confocal analysis revealed that sialylated variants were distributed throughout the biofilm, while variants expressing phosphorylcholine (PCho) were found within the biofilm. Consistent with this observation, PCho content of LOS purified from NTHI biofilms was increased compared to LOS from planktonic cultures. Hypothesizing that the observed changes in endotoxin composition could affect bioactivity, we compared inflammatory responses to NTHI LOS purified from biofilm and planktonic cultures. Our results show that endotoxins from biofilms induced weaker host innate responses. While we observed a minimal effect of sialylation on LOS bioactivity, there was a significant decrease in bioactivity associated with PCho substitutions. We thus conclude that biofilm growth increases the proportion of PCho+ variants in an NTHI population, resulting in a net decrease in LOS bioactivity. Thus, in addition to their well-documented resistance phenotypes, our data show that biofilm communities of NTHI bacteria contain variants that evoke less potent host responses.
Article Reference Novel sialic acid transporter of Haemophilus influenzae.
Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. N-Acetylneuraminic acid (sialic acid) can be incorporated into the lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into lipooligosaccharides is understood, the transporter responsible for N-acetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATP-independent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth.
Article Reference Haemophilus influenzae luxS mutants form a biofilm and have increased virulence.
To gain insight into the role of luxSHi in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxSHi mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB170 bioassay. Culture filtrates from Escherichia coli strain DH5alpha expressing a cloned luxSHi contained 350-fold more AI-2 activity per cell than E. coli DH5alpha containing the vector alone. The growth rate in several liquid media, and the cell density after overnight growth were not significantly different between the parents and the luxSHi mutants. Two clinical H. influenzae and their luxSHi mutants produced an identical biofilm in a flow system. Invasion of human cells by the luxSHi mutants, in comparison to the wild-type parents was strain-dependent, and cell type-dependent, but the luxSHi mutants tended to be more invasive. The luxSHi mutant of an otitis media isolate, strain R3157 appeared more virulent in the chinchilla model of otitis media: there were more bacteria in the middle ear, a greater inflammatory response and more goblet cell hyperplasia 10 days after the inoculation. We conclude that the H. influenzae homologue of luxS modulates certain virulence traits.
Article Reference Role of sialic acid and complex carbohydrate biosynthesis in biofilm formation by nontypeable Haemophilus influenzae in the chinchilla middle ear.
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.
Article Reference Expression of a peroxiredoxin-glutaredoxin by Haemophilus influenzae in biofilms and during human respiratory tract infection.
Evidence is mounting that nontypeable Haemophilus influenzae grows as a biofilm in the middle ear of children with otitis media and the airways of adults with chronic obstructive pulmonary disease. To begin to assess antigens expressed by H. influenzae in biofilms, cell envelopes of bacteria grown as a biofilm were compared to those grown planktonically. A approximately 30kDa peroxiredoxin-glutaredoxin was present in greater abundance during growth in biofilms. Mutants deficient in expression of peroxiredoxin-glutaredoxin were constructed by homologous recombination in four clinical isolates. The mutants showed a 25-50% reduction in biofilm formation compared to the corresponding parent strains. To study in vivo expression of peroxiredoxin-glutaredoxin during human respiratory tract infection, paired pre- and post-exacerbation serum from adults with chronic obstructive pulmonary disease and H. influenzae in sputum were assayed using an enzyme-linked immunosorbent assay and purified recombinant peroxiredoxin-glutaredoxin. Eight from 18 (44.4%) paired serum samples showed a significant increase in antibody to peroxiredoxin-glutaredoxin from pre- to post-infection. These results indicate that (1) peroxiredoxin-glutaredoxin is present in greater abundance in H. influenzae biofilms compared to planktonically grown bacteria; (2) peroxiredoxin-glutaredoxin is involved in biofilm formation by H. influenzae and the degree of involvement varies among strains; and (3) peroxiredoxin-glutaredoxin is expressed by H. influenzae during infection of the human respiratory tract and is recognized by the human immune system.
Article Reference Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans.
Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with alpha2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.
Article Reference Sialylation of lipooligosaccharides promotes biofilm formation by nontypeable Haemophilus influenzae.
Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.
Article Reference Biofilm formation by nontypeable Haemophilus influenzae: strain variability, outer membrane antigen expression and role of pili.
Nontypeable Haemophilus influenzae is an important cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Several lines of evidence suggest that the bacterium grows as a biofilm in the human respiratory tract.
Article Reference Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.
Nontypeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen, can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.
Article Reference Nontypeable Haemophilus influenzae inhibits autolysis and fratricide of Streptococcus pneumoniae in vitro.
Streptococcus pneumoniae (SP) and nontypeable Haemophilus influenzae (NTHi) are common commensals of the human airway and major bacterial pathogens of otitis media (OM) and other upper airway infections. The interaction between them may play an important role in the pathogenesis of polymicrobial infections. Although previous studies suggested NTHi could promote pneumococcal survival and biofilm formation, how NTHi affects pneumococcal activities has not been defined. Our data in the present studies indicated that the outcome of the interaction between SP and NTHi was in a cell-density-dependent manner and the enhancement of pneumococcal survival happened at the later stages of culturing. Using quantitative PCR, we found that the expression of pneumococcal genes regulating autolysis and fratricide, lytA and cbpD, were significantly down-regulated in co-culture with NTHi. We further observed that influence of NTHi was not on direct cell-to-cell contact, but that this contact may contribute to the interaction between these two microorganisms. These results suggest that pneumococcal survival and biofilm formation can be enhanced by down-regulating pneumococcal cell wall hydrolase production thereby inhibiting pneumococcal autolysis and fratricide in the presence of NTHi.
Article Reference Frequent carriage of resistance mechanisms to β-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children.
Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to β-lactams.