Nontypeable Haemophilus influenzae infection impedes Pseudomonas aeruginosa colonization and persistence in mouse respiratory tract

Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature, and the predominant bacterial species undergo a predictable series of changes as patients age. Young patients have populations dominated by opportunists that are typically found within the microbiome of the human nasopharynx, such as nontypeable Haemophilus influenzae (NTHi); these are eventually supplanted and the population within the CF lung is later dominated by pathogens such as Pseudomonas aeruginosa ( Pa ). In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract. Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential respiratory infection of mice with NTHi followed by Pa resulted in significantly lower Pa as compared to infection with Pa alone. Coinfected mice also had reduced airway tissue damage and lower levels of inflammatory cytokines as compared with Pa infected mice. Similar results were observed after instillation of heat-inactivated NTHi bacteria or purified NTHi lipooligosaccharide (LOS) endotoxin prior to Pa introduction. Based on these results, we conclude that NTHi significantly reduces susceptibility to subsequent Pa infection, most likely due to priming of host innate immunity rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early life infections in patients with CF.

Lipoprotein(a), an Opsonin, Enhances the Phagocytosis of Nontypeable Haemophilus influenzae by Macrophages

We recently showed that both nontypeable Haemophilus influenzae (NTHi) and its surface plasminogen- (Plg-) binding proteins interact with lipoprotein(a) (Lp(a)) in a lysine-dependent manner. Because Lp(a) can be taken up by macrophages, we postulated that it serves as an opsonin to enhance phagocytosis of NTHi by macrophages. Based on colony-forming unit (CFU) counts, Lp(a) was found to increase U937 macrophage-mediated phagocytosis of NTHi49247 and NTHi49766 by 34% and 43%, respectively, after 120 min. In contrast, Lp(a) did not enhance phagocytosis of Escherichia coli BL21 or E. coli JM109, which were unable to bind to Lp(a). As with U937 macrophages, Lp(a) was capable of increasing phagocytosis of NTHi49247 by peripheral blood mononuclear cell-derived macrophages. Opsonic phagocytosis by Lp(a) was inhibited by the addition of recombinant kringle IV type 10 (rKIV10), a lysine-binding competitor; moreover, Lp(a) did not increase phagocytosis of NTHi by U937 macrophages that were pretreated with a monoclonal antibody against the scavenger receptor CD36. Taken together, our observation suggests that Lp(a) might serve as a lysine-binding opsonin to assist macrophages in rapid recognition and phagocytosis of NTHi.

In Vivo Secretion of β-Lactamase-Carrying Outer Membrane Vesicles as a Mechanism of β-Lactam Therapy Failure

Outer membrane vesicles carrying β-lactamase (βLOMVs) protect bacteria against β-lactam antibiotics under experimental conditions, but their protective role during a patient’s treatment leading to the therapy failure is unknown. We investigated the role of βLOMVs in amoxicillin therapy failure in a patient with group A Streptococcus pyogenes (GAS) pharyngotonsillitis. The patient’s throat culture was examined by standard microbiological procedures. Bacterial vesicles were analyzed for β-lactamase by immunoblot and the nitrocefin assay, and in vivo secretion of βLOMVs was detected by electron microscopy. These analyses demonstrated that the patient’s throat culture grew, besides amoxicillin-susceptible GAS, an amoxicillin-resistant nontypeable Haemophilus influenzae (NTHi), which secreted βLOMVs. Secretion and β-lactamase activity of NTHi βLOMVs were induced by amoxicillin concentrations reached in the tonsils during therapy. The presence of NTHi βLOMVs significantly increased the minimal inhibitory concentration of amoxicillin for GAS and thereby protected GAS against bactericidal concentrations of amoxicillin. NTHi βLOMVs were identified in the patient’s pharyngotonsillar swabs and saliva, demonstrating their secretion in vivo at the site of infection. We conclude that the pathogen protection via βLOMVs secreted by the flora colonizing the infection site represents a yet underestimated mechanism of β-lactam therapy failure that warrants attention in clinical studies.

Nontypeable Haemophilus influenzae infection impedes Pseduomonas aeruginosa colonization and persistence in mouse respiratory tract

Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature wherein the predominant bacterial species changes as patients age. Young patients have populations dominated by pathobiont such as nontypeable Haemophilus influenzae (NTHi), that are eventually supplanted by pathogens such as Pseudomonas aeruginosa (Pa), which are more typical of late-stage CF disease. In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract. Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential infection of mice with NTHi followed by Pa showed significant reduction in Pa in the lungs as compared to infection with Pa alone. Coinfected mice also had reduced severity of airway tissue damage and lower levels of inflammatory cytokines as compared mice infected with Pa alone. Similar results were observed using heat-inactivated NTHi bacteria prior to Pa introduction. Based on these results, we conclude that NTHi can significantly reduce susceptibility to subsequent Pa infection, most likely due to immune priming rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early life infections in patients with CF

Immunization with HMW1 and HMW2 adhesins protects against colonization by heterologous strains of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and results in significant morbidity. The pathogenesis of NTHi disease begins with nasopharyngeal colonization, and therefore, the prevention of colonization represents a strategy to prevent disease. The NTHi HMW1 and HMW2 proteins are a family of conserved adhesins that are present in 75 to 80% of strains and have been demonstrated to play a critical role in colonization of the upper respiratory tract in rhesus macaques. In this study, we examined the vaccine potential of HMW1 and HMW2 using a mouse model of nasopharyngeal colonization. Immunization with HMW1 and HMW2 by either the subcutaneous or the intranasal route resulted in a strain-specific antibody response associated with agglutination of bacteria and restriction of bacterial adherence. Despite the specificity of the antibody response, immunization resulted in protection against colonization by both the parent NTHi strain and heterologous strains expressing distinct HMW1 and HMW2 proteins. Pretreatment with antibody against IL-17A eliminated protection against heterologous strains, indicating that heterologous protection is IL-17A dependent. This work demonstrates the vaccine potential of the HMW1 and HMW2 proteins and highlights the importance of IL-17A in protection against diverse NTHi strains.

Oropharyngeal Carriage of hpl-Containing Haemophilus haemolyticus Predicts Lower Prevalence and Density of NTHi Colonisation in Healthy Adults

Nontypeable Haemophilus influenzae (NTHi) is a major respiratory pathogen that initiates infection by colonising the upper airways. Strategies that interfere with this interaction may therefore have a clinically significant impact on the ability of NTHi to cause disease. We have previously shown that strains of the commensal bacterium Haemophilus haemolyticus (Hh) that produce a novel haem-binding protein, haemophilin, can prevent NTHi growth and interactions with host cells in vitro. We hypothesized that natural pharyngeal carriage of Hh strains with the hpl open reading frame (Hh-hpl+) would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Oropharyngeal swabs were collected from 257 healthy adults in Australia between 2018 and 2019. Real-time PCR was used to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl+ or Hh-hpl− genotype. The likelihood of acquiring/maintaining NTHi colonisation status over a two- to six-month period was assessed in individuals that carried either Hh-hpl− (n = 25) or Hh-hpl+ (n = 25). Compared to carriage of Hh-hpl− strains, adult (18–65 years) and elderly (>65 years) participants that were colonised with Hh-hpl+ were 2.43 or 2.67 times less likely to carry NTHi in their oropharynx, respectively. Colonisation with high densities of Hh-hpl+ correlated with a low NTHi carriage load and a 2.63 times lower likelihood of acquiring/maintaining NTHi colonisation status between visits. Together with supporting in vitro studies, these results encourage further investigation into the potential use of Hh-hpl+ as a respiratory probiotic candidate for the prevention of NTHi infection.