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.

Parental views and the key role of nurses for high vaccine acceptance in Sweden - a focus group study.

BACKGROUND: In Sweden, vaccine uptake is exceptionally high due to an efficient child immunization program. More than 97% of Swedish children were vaccinated at child health care centers (CHCs) according to the schedule at 2 years of age in 2021. From the age of 6 years, vaccinations are given within the school health care. Maintaining high vaccination coverage over time is one of the central motives to explore and understand drivers for vaccine acceptance. The current study aimed to assess parental vaccine acceptance concerning the national immunization program and explore factors contributing to the high vaccine acceptance in Sweden. METHODS: Parents of children aged 1-2 years and 8-12 years were recruited through purposive sampling and asked to participate in focus groups held in three cities in Sweden, in February and March 2019. In total, 47 parents participated in two focus groups per city, one session for parents of younger (1-2 years) and older (8-12 years) children respectively. The focus group discussions were analyzed using qualitative content analysis. RESULTS: Parents of children aged 1-2 years expressed the themes; strong compliance to and protection of the value of vaccinations; parents feel safe with an attentive relationship with their nurse; the spectrum of communication needs is essential to meet. For parents to children aged 8-12 years, the themes expressed were; vaccinate to do good for the individual and society; a foundation of trust is built at CHCs for decisions later on; decisions for vaccination become more complex as children get older; communication changes as children get older and need to be explicit and tailored to the situation. CONCLUSION: Both individual and societal perspectives were shown to influence the vaccination decision for childhood immunizations, as manifested in parental reflections and experiences. As nurses have a key role, it is important to provide them with continued support and tools to facilitate their support for parents in making informed decisions. Continuous work for supporting driving factors for vaccination over time is needed to maintain high vaccine acceptance in Sweden.

Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils.

Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

Heparin-binding protein in lower airway samples as a biomarker for pneumonia.

OBJECTIVES: Ventilator-associated pneumonia (VAP) is difficult to diagnose using clinical criteria and no biomarkers have yet been proved to be sufficiently accurate. The use of the neutrophil-derived Heparin-binding protein (HBP) as a biomarker for pneumonia was investigated in this exploratory case-control study in two intensive care units at a tertiary referral hospital. METHODS: Patients with clinical signs of pneumonia were recruited and bronchoalveolar lavage fluid (BALF) or bronchial wash (BW) samples were collected. Mechanically ventilated and lung healthy subjects were recruited as controls. HBP was measured with enzyme-linked immunosorbent assay. RESULTS: BALF was collected from 14 patients with pneumonia and 14 healthy controls. Median HBP in BALF pneumonia samples was 14,690 ng/ml and controls 16.2 ng/ml (p < 0.0001). BW was collected from 10 pneumonia patients and 10 mechanically ventilated controls. Median HBP in BW pneumonia was 9002 ng/ml and controls 7.6 ng/ml (p < 0.0001). CONCLUSIONS: These data indicate that HBP concentrations is significantly higher in lower airway samples from patients with pneumonia than control subjects and is a potentially useful biomarker for diagnosis of VAP.

A Role of Epithelial Cells and Virulence Factors in Biofilm Formation by Streptococcus pyogenes In Vitro.

Biofilm formation by Streptococcus pyogenes (group A streptococcus [GAS]) in model systems mimicking the respiratory tract is poorly documented. Most studies have been conducted on abiotic surfaces, which poorly represent human tissues. We have previously shown that GAS forms mature and antibiotic-resistant biofilms on physiologically relevant epithelial cells. However, the roles of the substratum, extracellular matrix (ECM) components, and GAS virulence factors in biofilm formation and structure are unclear. In this study, biofilm formation was measured on respiratory epithelial cells and keratinocytes by determining biomass and antibiotic resistance, and biofilm morphology was visualized using scanning electron microscopy. All GAS isolates tested formed biofilms that had similar, albeit not identical, biomass and antibiotic resistance for both cell types. Interestingly, functionally mature biofilms formed more rapidly on keratinocytes but were structurally denser and coated with more ECM on respiratory epithelial cells. The ECM was crucial for biofilm integrity, as protein- and DNA-degrading enzymes induced bacterial release from biofilms. Abiotic surfaces supported biofilm formation, but these biofilms were structurally less dense and organized. No major role for M protein, capsule, or streptolysin O was observed in biofilm formation on epithelial cells, although some morphological differences were detected. NAD-glycohydrolase was required for optimal biofilm formation, whereas streptolysin S and cysteine protease SpeB impaired this process. Finally, no correlation was found between cell adherence or autoaggregation and GAS biofilm formation. Combined, these results provide a better understanding of the role of biofilm formation in GAS pathogenesis and can potentially provide novel targets for future treatments against GAS infections.

Serologic markers of Chlamydia trachomatis and other sexually transmitted infections and subsequent ovarian cancer risk: Results from the EPIC cohort.

A substantial proportion of epithelial ovarian cancer (EOC) arises in the fallopian tube and other epithelia of the upper genital tract; these epithelia may incur damage and neoplastic transformation after sexually transmitted infections (STI) and pelvic inflammatory disease. We investigated the hypothesis that past STI infection, particularly Chlamydia trachomatis, is associated with higher EOC risk in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort including 791 cases and 1669 matched controls. Serum antibodies against C. trachomatis, Mycoplasma genitalium, herpes simplex virus type 2 (HSV-2) and human papillomavirus (HPV) 16, 18 and 45 were assessed using multiplex fluorescent bead-based serology. Conditional logistic regression was used to estimate relative risks (RR) and 95% confidence intervals (CI) comparing women with positive vs. negative serology. A total of 40% of the study population was seropositive to at least one STI. Positive serology to C. trachomatis Pgp3 antibodies was not associated with EOC risk overall, but with higher risk of the mucinous histotype (RR = 2.30 [95% CI = 1.22-4.32]). Positive serology for chlamydia heat shock protein 60 (cHSP60-1) was associated with higher risk of EOC overall (1.36 [1.13-1.64]) and with the serous subtype (1.44 [1.12-1.85]). None of the other evaluated STIs were associated with EOC risk overall; however, HSV-2 was associated with higher risk of endometrioid EOC (2.35 [1.24-4.43]). The findings of our study suggest a potential role of C. trachomatis in the carcinogenesis of serous and mucinous EOC, while HSV-2 might promote the development of endometrioid disease.

A Cross-Sectional Cohort Study of Extended-Spectrum-Beta-Lactamase-Producing Enterobacterales in Patients with Traveler's Diarrhea.

Patients with traveler's diarrhea (TD) can acquire extended-spectrum-beta-lactamase (ESBL)-producing members of the Enterobacterales (EPE) during travel to areas of endemicity. The aim of the present study was to investigate the prevalence and characteristics of EPE carriage in travelers from southern Sweden who were sampled for bacterial diagnostics of TD compared to those of EPE carriage 10 years ago. Clinical samples sent for culture of common causes of bacterial enterocolitis, if the referral stated foreign travel, were included in the study. Antimicrobial susceptibility testing was done according to the EUCAST disk diffusion test method. EPE strains were subjected to whole-genome sequencing (WGS). Eighty-four of 303 patients carried a total of 92 ESBL-producing members of the Enterobacterales The overall prevalence of EPE in tested samples was thus 28%, compared to 24% 10 years earlier (P = 0.33). Among 86 strains available for WGS, 47 different sequence types (STs) were identified, and there were only 5 ST131 strains. Of the 79 Escherichia coli isolates, 76% carried at least one fim (type 1 fimbria) gene, 29% carried at least one pap (p-fimbriae) gene, and 43% were extraintestinal pathogenic E. coli (ExPEC) or uropathogenic E. coli (UPEC). Over half of the E. coli strains (57%) were intestinal pathogenic E. coli, most commonly enteroaggregative E. coli (EAEC) (33%), and enteroinvasive E. coli EIEC (22%). A relatively high proportion of patients with traveler's diarrhea carry EPE, but there was no significant increase compared to 10 years ago. Most E. coli strains were intestinal pathogenic strains. A comparatively high proportion of the strains were ExPEC/UPEC, many expressing the virulence genes pap and/or fim (This project was assigned ClinicalTrials.gov number NCT03866291.).

Anti-EF-Tu IgG titers increase with age and may contribute to protection against the respiratory pathogen Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) is a pathogen that commonly colonizes the nasopharynx of preschool children, causing opportunistic infections including acute otitis media (AOM). Patients suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized with NTHi and occasionally suffer from exacerbations by the bacterium leading to increased morbidity. Elongation-factor thermo unstable (EF-Tu), a protein critical for bacterial protein synthesis, has been found to moonlight on the surface of several bacteria. Here, we show that antibodies against NTHi EF-Tu were present in children already at 18 months of age, and that the IgG antibody titers increased with age. Children harboring NTHi in the nasopharynx also displayed significantly higher IgG concentrations. Interestingly, children suffering from AOM had significantly higher anti-EF-Tu IgG levels when NTHi was the causative agent. Human sera recognized mainly the central and C-terminal part of the EF-Tu molecule and peptide-based epitope mapping confirmed similar binding patterns for sera from humans and immunized mice. Immunization of BALB/c and otitis-prone Junbo (C3H/HeH) mice promoted lower infection rates in the nasopharynx and middle ear, respectively. In conclusion, our results suggest that IgG directed against NTHi EF-Tu may play an important role in the host immune response against NTHi.

Bacteremia with ESBL-producing Enterobacterales is associated with IgG antibodies reacting with CTX-M-15 and/or CTX-M-27.

OBJECTIVES: The adaptive humoral immune response following clinical infection with extended spectrum beta-lactamase (ESBL)-producing Enterobacterales (EPE) has not been thoroughly investigated. The aim of this study was to investigate the presence of anti-CTX-M-15 and/or anti-CTX-M-27 IgG antibodies in bacteremia patients diagnosed with EPE compared to a control group consisting of patients suffering from bacteremia with third generation cephalosporin-susceptible Escherichia coli (3GCSE). METHODS: Patientswith EPE (n = 59) or 3GCSE (n = 42) bacteremia were recruited in this case control study in the Skåne County (South of Sweden). Sera were collected 1-26 months after bacteremia. Enzyme-linked immunosorbent assay (ELISA) was used for detection of specific IgG antibodies directed against recombinant beta-lactamases CTX-M-15 and CTX-M-27. The beta-lactamase resistance genes of the corresponding EPE blood isolates were determined by DNA sequencing. RESULTS: The majority (n = 47; 80 %) of the 59 EPE blood isolates carried blaCTX-M-15 or blaCTX-M-27 genes. IgG antibodies reacting to the corresponding CTX-M enzyme was seen in 28 % (13/47) of patients suffering from EPE-bacteremia, while antibodies were detected in only 9.5 % (4/42) of patients with 3GCSE (p = 0.03). Patients with EPE had a statistically significantly higher median Charlson comorbidity index and prevalence of renal disease (p = 0.01), compared to the 3GCSE control group. CONCLUSION: This study implies that EPE bacteremia can trigger production of IgG antibodies targeting ESBL. Further investigations are required to determine the functional role of anti-ESBL antibodies against EPE bacteremia.

CEACAM1 inhibits Toll-like receptor 2-triggered antibacterial responses of human pulmonary epithelial cells.

Although Moraxella catarrhalis and Neisseria meningitidis are important human pathogens, they often colonize the human respiratory tract without causing overt clinical symptoms. Both pathogens express structurally unrelated proteins that share the ability to stimulate the adhesion molecule CEACAM1 expressed on human cells. Here we demonstrate that the interaction of CEACAM1 with ubiquitous surface protein A1 expressed on M. catarrhalis or with opacity-associated proteins on N. meningitidis resulted in reduced Toll-like receptor 2-initiated transcription factor NF-kappaB-dependent inflammatory responses of primary pulmonary epithelial cells. These inhibitory effects were mediated by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of CEACAM1 and by recruitment of the phosphatase SHP-1, which negatively regulated Toll-like receptor 2-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway. Our results identify a CEACAM1-dependent immune-evasion strategy.

Suppurative otitis media in Angola: clinical and demographic features.

OBJECTIVE: To describe the demographics and clinical findings in patients with otorrhoea in Angola. METHODS: A total of 411 patients with otorrhoea presenting in the ear, nose and throat clinic in Luanda and healthcare centres in other Angolan provinces underwent interview and clinical examination. We describe the demographics and clinical characteristics of the patients. RESULTS: The majority (64%) of patients were children <15 years (age ranged from 1 month to 77 years; median age 10.9 years) while 31% were children <5 years. In 83% of the patients, otorrhoea had lasted >14 days at the time of the examination indicating chronic suppurative otitis media (CSOM), which was confirmed with otoscopy in 72% of patients. Acute otitis media occurred in 16% of patients and was more common in children than in adults (22% vs. 10%; P = 0.007). Median duration of otorrhoea was >12 months. Earache (67%), fever (20%), dizziness (17%), nausea and/or vomiting (6%) were the main symptoms. Adult patients reported noticing hearing impairment (HI) more often than the parents of child patients (72% vs. 50%; P < 0.0001). Reported HI correlated with otorrhoea duration (P < 0.0001), presence of earache, dizziness, and measles or meningitis in history. The level of education in the family did not correlate with symptom duration. CONCLUSIONS: Otorrhoea is mainly due to CSOM and affects patients long-term in Angola. Otorrhoea duration is the strongest predictor of HI. Education on OM and its treatment is needed to prevent HI.

OBJECTIF: Décrire les données démographiques et les résultats cliniques chez les patients atteints d'otorrhée en Angola. MÉTHODES: Au total, 411 patients atteints d'otorrhée visitant la clinique ORL à Luanda et dans les centres de soins de santé d'autres provinces angolaises ont subi un entretien et un examen clinique. Nous décrivons les caractéristiques démographiques et cliniques des patients. RÉSULTATS: La majorité (64%) des patients étaient des enfants <15 ans (tranche d'âge de 1 mois à 77 ans; âge médian de 10,9 ans) tandis que 31% étaient des enfants <5 ans. Chez 83% des patients, l'otorrhée avait duré >14 jours au moment de l'examen indiquant une otite moyenne suppurée chronique (OMSC), qui a été confirmée par otoscopie chez 72% des patients. Une otite moyenne aiguë est survenue chez 16% des patients et était plus fréquente chez les enfants que chez les adultes (22% vs 10%; P = 0,007). La durée médiane de l'otorrhée était >12 mois. Les maux d'oreille (67%), la fièvre (20%), les étourdissements (17%), les nausées et/ou les vomissements (6%) étaient les principaux symptômes. Les patients adultes ont rapporté avoir remarqué une déficience auditive (DA) plus souvent que les parents d'enfants patients (72% vs 50%; P < 0,0001). La DA rapportée était en corrélation avec la durée de l'otorrhée (P < 0,0001), la présence de maux d'oreille, d'étourdissements et de rougeole ou de méningite dans les antécédents. Le niveau d'éducation dans la famille n'était pas corrélé à la durée des symptômes. CONCLUSIONS: L'otorrhée est principalement due à l'OMSC et affecte les patients à long terme en Angola. La durée de l'otorrhée est le meilleur prédicteur de la DA. L'éducation sur l'OM et son traitement est nécessaire pour prévenir la DA.

Feeding Honeybee Colonies with Honeybee-Specific Lactic Acid Bacteria (Hbs-LAB) Does Not Affect Colony-Level Hbs-LAB Composition or Paenibacillus larvae Spore Levels, Although American Foulbrood Affected Colonies Harbor a More Diverse Hbs-LAB Community.

The main current methods for controlling American Foulbrood (AFB) in honeybees, caused by the bacterial pathogen Paenibacillus larvae, are enforced incineration or prophylactic antibiotic treatment, neither of which is fully satisfactory. This has led to an increased interest in the natural relationships between the pathogenic and mutualistic microorganisms of the honeybee microbiome, in particular, the antagonistic effects of Honeybee-Specific Lactic Acid Bacteria (hbs-LAB) against P. larvae. We investigated whether supplemental administration of these bacteria affected P. larvae infection at colony level over an entire flowering season. Over the season, the supplements affected neither colony-level hbs-LAB composition nor naturally subclinical or clinical P. larvae spore levels. The composition of hbs-LAB in colonies was, however, more diverse in apiaries with a history of clinical AFB, although this was also unrelated to P. larvae spore levels. During the experiments, we also showed that qPCR could detect a wider range of hbs-LAB, with higher specificity and sensitivity than mass spectrometry. Honeybee colonies are complex super-organisms where social immune defenses, natural homeostatic mechanisms, and microbiome diversity and function play a major role in disease resistance. This means that observations made at the individual bee level cannot be simply extrapolated to infer similar effects at colony level. Although individual laboratory larval assays have clearly demonstrated the antagonistic effects of hbs-LAB on P. larvae infection, the results from the experiments presented here indicate that direct conversion of such practice to colony-level administration of live hbs-LAB is not effective.

Short Leucine-Rich Proteoglycans Modulate Complement Activity and Increase Killing of the Respiratory Pathogen Moraxella catarrhalis.

The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis, resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs.

The Laminin Interactome: A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization.

Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilus influenzae (NTHi) colonization in the airway mucosa. In this study, we identified elongation factor thermo-unstable (EF-Tu), l-lactate dehydrogenase (LDH), protein D (PD), and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (protein 4 [P4], protein E [PE], protein F [PF], and Haemophilus adhesion and penetration protein [Hap]), EF-Tu, LDH, PD, and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. More importantly, the NTHi laminin interactome consisting of the well-studied and novel Lbps recognized laminin LG domains from the subunit α chains of laminin-111 and -332, the latter isoform of which is the main laminin in the airway BM. The NTHi interactome mainly targeted multiple heparin-binding domains of laminin. In conclusion, the NTHi interactome exhibited a high plasticity of interactions with different laminin isoforms via multiple heparin-binding sites.

HAMLET, a protein complex from human milk has bactericidal activity and enhances the activity of antibiotics against pathogenic Streptococci.

HAMLET is a protein-lipid complex derived from human milk that was first described for its tumoricidal activity. Later studies showed that HAMLET also has direct bactericidal activity against select species of bacteria, with highest activity against Streptococcus pneumoniae Additionally, HAMLET in combination with various antimicrobial agents can make a broader range of antibiotic-resistant bacterial species sensitive to antibiotics. Here, we show that HAMLET has direct antibacterial activity not only against pneumococci, but also against Streptococcus pyogenes (GAS) and Streptococcus agalactiae (GBS). Analogous to pneumococci, HAMLET-treatment of GAS and GBS resulted in depolarization of the bacterial membrane followed by membrane permeabilization and death that could be inhibited by calcium and sodium transport inhibitors. Treatment of clinical antibiotic-resistant isolates of S. pneumoniae, GAS, and GBS with sublethal concentrations of HAMLET in combination with antibiotics decreased the minimal inhibitory concentrations of the respective antibiotic into the sensitive range. This effect could also be blocked by ion transport inhibitors, suggesting that HAMLET's bactericidal and combination treatment effects used similar mechanisms. Finally, we show that HAMLET potentiated the effects of erythromycin against erythromycin-resistant bacteria more effectively than it potentiated killing by penicillin G of bacteria resistant to penicillin G. These results show for the first time that HAMLET effectively kills three different species of pathogenic Streptococci using similar mechanisms and also potentiate the activity of macrolides and lincosamides more effectively than combination treatment with beta-lactams. These findings suggest a potential therapeutic role for HAMLET in repurposing antibiotics currently causing treatment failures in patients.

Honeybee-Specific Lactic Acid Bacterium Supplements Have No Effect on American Foulbrood-Infected Honeybee Colonies.

Paenibacillus larvae, the causative agent of American foulbrood (AFB), is the primary bacterial pathogen affecting honeybees and beekeeping. The main methods for controlling AFB are incineration of diseased colonies or prophylactic antibiotic treatment (e.g., with tylosin), neither of which is fully satisfactory. The search for superior means for controlling AFB has led to an increased interest in the natural relationships between the honeybee-pathogenic and mutualistic microorganisms and, in particular, the antagonistic effects of honeybee-specific lactic acid bacteria (hbs-LAB) against P. larvae These effects have been demonstrated only on individual larvae in controlled laboratory bioassays. Here we investigated whether supplemental administration of hbs-LAB had a similar beneficial effect on P. larvae infection at colony level. We compared experimentally AFB-infected colonies treated with hbs-LAB supplements to untreated and tylosin-treated colonies and recorded AFB symptoms, bacterial spore levels, and two measures of colony health. To account for the complexity of a bee colony, we focused on (Bayesian) probabilities and magnitudes of effect sizes. Tylosin reduced AFB disease symptoms but also had a negative effect on colony strength. The tylosin treatment did not, however, affect P. larvae spore levels and might therefore "mask" the potential for disease. hbs-LAB tended to reduce brood size in the short term but was unlikely to affect AFB symptoms or spores. These results do not contradict demonstrated antagonistic effects of hbs-LAB against P. larvae at the individual bee level but rather suggest that supplementary administration of hbs-LAB may not be the most effective way to harness these beneficial effects at the colony level.IMPORTANCE The previously demonstrated antagonistic effects of honeybee-derived bacterial microbiota on the infectivity and pathogenicity of P. larvae in laboratory bioassays have identified a possible new approach to AFB control. However, honeybee colonies are complex superorganisms where social immune defenses play a major role in resistance against disease at the colony level. Few studies have investigated the effect of beneficial microorganisms on bee diseases at the colony level. Effects observed at the individual bee level do not necessarily translate into similar effects at the colony level. This study partially fills this gap by showing that, unlike at the individual level, hbs-LAB supplements did not affect AFB symptoms at the colony level. The inference is that the mechanisms regulating the honeybee microbial dynamics within a colony are too strong to manipulate positively through supplemental feeding of live hbs-LAB and that new potential remedies identified through laboratory research have to be tested thoroughly in situ, in colonies.

PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis.

Respiratory tract infections are one of the leading causes of mortality worldwide urging better understanding of interactions between pathogens causing these infections and the host. Here we report that an extracellular matrix component proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel antibacterial component of innate immunity. We detected the presence of PRELP in human bronchoalveolar lavage fluid and showed that PRELP can be found in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and the adventitia of blood vessels in lung tissue. PRELP specifically binds respiratory tract pathogens Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae, but not other bacterial pathogens tested. We focused our study on M. catarrhalis and found that PRELP binds the majority of clinical isolates of M. catarrhalis (n = 49) through interaction with the ubiquitous surface protein A2/A2H. M. catarrhalis usually resists complement-mediated serum killing by recruiting to its surface a complement inhibitor C4b-binding protein, which is also a ligand for PRELP. We found that PRELP competitively inhibits binding of C4b-binding protein to bacteria, which enhances membrane attack complex formation on M. catarrhalis and thus leads to increased serum sensitivity. Furthermore, PRELP enhances phagocytic killing of serum-opsonized M. catarrhalis by human neutrophils in vitro. Moreover, PRELP reduces Moraxella adherence to and invasion of human lung epithelial A549 cells. Taken together, PRELP enhances host innate immunity against M. catarrhalis through increasing complement-mediated attack, improving phagocytic killing activity of neutrophils, and preventing bacterial adherence to lung epithelial cells.

Capsule Typing of Haemophilus influenzae by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Encapsulated Haemophilus influenzae strains belong to type-specific genetic lineages. Reliable capsule typing requires PCR, but a more efficient method would be useful. We evaluated capsule typing by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Isolates of all capsule types (a-f and nontypeable; n = 258) and isogenic capsule transformants (types a-d) were investigated. Principal component and biomarker analyses of mass spectra showed clustering, and mass peaks correlated with capsule type-specific genetic lineages. We used 31 selected isolates to construct a capsule typing database. Validation with the remaining isolates (n = 227) showed 100% sensitivity and 92.2% specificity for encapsulated strains (a-f; n = 61). Blinded validation of a supplemented database (n = 50) using clinical isolates (n = 126) showed 100% sensitivity and 100% specificity for encapsulated strains (b, e, and f; n = 28). MALDI-TOF mass spectrometry is an accurate method for capsule typing of 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.

Genetic predisposition to infection in a case of atypical hemolytic uremic syndrome.

Most cases of hemolytic uremic syndrome (HUS) are caused by infection with enterohemorrhagic Escherichia coli (EHEC). Genetic defects causing uncontrolled complement activation are associated with the more severe atypical HUS (aHUS). Non-EHEC infections can trigger the disease, however, complement defects predisposing to such infections have not yet been studied. We describe a 2-month-old patient infected with different Gram-negative bacterial species resulting in aHUS. Serum analysis revealed slow complement activation kinetics. Rare variant R229C was found in complement inhibitor vitronectin. Recombinant mutated vitronectin showed enhanced complement inhibition in vitro and may have been a predisposing factor for infection. Our work indicates that genetic changes in aHUS can not only result in uncontrolled complement activation but also increase vulnerability to infections contributing to aHUS.