Alloiococcus otitidis Forms Multispecies Biofilm with Haemophilus influenzae: Effects on Antibiotic Susceptibility and Growth in Adverse Conditions.

Otitis media with effusion (OME) is a biofilm driven disease and commonly accepted otopathogens, such as Haemophilus influenzae, Streptococcus pneumonia, and Moraxella catarrhalis, have been demonstrated to form polymicrobial biofilms within the middle ear cleft. However, Alloiococcus otitidis (A. otitidis), which is one of the most commonly found bacteria within middle ear aspirates of children with OME, has not been described to form biofilms. The aim of this study was to investigate whether A. otitidis can form biofilms and investigate the impact on antibiotic susceptibility and survivability in polymicrobial biofilms with H. influenzae in vitro. The ability of A. otitidis to form single-species and polymicrobial biofilms with H. influenzae was explored. Clinical and commercial strains of A. otitidis and H. influenzae were incubated in brain heart infusion with and without supplementation. Biofilm was imaged using confocal laser scanning microscopy and scanning electron microscopy. Quantification of biofilm biomass and viable bacterial number was assessed using crystal violet assays and viable cell counting in both optimal growth conditions and in adverse growth conditions (depleted media and sub-optimal growth temperature). Antimicrobial susceptibility and changes in antibiotic resistance of single-species and multi-species co-culture were assessed using a microdilution method to assess minimal bactericidal concentration and E-test for amoxicillin and ciprofloxacin. A. otitidis formed single-species and polymicrobial biofilms with H. influenzae. Additionally, whilst strain dependent, combinations of polymicrobial biofilms decreased antimicrobial susceptibility, albeit a small magnitude, in both planktonic and polymicrobial biofilms. Moreover, A. otitidis promoted H. influenzae survival by increasing biofilm production in depleted media and at suboptimal growth temperature. Our findings suggest that A. otitidis may play an indirect pathogenic role in otitis media by altering H. influenzae antibiotic susceptibility and enhancing growth under adverse conditions.

Inactivation of the Thymidylate Synthase thyA in Non-typeable Haemophilus influenzae Modulates Antibiotic Resistance and Has a Strong Impact on Its Interplay with the Host Airways.

Antibacterial treatment with cotrimoxazol (TxS), a combination of trimethoprim and sulfamethoxazole, generates resistance by, among others, acquisition of thymidine auxotrophy associated with mutations in the thymidylate synthase gene thyA, which can modify the biology of infection. The opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently encountered in the lower airways of chronic obstructive pulmonary disease (COPD) patients, and associated with acute exacerbation of COPD symptoms. Increasing resistance of NTHi to TxS limits its suitability as initial antibacterial against COPD exacerbation, although its relationship with thymidine auxotrophy is unknown. In this study, the analysis of 2,542 NTHi isolates recovered at Bellvitge University Hospital (Spain) in the period 2010-2014 revealed 119 strains forming slow-growing colonies on the thymidine low concentration medium Mueller Hinton Fastidious, including one strain isolated from a COPD patient undergoing TxS therapy that was a reversible thymidine auxotroph. To assess the impact of thymidine auxotrophy in the NTHi-host interplay during respiratory infection, thyA mutants were generated in both the clinical isolate NTHi375 and the reference strain RdKW20. Inactivation of the thyA gene increased TxS resistance, but also promoted morphological changes consistent with elongation and impaired bacterial division, which altered H. influenzae self-aggregation, phosphorylcholine level, C3b deposition, and airway epithelial infection patterns. Availability of external thymidine contributed to overcome such auxotrophy and TxS effect, potentially facilitated by the nucleoside transporter nupC. Although, thyA inactivation resulted in bacterial attenuation in a lung infection mouse model, it also rendered a lower clearance upon a TxS challenge in vivo. Thus, our results show that thymidine auxotrophy modulates both the NTHi host airway interplay and antibiotic resistance, which should be considered at the clinical setting for the consequences of TxS administration.

Flagellar expression in clinical isolates of non-typeable Haemophilus influenzae.

PURPOSE: Haemophilus influenzae is a commensal organism found in the upper respiratory tract of humans. When H. influenzae becomes a pathogen, these bacteria can move out of their commensal niche and cause multiple respiratory tract diseases such as otitis media, sinusitis, conjunctivitis and bronchitis in children, and chronic obstructive pulmonary disease in adults. However, H. influenzae is currently considered a non-flagellate bacterium. METHODOLOGY AND RESULTS: In this study, 90 clinical isolates of H. influenzae strains (typeable and non-typeable) showed different degrees of the swarm-motility phenotype in vitro.Keys findings. One of these strains, NTHi BUAP96, showed the highest motility rate and its flagella were revealed using transmission electron microscopy and Ryu staining. Moreover, the flagellar genes fliC and flgH exhibited high homology with those of Actinobacillus pleuropneumoniae, Escherichia coli and Shigella flexneri. Furthermore, Western blot analysis, using anti-flagellin heterologous antibodies from E. coli, demonstrated cross-reaction with a protein present in NTHi BUAP96. CONCLUSION: This study provides, for the first time, information on flagellar expression in H. influenzae, representing an important finding related to its evolution and pathogenic potential.

Culture of non-typeable Haemophilus influenzae from the nasopharynx: Not all media are equal.

The efficacy of chocolate agar, versus bacitracin, vancomycin, clindamycin, chocolate agar (BVCCA) for the isolation of non-typeable Haemophilus influenzae (NTHi) from nasopharyngeal swabs was determined. BVCCA cultured NTHi from 97.3% of NTHi-positive swabs, compared to 87.1% for chocolate agar. To maximise culture sensitivity, the use of both media is recommended.

Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection.

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Ultrasound imaging and characterization of biofilms based on wavelet de-noised radiofrequency data.

The ability to non-invasively image and characterize bacterial biofilms in children during nasopharyngeal colonization with potential otopathogens and during acute otitis media would represent a significant advance. We sought to determine if quantitative high-frequency ultrasound techniques could be used to achieve that goal. Systematic time studies of bacterial biofilm formation were performed on three preparations of an isolated Haemophilus influenzae (NTHi) strain, a Streptococcus pneumoniae (Sp) strain and a combination of H. influenzae and S. pneumoniae (NTHi + Sp) in an in vitro environment. The process of characterization included conditioning of the acquired radiofrequency data obtained with a 15-MHz focused, piston transducer by using a seven-level wavelet decomposition scheme to de-noise the individual A-lines acquired. All subsequent spectral parameter estimations were done on the wavelet de-noised radiofrequency data. Various spectral parameters-peak frequency shift, bandwidth reduction and integrated backscatter coefficient-were recorded. These parameters were successfully used to map the progression of the biofilms in time and to differentiate between single- and multiple-species biofilms. Results were compared with those for confocal microscopy and theoretical evaluation of form factor. We conclude that high-frequency ultrasound may prove a useful modality to detect and characterize bacterial biofilms in humans as they form on tissues and plastic materials.

Haemophilus influenzae OxyR: characterization of its regulation, regulon and role in fitness.

To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a crucial role for OxyR in bacterial fitness.

Synergistic induction of MUC5AC mucin by nontypeable Haemophilus influenzae and Streptococcus pneumoniae.

Mucin overproduction is a hallmark of chronic respiratory diseases (CRD) such as chronic obstructive pulmonary disease and asthma, and otitis media. Despite the fact that nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae are co-existing under these disease conditions, little is known about how NTHi and S. pneumoniae induce mucin overproduction. Here we show that NTHi and S. pneumoniae, when present together, synergistically induce MUC5AC mucin transcription. TLR2/4-MyD88-TAK1 signaling cascade transmits signal to regulate the synergistic induction of MUC5AC. The activation of MKK3/6-p38 and ERK MAPK pathways are required for the synergistic induction of MUC5AC. Moreover, S. pneumoniae synergizes with NTHi to induce MUC5AC expression via AP-1-dependent mechanism. Thus, our studies provide direct evidence for the synergistic induction of MUC5AC in mixed infections and bring novel insights into our understanding of molecular mechanisms underlying polymicrobial infections in CRD and OM.

Surface anchoring of a bacterial adhesin secreted by the two-partner secretion pathway.

In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. In the present study we sought to determine the mechanism by which HMW1 is anchored to the bacterial surface. In initial experiments we found that HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. Further analysis established that HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Mutagenesis and polyethylene glycol maleimide labelling revealed that anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region. Immunolabelling studies demonstrated that the immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains in the periplasm or is buried in HMW1B. Coexpression of HMW1 lacking the C-terminal 20 amino acids and wild-type HMW1 supported the conclusion that the C-terminus of HMW1 occupies the HMW1B pore. These observations may have broad relevance to proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.

The phasevarion: a genetic system controlling coordinated, random switching of expression of multiple genes.

Several host-adapted bacterial pathogens contain methyltransferases associated with type III restriction-modification (R-M) systems that are subject to reversible, high-frequency on/off switching of expression (phase variation). To investigate the role of phase-variable expression of R-M systems, we made a mutant strain lacking the methyltransferase (mod) associated with a type III R-M system of Haemophilus influenzae and analyzed its phenotype. By microarray analysis, we identified a number of genes that were either up- or down-regulated in the mod mutant strain. This system reports the coordinated random switching of a set of genes in a bacterial pathogen and may represent a widely used mechanism.

Destabilization of tetranucleotide repeats in Haemophilus influenzae mutants lacking RnaseHI or the Klenow domain of PolI.

A feature of Haemophilus influenzae genomes is the presence of several loci containing tracts of six or more identical tetranucleotide repeat units. These repeat tracts are unstable and mediate high frequency, reversible alterations in the expression of surface antigens. This process, termed phase variation (PV), enables H.influenzae to rapidly adapt to fluctuations in the host environment. Perturbation of lagging strand DNA synthesis is known to destabilize simple sequence repeats in yeast and Escherichia coli. By using a chromosomally located reporter construct, we demonstrated that the mutation of an H.influenzae rnhA (encoding RnaseHI) homologue increases the mutation rates of tetranucleotide repeats approximately 3-fold. Additionally, deletion of the Klenow domain of DNA polymerase I (PolI) resulted in a approximately 35-fold increase in tetranucleotide repeat-mediated PV rates. Deletion of the PolI 5'>3' exonuclease domain appears to be lethal. The phenotypes of these mutants suggest that delayed or mutagenic Okazaki fragment processing destabilizes H.influenzae tetranucleotide repeat tracts.

FtsK activities in Xer recombination, DNA mobilization and cell division involve overlapping and separate domains of the protein.

Escherichia coli FtsK is a multifunctional protein that couples cell division and chromosome segregation. Its N-terminal transmembrane domain (FtsK(N)) is essential for septum formation, whereas its C-terminal domain (FtsK(C)) is required for chromosome dimer resolution by XerCD-dif site-specific recombination. FtsK(C) is an ATP-dependent DNA translocase. In vitro and in vivo data point to a dual role for this domain in chromosome dimer resolution (i) to directly activate recombination by XerCD-dif and (ii) to bring recombination sites together and/or to clear DNA from the closing septum. FtsK(N) and FtsK(C) are separated by a long linker region (FtsK(L)) of unknown function that is highly divergent between bacterial species. Here, we analysed the in vivo effects of deletions of FtsK(L) and/or of FtsK(C), of swaps of these domains with their Haemophilus influenzae counterparts and of a point mutation that inactivates the walker A motif of FtsK(C). Phenotypic characterization of the mutants indicated a role for FtsK(L) in cell division. More importantly, even though Xer recombination activation and DNA mobilization both rely on the ATPase activity of FtsK(C), mutants were found that can perform only one or the other of these two functions, which allowed their separation in vivo for the first time.

Global versus local regulatory roles for Lrp-related proteins: Haemophilus influenzae as a case study.

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp(+) and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.

Preservation of Haemophilus influenzae and Haemophilus parainfluenzae at -70 degrees C.

We have studied the viability of Haemophilus spp. preserved for 5 to 12 months at -70 degrees C. The following media were used: Laboratoire de Santé Publique du Québec (LSPQ) preservation medium, trypticase soy broth with 10 degrees C (vol/vol) glycerol and 40 degrees C (vol/vol) horse serum (TSBG), and Levinthal's broth (LB) medium. Three clinical isolates of both H. influenzae and H. parainfluenzae were used. After 5 months no differences in viability were observed between strains preserved in TSBG and strains preserved in LB, but a significant loss of viability was observed in strains preserved in LSPQ medium. No significant changes in antimicrobial susceptibility were observed after 5-month storage in any medium. After 12 months, TSBG appeared to be the most suitable cryopreservation medium for the six strains tested. We conclude that TSBG represents a good medium for the maintenance of Haemophilus spp. at -70 degrees C for up to 1 year.

Evaluation of phase variation of nontypeable Haemophilus influenzae lipooligosaccharide during nasopharyngeal colonization and development of otitis media in the chinchilla model.

Nontypeable Haemophilus influenzae (NTHI) has four loci, lic-1 to lic-3 and lgtC, that generate phase-variable lipooligosaccharide (LOS) structures. lic-1, which is required for the expression of phosphorylcholine (ChoP), is the best characterized and is associated with an enhanced ability of H. influenzae to persist within the nasopharynges of infant rats. Recent data indicate that LOS impacts various aspects of NTHI virulence in the chinchilla model of nasopharyngeal colonization and otitis media (OM). In this study the effects of ChoP expression and the sequences of lic-1 to lic-3 and lgtC of NTHI strain 2019 were evaluated in the chinchilla OM model. Nasopharyngeal colonization data showed that a switch from the ChoP(-) to the ChoP(+) phenotype was observed as early as day 3 after intranasal inoculation. Chinchillas colonized by strains with the ChoP(+) phenotype demonstrated a significantly higher level of NTHI 2019 per milliliter of nasal lavage fluid than chinchillas colonized with predominantly the ChoP(-) variant (P < 0.05). The concentration of cells with the ChoP(+) phenotype in the middle ear was 3 log units higher than that of cells with the ChoP(-) variant (P < 0.01). There was a statistically significant association between ChoP(+) expression in the nasal lavage and the development of OM with culture-positive middle ear fluids in this model. These data suggest that expression of the ChoP(+) phenotype promotes enhanced nasopharyngeal colonization and development of OM.

Quantitation of bacterial adherence by image analysis.

In studies of the adherence of pathogenic bacteria to host eukaryotic cells in vitro, the counting of the bacteria is often challenging, especially if many experiments are involved. We developed a method to use digital imaging and computer-aided recognition for the quantitation of bacteria attached to cultured cells. We employed an immunocytochemical method to stain the bacteria and leave the hosts cells relatively unstained. We describe this method for use with five species of bacteria, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, and Chlamydia pneumoniae. To demonstrate an application of this method, we studied the attachment of H. influenzae and S. pneumoniae to target epithelial cell lines derived from the respiratory tract.

The identification a novel gene required for lipopolysaccharide biosynthesis by Haemophilus influenzae RM7004, using transposon Tn916 mutagenesis.

Mutagenesis with the transposon Tn916 was used as a strategy to identify genes required for synthesis of the Gal alpha (1-4) beta Gal component of Haemophilus influenzae strain RM7004 lipopolysaccharide. Insertion of Tn916 into an open reading frame (ORF) encoding a protein with 75% homology to the Escherichia coli methionine related protein (Mrp) is described. Mutations in mrp resulted in loss of reactivity with monoclonal antibody (mAb) 4C4, which recognises Gal alpha (1-4) beta Gal, and expression of LPS with a different electrophoretic profile to that of wild-type RM7004. An unexpected feature of this mutation was that it appeared to influence the number of copies of 5'-CAAT-3' present in lic2A, a gene which is also required for biosynthesis and phase variable expression of the Gal alpha (1-4) beta Gal LPS epitope.

Neutrophil response to nontypable Haemophilus influenzae in respiratory infections.

Sputa from patients with respiratory infections by nontypable Haemophilus influenzae (H. influenzae) were investigated by electron microscopy. The cell wall of H. influenzae appeared wavy and nonwavy. In the cell wall the peptidoglycan layer was ill-defined. These patients had adequate IgG response in the serum against H. influenzae. However neither capsule nor fimbriae were found. Different stages of phagocytosis and destruction of the bacteria by polymorphonuclear neutrophils (PMN) were observed. PMNs were also found to phagocytose the debris. Evidences were found that the debris is formed mainly by the destruction of polymorphonuclear neutrophil. Extracellular lysosomes were also observed, which may have a role in destruction of both bacteria and host tissue. It was concluded that nontypable H. influenzae are nonfimbriated and noncapsulated during infection. Debris are the end product of PMN destruction, and phagocytosis of debris by PMNs has a role in the pathogenesis of chronic respiratory diseases.

Adherence of Haemophilus influenzae to middle ear mucosa injured by killed H. influenzae.

Repetitive acute otitis media is due to recurrent bacterial infection of middle ear superimposed on chronic otitis media with effusion. Endotoxin, one of the constituents of Haemophilus influenzae, is present in some cases in the middle ear effusion of otitis media with effusion and has been demonstrated experimentally to damage the middle ear mucosa. The aim of this study was to determine the effect of killed H. influenzae on the adherence of H. influenzae and H. parainfluenzae to the middle ear epithelial cells. The numbers of adherent organisms per epithelial cell in ears inoculated previously with killed H. influenzae or with normal saline (0.9% NaCl) were compared. Prior middle ear inoculation of killed H. influenzae enhanced the adherence of H. influenzae to middle ear epithelial cells, but it had little effect on the adherence of H. parainfluenzae. H. influenzae adhered to middle ear epithelial cells in greater numbers than H. parainfluenzae. Results demonstrate that a middle ear pathogen adheres to middle ear epithelial cells presumably damaged by killed H. influenzae, whereas a non-pathogen does not. These findings might partly explain the increased susceptibility of an ear with chronic otitis media with effusion to recurrent infection with H. influenzae.

Effect of fusafungine on adherence of Haemophilus influenzae type b to human epithelial cells in vitro.

Haemophilus influenzae, a normal host of the nasopharynx of humans, may become a pathogen. The first step of infection is adherence to epithelial cells of the nasopharynx through glycopeptidic adhesins, or pili. Adherence to human epithelial cells in continuous lines, HeLa and Hep2, of 8 piliated strains of Haemophilus influenzae isolated from human infections of the respiratory tract was studied in vitro in the presence of fusafungine, a local bacteriostatic antibiotic. When the bacteria were grown in the presence of 0.5 x the MIC, fusafungine afforded 45-75% of adherence inhibition, but this inhibitory effect did not parallel the MICs. In contrast, no significant effect could be observed either when epithelial cells were exposed to 0.5 x the MIC before use in the adherence assay, or when this assay was performed in the presence of 0.5 x the MIC of fusafungine. The partial adherence inhibition observed suggests that fusafungine interacts with the bacterial binding sites but that other mechanisms may contribute to the inhibitory process. This effect of fusafungine should prevent but not eradicate colonization of the nasopharyngeal mucosa by Haemophilus influenzae and may account for the therapeutic efficacy reported in infections of the respiratory tract due to Haemophilus influenzae.