Lauren O. Bakaletz

Nontypeable Haemophilus influenzae as a pathogen in children.

Nontypeable Haemophilus influenzae is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. H. influenzae type b conjugate vaccines have no effect on infections caused by nontypeable strains because nontypeable strains are nonencapsulated. Approximately, one-third of episodes of otitis media are caused by nontypeable H. influenzae and the bacterium is the most common cause of recurrent otitis media. Recent progress in elucidating molecular mechanisms of pathogenesis, understanding the role of biofilms in otitis media and an increasing understanding of immune responses have potential for development of novel strategies to improve prevention and treatment of otitis media caused by nontypeable H. influenzae. Feasibility of vaccination for prevention of otitis media due to nontypeable H. influenzae was recently demonstrated in a clinical trial with a vaccine that included the surface virulence factor, protein D.

Genomic Sequence of an Otitis Media Isolate of Nontypeable Haemophilus influenzae: Comparative Study with H. influenzae Serotype d, Strain KW20

In 1995, the Institute for Genomic Research completed the genome sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. Although extremely useful in understanding the basic biology of H. influenzae, these data have not provided significant insight into disease caused by nontypeable H. influenzae, as serotype d strains are not pathogens. In contrast, strains of nontypeable H. influenzae are the primary pathogens of chronic and recurrent otitis media in children. In addition, these organisms have an important role in acute otitis media in children as well as other respiratory diseases. Such strains must therefore contain a gene repertoire that differs from that of strain Rd. Elucidation of the differences between these genomes will thus provide insight into the pathogenic mechanisms of nontypeable H. influenzae. The genome of a representative nontypeable H. influenzae strain, 86-028NP, isolated from a patient with chronic otitis media was therefore sequenced and annotated. Despite large regions of synteny with the strain Rd genome, there are large rearrangements in strain 86-028NPs genome architecture relative to the strain Rd genome. A genomic island similar to an island originally identified in H. influenzae type b is present in the strain 86-028NP genome, while the mu-like phage present in the strain Rd genome is absent from the strain 86-028NP genome. Two hundred eighty open reading frames were identified in the strain 86-028NP genome that were absent from the strain Rd genome. These data provide new insight that complements and extends the ongoing analysis of nontypeable H. influenzae virulence determinants.

Differential Type I Interferon Induction by Respiratory Syncytial Virus and Influenza A Virus In Vivo

ABSTRACT Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-α/β induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-α/β production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-α/β in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.

Viral potentiation of bacterial superinfection of the respiratory tract

Virus infections of the respiratory tract predispose it to bacterial superinfections. Epidemiological studies, clinical evidence of viral-bacterial co-infection and animal models of such interactions suggest a time course of events and several mechanisms by which viral potentiation may occur. It appears that structural and functional disruption of the respiratory mucosal epithelium is a major contributor to the synergistic effects of superinfection.

Particulate Matter in the Posterior Semicircular Canal

The pathoetiology of benign paroxysmal positional vertigo (BPPV) is controversial. Particulate matter within the posterior semicircular canal has been identified intraoperatively in patients with BPPV but has also been reported in non‐BPPV patients at the time of translabyrinthine surgery (Parnes LS, McClure JA. Free‐floating endolymphatic particles: a new operative finding during posterior semicircular canal occlusion. Laryngoscope 1992;102:988‐92; Schuknecht HF, Ruby RRF. Cupulolithiasis. Adv Otorhinolaryngol 1973;20:434‐43; Kveton JF, Kashgarian M. Particulate matter within the membranous labyrinth: pathologic or normal? Am J Otol 1994;15:173‐6). The nature of the particulate matter remains unknown.

Microbial interactions in the respiratory tract.

Upper respiratory tract infections are caused by the synergistic and antagonistic interactions between upper respiratory tract viruses and 3 predominant bacterial pathogens: Streptococcus pneumoniae, nontypeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis, which are members of the commensal flora of the nasopharynx. For many bacterial pathogens, colonization of host mucosal surfaces is a first and necessary step in the infectious process. S. pneumoniae and H. influenzae have intricate interactions in the nasopharynx. The host innate immune response may influence these interactions and therefore influence the composition of the colonizing flora and the invading bacteria. S. pneumoniae, nontypeable H. influenzae, and M. catarrhalis can behave as opportunistic pathogens of the middle ear when conditions are optimal. Chronic otitis media (OM) and recurrent OM include a biofilm component. Each of the 3 predominant pathogens of OM can form a biofilm and have been shown to comprise biofilms present on middle ear mucosa specimens recovered from children with recurrent or chronic OM. Some of these characterized biofilms are of mixed bacterial etiology, suggesting that progress made on single-microbe directed strategies for treatment and/or prevention of OM, although highly encouraging, are likely to be inadequate. A significantly greater understanding about microbial physiology is required as it relates to the involvement of biofilms in OM, to identify points in the natural course of the disease that are perhaps more amenable to treatment strategies, as well as to identify biofilm-relevant antigenic targets that would be helpful in the rational design of vaccines to prevent OM.

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.

ABSTRACT 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.

Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins.

Bacteria that cause chronic and/or recurrent diseases often rely on a biofilm lifestyle. The foundation of the biofilm structure is the extracellular polymeric substance (EPS) that acts as a barrier to both effectors of the immune system and antimicrobial agents. Recent work has highlighted extracellular DNA (eDNA) as a key component common to many pathogenic biofilms. Here, we show that the DNABII family of proteins, well known for their strong structural influences on intracellular DNA, was also critical for the integrity of the EPS matrix of biofilms that contain eDNA. In fact, antisera derived against a purified Escherichia coli DNABII family member rapidly disrupts the biofilm EPS formed by multiple human pathogens in vitro. In addition, when a member of this family of proteins was used as an immunogen in an animal model in which the bacteria had already formed a robust biofilm at the site of infection, the resultant targeted immune response strongly ameliorated this biofilm disease in vivo. Finally, this methodology to debulk the biofilm of EPS was shown to work synergistically with otherwise ineffective traditional anti-microbial approaches in vitro. We discuss the prospects for targeting DNABII family members as a potential universal strategy for treating biofilm diseases.

Developing a nontypeable Haemophilus influenzae (NTHi) vaccine

There is a current high demand for nontypable Haemophilus influenzae (NTHi) vaccines. Various options for the composition of such vaccines are possible. Decisions about the vaccine composition have to take into account the antigenic variability of NTHi, so even complex immunogens such as whole bacteria would preferentially have a tailor-made antigenic composition. We will present a summary of NTHi vaccine development, describing research efforts from SmithKline Beecham and other laboratories. Currently, major (P1, P2, P4, P5) and minor (P6, D15, TbpA/B, ellipsis) outer membrane proteins, LPS, adhesins (HMW, Hia, pili, P5) are being studied. Preclinical results with LPD, P5 (LB1) and OMP26 from our laboratories will be described including the use of animal models of otitis and lung infection.

The PilA protein of non‐typeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract

We recently described the expression of type IV pili (Tfp) by non‐typeable Haemophilus influenzae (NTHI), a common respiratory tract pathogen. Prior to that report, Tfp were not thought to be produced by NTHI as they are not observed on NTHI when grown on chocolate agar or other commonly used growth media. To further characterize growth conditions permissive for the expression of NTHI Tfp, as well as determine their role in colonization and virulence, we transformed an NTHI otitis media isolate with a reporter plasmid containing the lux gene cluster driven by the pilA promoter. Transcription from the pilA promoter was demonstrated under a variety of in vitro growth conditions and, importantly, by ex vivo imaging of luciferase‐producing NTHI in infected chinchillas. Luciferase‐producing NTHI were also identified within a biofilm formed by NTHI in vivo. We further demonstrated a role for NTHI PilA in adherence to human respiratory epithelial cells, in colonization of the chinchilla respiratory tract as well as a requirement for PilA in biofilm development, both in vitro and in vivo. Collectively, our data demonstrate that NTHI express PilA in vivo, and that PilA plays an important role in the pathogenesis of an upper respiratory tract infection induced by NTHI.