Nontypeable Haemophilus influenzae P5 Binds Human C4b-Binding Protein, Promoting Serum Resistance.

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes infections mainly in the upper and lower respiratory tract. The bacterium is associated with bronchitis and exacerbations in patients suffering from chronic obstructive pulmonary disease and frequently causes acute otitis media in preschool children. We have previously demonstrated that the binding of C4b binding protein (C4BP) is important for NTHi complement evasion. In this study, we identified outer membrane protein 5 (P5) of NTHi as a novel ligand of C4BP. Importantly, we observed significantly lower C4BP binding and decreased serum resistance in P5-deficient NTHi mutants. Surface expression of recombinant P5 on Escherichia coli conferred C4BP binding and consequently increased serum resistance. Moreover, P5 expression was positively correlated with C4BP binding in a series of clinical isolates. We revealed higher levels of P5 surface expression and consequently more C4BP binding in isolates from the lower respiratory tract of chronic obstructive pulmonary disease patients and tonsil specimens compared with isolates from the upper respiratory tract and the bloodstream (invasive strains). Our results highlight P5 as an important protein for protecting NTHi against complement-mediated killing.

The Interplay Between Immune Response and Bacterial Infection in COPD: Focus Upon Non-typeable Haemophilus influenzae.

Chronic obstructive pulmonary disease (COPD) is a debilitating respiratory disease and one of the leading causes of morbidity and mortality worldwide. It is characterized by persistent respiratory symptoms and airflow limitation due to abnormalities in the lower airway following consistent exposure to noxious particles or gases. Acute exacerbations of COPD (AECOPD) are characterized by increased cough, purulent sputum production, and dyspnea. The AECOPD is mostly associated with infection caused by common cold viruses or bacteria, or co-infections. Chronic and persistent infection by non-typeable Haemophilus influenzae (NTHi), a Gram-negative coccobacillus, contributes to almost half of the infective exacerbations caused by bacteria. This is supported by reports that NTHi is commonly isolated in the sputum from COPD patients during exacerbations. Persistent colonization of NTHi in the lower airway requires a plethora of phenotypic adaptation and virulent mechanisms that are developed over time to cope with changing environmental pressures in the airway such as host immuno-inflammatory response. Chronic inhalation of noxious irritants in COPD causes a changed balance in the lung microbiome, abnormal inflammatory response, and an impaired airway immune system. These conditions significantly provide an opportunistic platform for NTHi colonization and infection resulting in a "vicious circle." Episodes of large inflammation as the consequences of multiple interactions between airway immune cells and NTHi, accumulatively contribute to COPD exacerbations and may result in worsening of the clinical status. In this review, we discuss in detail the interplay and crosstalk between airway immune residents and NTHi, and their effect in AECOPD for better understanding of NTHi pathogenesis in COPD patients.

Benzylpenicillin versus wide-spectrum beta-lactam antibiotics as empirical treatment of Haemophilus influenzae-associated lower respiratory tract infections in adults; a retrospective propensity score-matched study.

There is consensus that definitive therapy for infections with H. influenzae should include antimicrobial agents with clinical breakpoints against the bacterium. In Scandinavia, benzylpenicillin is the recommended empirical treatment for community-acquired pneumonia (CAP) except in very severe cases. However, the effect of benzylpenicillin on H. influenzae infections has been debated. The aim of this study was to compare the outcomes of patients given benzylpenicillin with patients given wide-spectrum beta-lactams (WSBL) as empirical treatment of lower respiratory tract H. influenzae infections requiring hospital care. We identified 481 adults hospitalized with lower respiratory tract infection by H. influenzae, bacteremic and non-bacteremic. Overall, 30-day mortality was 9% (42/481). Thirty-day mortality, 30-day readmission rates, and early clinical response rates were compared in patients receiving benzylpenicillin (n = 199) and a WSBL (n = 213) as empirical monotherapy. After adjusting for potential confounders, empirical benzylpenicillin treatment was not associated with higher 30-day mortality neither in a multivariate logistic regression (aOR 2.03 for WSBL compared to benzylpenicillin, 95% CI 0.91-4.50, p = 0.082), nor in a propensity score-matched analysis (aOR 2.14, 95% CI 0.93-4.92, p = 0.075). Readmission rates did not significantly differ between the study groups, but early clinical response rates were significantly higher in the WSBL group (aOR 2.28, 95% CI 1.21-4.31, p = 0.011), albeit still high in both groups (84 vs 81%). In conclusion, despite early clinical response rates being slightly lower for benzylpenicillin compared to WSBL, we found no support for increased mortality or readmission rates in patients empirically treated with benzylpenicillin for lower respiratory tract infections by H. influenzae.

A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility.

BACKGROUND: Identification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to ß-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93-57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined. RESULTS: Site-directed mutagenesis of a ß-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion screening and broth microdilution determination of MICs for ß-lactam agents were done with the NTHi3655-PBP3Y528H mutant and were compared with the NTHi3655 wild-type as well as the original clinical isolate NTHi93-57485. Introduction of the Y528H substitution in NTHi3655 resulted in positive screening for ß-lactam resistance. MICs for aminopenicillins were increased in the mutant compared to the wild-type. However, the mutant remained susceptible to aminopenicillins according to EUCAST clinical breakpoints (assuming intravenous treatment) and the introduction of Y528H alone did not increase the resistance to the same level as NTHi93-57485. None of the isolates had frame shift insertions in the acrR gene regulating the AcrAB efflux pump. CONCLUSIONS: In parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility.

Aerobic bacteria associated with chronic suppurative otitis media in Angola.

BACKGROUND: Chronic suppurative otitis media (CSOM) is an important cause of hearing loss in children and constitutes a serious health problem globally with a strong association to resource-limited living conditions. Topical antibiotics combined with aural toilet is the first-hand treatment for CSOM but antimicrobial resistance and limited availability to antibiotics are obstacles in some areas. The goal of this study was to define aerobic pathogens associated with CSOM in Angola with the overall aim to provide a background for local treatment recommendations. METHODS: Samples from ear discharge and the nasopharynx were collected and cultured from 152 patients with ear discharge and perforation of the tympanic membrane. Identification of bacterial species was performed with matrix-assisted laser desorption/ionization-time of flight mass spectrometry and pneumococci were serotyped using multiplex polymerase chain reactions. Antimicrobial susceptibility testing was done according to EUCAST. RESULTS: One hundred eighty-four samples from ear discharge and 151 nasopharyngeal swabs were collected and yielded 534 and 289 individual isolates, respectively. In all patients, correspondence rate of isolates from 2 ears in patients with bilateral disease was 27.3% and 9.3% comparing isolates from the nasopharynx and ear discharge, respectively. Proteus spp. (14.7%), Pseudomonas aeruginosa (13.2%) and Enterococcus spp. (8.8%) were dominating pathogens isolated from ear discharge. A large part of the remaining species belonged to Enterobacteriaceae (23.5%). Pneumococci and Staphylococcus aureus were detected in approximately 10% of nasopharyngeal samples. Resistance rates to quinolones exceeded 10% among Enterobacteriaceae and was 30.8% in S. aureus, whereas 6.3% of P. aeruginosa were resistant. CONCLUSIONS: The infection of the middle ear in CSOM is highly polymicrobial, and isolates found in nasopharynx do not correspond well with those found in ear discharge. Pathogens associated with CSOM in Angola are dominated by gram-negatives including Enterobacteriaceae and P. aeruginosa, while gram-positive enterococci also are common. Based on the results of antimicrobial susceptibility testing topical quinolones would be the preferred antibiotic therapy of CSOM in Angola. Topical antiseptics such as aluminium acetate, acetic acid or boric acid, however, may be more feasible options due to a possibly emerging antimicrobial resistance.

The prevalence, population structure and screening test specificity of penicillin-susceptible Staphylococcus aureus bacteremia isolates in Malmö, Sweden.

OBJECTIVES: The objectives of this study were to examine the prevalence of penicillin-susceptible bacteremic Staphylococcus aureus in the Malmö area in 2014, to re-evaluate the phenotypic methods of penicillinase detection on these isolates, and to investigate the clonal distribution of penicillin-susceptible isolates. METHODS: All non-redundant S. aureus from blood in the Malmö catchment area in southern Sweden 2014 were screened for penicillin susceptibility using PcG 1U disk diffusion, E-test PcG and the nitrocefin test. All isolates screened as likely susceptible were subjected to PCR for detection of penicillinase (blaZ) and spa-typing. RESULTS: Almost one out of three bacteremic isolates (80/257; 31.1%) were susceptible to penicillin. All screening methods except for the nitrocefin test alone had a low proportion of isolates falsely tested as susceptible, but no method used in the study had perfect specificity compared with PCR. Penicillin-susceptible isolates had a distinct phylogenetic distribution, and two clonal complexes (CC5 and CC45) constituted half of the isolates. CONCLUSION: Almost one third of S. aureus isolated from blood in southern Sweden in 2014 was susceptible to penicillin. Considering that intravenous penicillin has theoretical advantages compared with the standard treatment in the study area, we argue that routine testing of penicillin susceptibility should be reconsidered.