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Background: Concerns about non-typeable Haemophilus influenzae (NTHi) in otitis media (OM) have grown after the introduction of pneumococcal conjugate vaccine (PCV). We aim to better understand the clinical role of NTHi in pediatric OM. Methods: Middle ear fluid samples from children

BACKGROUND: Persistence of bacterial pathogens in the airways has profound consequences on the course and pathogenesis of chronic obstructive pulmonary disease (COPD). Patients with COPD continuously acquire and clear strains of Moraxella catarrhalis, a major pathogen in COPD. Some strains are cleared quickly and some persist for months to years. The mechanism of the variability in duration of persistence is unknown. METHODS: Guided by genome sequences of selected strains, we studied the expression of Hag/MID, hag/mid gene sequences, adherence to human cells, and autoaggregation in longitudinally collected strains of M. catarrhalis from adults with COPD. RESULTS: Twenty-eight of 30 cleared strains of M. catarrhalis expressed Hag/MID whereas 17 of 30 persistent strains expressed Hag/MID upon acquisition by patients. All persistent strains ceased expression of Hag/MID during persistence. Expression of Hag/MID in human airways was regulated by slipped-strand mispairing. Virulence-associated phenotypes (adherence to human respiratory epithelial cells and autoaggregation) paralleled Hag/MID expression in airway isolates. CONCLUSIONS: Most strains of M. catarrhalis express Hag/MID upon acquisition by adults with COPD and all persistent strains shut off expression during persistence. These observations suggest that Hag/MID is important for initial colonization by M. catarrhalis and that cessation of expression facilitates persistence in COPD airways.

Background The causal attribution of bacterial pathogens to severe acute respiratory infections (SARI) is challenging because many bacteria are frequently detected in the upper respiratory tract of asymptomatic persons. Quantification of pathogen load may help differentiate asymptomatic pathogen carriage from clinically significant infection. We aimed to determine whether real-time PCR (rt-PCR) cycle threshold (Ct) values, as a proxy for bacterial load, differ between adults with SARI and asymptomatic adults. Methods Adults with SARI (acute onset of fever and cough, requiring hospitalization) were frequency matched to asymptomatic adults (enrolled from trauma and orthopedic inpatient wards) by age group, catchment area, and enrollment date at three surveillance sites in Guatemala. Nasopharyngeal and oropharyngeal specimens were collected from all participants and tested for pathogens using rt-PCR. Using the Wilcoxon rank sum test, we compared the distributions and median Ct values between ill and asymptomatic adults in whom Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Streptococcus pneumoniae, and Klebsiella pneumoniae were detected. Results Between October 2013 and October 2015, 304 adults with SARI and 174 asymptomatic adults were enrolled (Table). M. catarrhalis, S. aureus, and S. pneumoniae were detected with similar frequency in both groups. H. influenzae and K. pneumoniae were detected more frequently in asymptomatic adults. We found the greatest difference in Ct value distributions between ill (median Ct=30.8) and asymptomatic adults (median Ct=35.6) with S. pneumoniae detections (p< 0.01) (Figure). Median Ct values of H. influenzae (29.3 vs 31.1, p=0.04) and M. catarrhalis (29.2 vs 31.5, p=0.05) were also lower among adults with SARI. Frequency of select bacterial pathogen detection among adults with SARI and among asymptomatic adults, Guatemala, 2013-2015 Distributions of Ct values among adults with SARI and asymptomatic adults in whom a given bacterial pathogen was detected Conclusion Pathogen loads of S. pneumoniae, H. influenzae, and M. catarrhalis were higher among adults with SARI than among asymptomatic adults, suggesting that Ct values may provide insight into SARI etiology for some pathogens, despite the similar frequency of detection among both ill and asymptomatic adults. Future work will normalize Ct values to account for variation in testing and analysis and explore the use of Ct values to estimate population attributable fractions of respiratory infections. Disclosures All Authors: No reported disclosures

Background and Objective: Respiratory syncytial virus (RSV) is the most significant cause of acute respiratory infection (ARI) in early life. RSV and other respiratory viruses are known to stimulate substantial outgrowth of potentially pathogenic bacteria in the upper airways of young children. However, the clinical significance of interactions between viruses and bacteria is currently unclear. The present study aimed to clarify the effect of viral and bacterial co-detections on disease severity during paediatric ARI. Methods: Nasopharyngeal aspirates from children under 2years of age presenting with ARI to the emergency department were screened by quantitative PCR for 17 respiratory viruses and the bacterial pathogens Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Associations between pathogen detection and clinical measures of disease severity were investigated. Results: RSV was the most common virus detected, present in 29 of 58 samples from children with ARI (50%). Detection of S. pneumoniae was significantly more frequent during RSV infections compared to other respiratory viruses (adjusted effect size: 1.8, P: 0.03), and co-detection of both pathogens was associated with higher clinical disease severity scores (adjusted effect size: 1.2, P: 0.03). Conclusion: Co-detection of RSV and S. pneumoniae in the nasopharynx was associated with more severe ARI, suggesting that S. pneumoniae colonization plays a pathogenic role in young children.

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from the patients in Japan was conducted by Japanese Society of Chemotherapy, Japanese association for infectious diseases and Japanese society for Clinical Microbiology in 2012. The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period between January and December in 2012 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical Laboratory Standard Institutes. Susceptibility testing was evaluated in 1236 strains (232 Staphylococcus aureus, 225 Streptococcus pneumoniae, 16 Streptococcus pyogenes, 231 Haemophilus influenzae, 147 Moraxella catarrhalis, 167 Klebsiella pneumoniae and 218 Pseudomonas aeruginosa). Ratio of methicillin-resistant S. aureus was 51.3%, and those of penicillin-intermediate S. pneumoniae was 0.4%. Among H. influenzae, 5.6% of them were found to be β-lactamase-producing ampicillin-resistant strains, and 37.2% to be β-lactamase-non-producing ampicillin-resistant strains. Extended spectrum β-lactamase-producing K. pneumoniae and multi-drug resistant P. aeruginosa with metallo β-lactamase were 4.2% and 3.2%, respectively. Continuous national surveillance is important to determine the actual situation of the resistance shown by bacterial respiratory pathogens to antimicrobial agents.

Summary Objectives We sought to understand how polymicrobial colonization varies during health, viral upper respiratory infection (URI) and acute upper respiratory bacterial infection to understand differences in infection-prone vs. non-prone patients. Methods Nasopharyngeal (NP) samples were collected from 74 acute otitis media (AOM) infection-prone and 754 non-prone children during 2094 healthy visits, 673 viral URI visits and 631 AOM visits. Three otopathogens Streptococcus pneumoniae (Spn) , Nontypeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis (Mcat) were identified by culture. Results NP colonization rates of multiple otopathogens during health were significantly lower than during viral URI, and during URI they were lower than at onset of upper respiratory bacterial infection in both AOM infection-prone and non-prone children. AOM infection-prone children had higher polymicrobial colonization rates than non-prone children during health, viral URI and AOM. Polymicrobial colonization rates of AOM infection-prone children during health were equivalent to that of non-prone children during viral URI, and during viral URI were equivalent to that of non-prone during AOM infection. Spn colonization was positively associated with NTHi and Mcat colonization during health, but negatively during AOM infection. Conclusion The infection-prone patients more frequently have multiple potential bacterial pathogens in the NP than the non-prone patients. Polymicrobial interaction in the NP differs during health and at onset of infection.

Background There is limited information on the association between colonization density of upper respiratory tract colonizers and pathogen-specific pneumonia. We assessed this association for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, and Pneumocystis jirovecii. Methods In 7 low- and middle-income countries, nasopharyngeal/oropharyngeal swabs from children with severe pneumonia and age-frequency matched community controls were tested using quantitative polymerase chain reaction (PCR). Differences in median colonization density were evaluated using the Wilcoxon rank-sum test. Density cutoffs were determined using receiver operating characteristic curves. Cases with a pathogen identified from lung aspirate culture or PCR, pleural fluid culture or PCR, blood culture, and immunofluorescence for P. jirovecii defined microbiologically confirmed cases for the given pathogens. Results Higher densities of H. influenzae were observed in both microbiologically confirmed cases and chest radiograph (CXR)–positive cases compared to controls. Staphylococcus aureus and P. jirovecii had higher densities in CXR-positive cases vs controls. A 5.9 log10 copies/mL density cutoff for H. influenzae yielded 86% sensitivity and 77% specificity for detecting microbiologically confirmed cases; however, densities overlapped between cases and controls and positive predictive values were poor ( Conclusions There is evidence for an association between H. influenzae colonization density and H. influenzae–confirmed pneumonia in children; the association may be particularly informative in epidemiologic studies. Colonization densities of M. catarrhalis, S. aureus, and P. jirovecii are unlikely to be of diagnostic value in clinical settings.

According to studies based on bacterial cultures of middle ear fluids, Streptococcus pneumoniae , Haemophilus influenzae , and Moraxella catarrhalis have been the most common pathogens in acute otitis media. However, bacterial culture can be affected by reduced viability or suboptimal growth of bacteria. PCR detects bacterial DNA from samples with greater sensitivity than culture. In the present study, we analyzed the middle ear pathogens with both conventional culture and semiquantitative real-time PCR in 90 middle ear fluid samples obtained from children aged 5 to 42 months during acute otitis media episodes. Samples were tested for the presence of S. pneumoniae , H. influenzae , M. catarrhalis , Alloiococcus otitidis , Staphylococcus aureus , and Pseudomonas aeruginosa . One or more bacterial pathogens were detected in 42 (47%) samples with culture and in 69 (77%) samples with PCR. According to PCR analysis, M. catarrhalis results were positive in 42 (47%) samples, H. influenzae in 30 (33%), S. pneumoniae in 27 (30%), A. otitidis in 6 (6.7%), S. aureus in 5 (5.6%), and P. aeruginosa in 1 (1.1%). Multibacterial etiology was seen in 34 (38%) samples, and M. catarrhalis was detected in most (85%) of those cases. Fifteen signals for M. catarrhalis were strong, suggesting a highly probable etiological role of the pathogen. In conclusion, even though M. catarrhalis is often a part of mixed flora in acute otitis media, a considerable proportion of cases may be primarily attributable to this pathogen.

Background Sinusitis is a complex involvement of the upper respiratory system by bacteria, viruses, fungi, or other allergens. Streptococcus pneumoniae , Haemophilus influenzae , and Moraxella catarrhalis are the dominant bacterial microorganisms involved in acute sinusitis, whereas in chronic sinusitis, Staphylococcus aureus and some anaerobic bacteria are the prevailing pathogens. Appropriate antibiotic treatment requires sinusitis bacteriology assessment. The aim of this study was to isolate bacteria in clinical samples from patients with chronic sinusitis. Methods A total of 55 samples were collected from patients with chronic sinusitis undergoing surgery at Imam Khomeini Hospital in Ahvaz, Iran. Samples were cultured in conventional medium, and for each culture, Gram staining, catalase, coagulase, oxidase, and DNAse tests were performed and isolates were stored for polymerase chain reaction analysis. Results Twenty-three isolates were obtained from five patients, including S. aureus (23.6%), Rhizomucor (1.8%), and Escherichia (1.8%) by the culture method and M. catarrhalis (3.6%) and S. Pneumoniae (7.2%) by the polymerase chain reaction method. Compared with acute sinusitis, the microbiology of chronic sinusitis remains controversial. Results are affected by many factors, including diversity of molecular and culture methods, sterilization of sampling area, sample transfer to laboratory, use of antibiotics prior to surgery, and nasal polyps. Conclusion In Iran, the causative agents of chronic sinusitis are similar to those in other countries. Compared with other bacteria, S. aureus was observed more often in asthmatic patients with sinusitis.

Viral infections of the upper respiratory tract may influence the commensal nasopharyngeal bacteria. Changes in the bacterial niche could affect transmission dynamics. Attenuated vaccine viruses can be used to investigate this empirically in humans.To study the effects of mild viral upper respiratory infections on nasopharyngeal bacterial colonization using live attenuated influenza vaccine (LAIV) as a surrogate.We used trivalent LAIV to evaluate the effects of viral infection on bacterial carriage and density of Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, and Staphylococcus aureus. A total of 151 healthy children were randomized 1:1 to receive the vaccine starting either at recruitment (n = 74) or 28 days later (n = 77) in a stepped wedge fashion, allowing comparisons between recipients and nonrecipients as well as whole-group comparisons pre- and postvaccination. Bacterial carriage and density were determined using quantitative polymerase chain reaction assays.A total of 151 children were recruited, 77 in the LAIV group and 74 in the control group. LAIV recipients (n = 63 analyzed) showed an apparent transient increase in H. influenzae carriage but no further significant differences in carriage prevalence of the four bacterial species compared with controls (n = 72 analyzed). S. pneumoniae density was substantially higher in vaccine recipients (16,687 vs. 1935 gene copies per milliliter) 28 days after the first dose (P 0.001). Whole-group multivariable analysis (prevaccine, after one dose, and after two doses) also showed increases in density of other species and H. influenzae carriage prevalence.In the absence of any safety signals despite widespread use of the vaccine, these findings suggest that bacterial density, and thus transmission rates among children and to people in other age groups, may rise following attenuated influenza infections without associated clinical disease. LAIV could therefore be used as an experimental tool to elucidate the dynamics of transmission of nasopharyngeal bacteria.

Background and Objective From mid February to the end of March, each year ∼30% of Japanese have Japanese cedar pollinosis. Moreover, 10–50% of patients with this pollinosis exhibit nasal manifestations in the preseason. These patients have a predominance of neutrophils but not eosinophils in nasal swabs and high carriage of Staphylococcus aureus. We hypothesized that S. aureus or other bacteria and associated neutrophilia were involved in preseasonal symptoms. Methods Cytology and bacterial colony growth were assessed in nasal swabs in the groups of asymptomatic patients in the preseason (PreAsP) (n = 53) and symptomatic patients in the preseason (PreSyP) (n = 60), and in group of symptomatic patients in season (InSyP) (n = 72). Results In the preseason, high neutrophilia was present in only 20% of the PreAsP group but in 47% of the PreSyP group (p < 0.01). Nasal carriage of S. aureus in the PreAsP and PreSyP groups were 79%, 75%, respectively, whereas, for Moraxella catarrhalis, these were 9% versus 25% (PreAsP versus PreSyP group; p < 0.05). In patients with positive results for S. aureus and M. catarrhalis, the degrees of neutrophilia (-, ∓, +, 2+, 3+) in the PreSyP group were larger than in the PreAsP groups (p < 0.01). In the PreSyP group, the magnitude of neutrophilia was greater (p < 0.05) in subgroups with more colonies of S. aureus than in subgroups with fewer colonies. Conclusion Nasal symptoms in the preseason are associated with neutrophilia and nasal colonization with S. aureus and M. catarrhalis. Patients with symptoms in the preseason had improved symptom scores when given prophylactic treatment early in season but had more-severe symptom scores late in season than asymptomatic patients in the preseason. Neutrophil-associated tissue damage related to bacterial colonization may underlie these associations.

Moraxella catarrhalis is an exclusively human pathogen that is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to prevent M. catarrhalis infections would have an enormous global impact in reducing morbidity resulting from these infections. Substrate binding protein 2 (SBP2) of an ABC transporter system has recently been identified as a promising vaccine candidate antigen on the bacterial surface of M. catarrhalis . In this study, we showed that SBP1, -2, and -3 individually bind different basic amino acids with exquisite specificity. We engineered mutants that each expressed a single SBP from this gene cluster and showed in growth experiments that SBP1, -2, and -3 serve a nutritional function through acquisition of amino acids for the bacterium. SBP2 mediates uptake of arginine, a strict growth requirement of M. catarrhalis . Adherence and invasion assays demonstrated that SBP1 and SBP3 play a role in invasion of human respiratory epithelial cells, consistent with a nutritional role in intracellular survival in the human respiratory tract. This work demonstrates that the SBPs of an ABC transporter system function in the uptake of basic amino acids to support growth of M. catarrhalis . The critical role of SBP2 in arginine uptake may contribute to its potential as a vaccine antigen.

Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are common causative agents of respiratory infections. Pneumococcal conjugate vaccines have been introduced recently, but their effect on the natural immunity against protein antigens from these pathogens has not been elucidated.This was an age-matched observational controlled study that evaluated the influence of 10-valent pneumococcal conjugate vaccines on the levels of antibodies and frequencies of antibody responses against proteins from S. pneumoniae, H. influenzae and M. catarrhalis in serum samples of children with community-acquired pneumonia. Eight pneumococcal proteins (pneumolysin, choline-binding protein A, pneumococcal surface protein A families 1 and 2, pneumococcal choline-binding protein A, pneumococcal histidine triad protein D, serine/threonine protein kinase, protein required for cell wall separation of group B streptococcus), 3 proteins from H. influenzae (including protein D) and 5 M. catarrhalis proteins were investigated.The study group comprised 38 vaccinated children and 114 age-matched controls (median age: 14.5 vs. 14.6 months, respectively; P = 0.997), all with community-acquired pneumonia. There was no difference on clinical baseline characteristics between vaccinated and unvaccinated children. Vaccinated children had significantly lower levels of antibodies against 4 of the studied pneumococcal antigens (P = 0.048 for Ply, P = 0.018 for pneumococcal surface protein A, P = 0.001 for StkP and P = 0.028 for PcsB) and higher levels of antibodies against M. catarrhalis (P = 0.015). Nevertheless, the vaccination status did not significantly affect the rates of antibody responses against S. pneumoniae, H. influenzae and M. catarrhalis.In spite of the differences that have been found on the level of natural antibodies, no effect from pneumococcal vaccination was observed on the rate of immune responses associated with community-acquired pneumonia against protein antigens from S. pneumoniae, H. influenzae and M. catarrhalis.

Versatile 1, 2, 3-triazole pharmacophore-based benzofused heterocycles containing halogen- substituted aromatic (9–17 and 25–28), 7- substituted coumarin (18–23 and 29–30) or penciclovir-like subunit(31a, b–38a) were designed and synthesized to evaluate their antibacterial activities against selected Gram- positive and Gram-negative bacteria. Hybridization approach using environmentally friendly Cu(I)-catalyzed click reaction under microwave irradiation was adopted in the synthesis of regioselective 1, 4- disubstituted 1, 2, 3-triazole tethered heterocycles (9–23 and 25–30), while post-N- alkylation of NH- 1, 2, 3-triazoles afforded both 2, 4- (31a–38a) and 1, 4-disubstituted (31b–33b, 35b–37b) 1, 2, 3- triazole regioisomers. The compounds 18–23 and 25– 30 revealed fluorescence in the violet region of the visible spectrum with a strong influence of phenyl spacer in 25–30 on both wavelength and emission intensity. Fusion of selected subunits led to new hybrid architecture, benzothiazole– 1, 2, 3-triazole– coumarin 29 that demonstrated extremely narrow spectrum activity towards fastidious Gram- negative bacteria Moraxella catarrhalis. Selected hybrid showed the potency against Moraxella catarrhalis (MIC < 0.25 ug/mL) comparable to that of reference antibiotic azithromycin, which suggested that further investigations are necessary to optimize this potential hit compound as a new anti-Moraxella catarrhalis agent.

This cross-sectional study assessed the prevalence of bacteria isolated from Spanish children with suspected chronic lower respiratory tract infection (LRTI) for whom bronchoalveolar lavage (BAL) was indicated. BAL fluid (BALF) was collected from 191 children (aged ≥ 6 months to 104 colony-forming units/mL) in 10.5, 8.9, and 6.3% of children, respectively. Clinical characteristics were similar among participants, regardless of positivity status for any of the bacteria. Approximately 26% of pneumococcal isolates were PCV13 serotypes, and 96% of H. influenzae isolates were non-typeable (NTHi). Concordance between BALF and NPS isolates was 51.0% for S. pneumoniae, 52.1% for H. influenzae, and 22.0% for M. catarrhalis. Conclusion: S. pneumoniae, NTHi, and M. catarrhalis were the main bacteria detected in BALF and NPS. Children with suspected chronic LRTI may benefit from a vaccine protecting against NTHi. What is Known: • Chronic lower respiratory tract infection (LRTI) in children can cause high morbidity and is a major use of healthcare resources worldwide. Despite this, their etiology or potential preventive measures are poorly assessed. • Bronchoalveolar lavage can be used to determine bacterial etiology of chronic LRTI. What is New: • We used conventional and molecular techniques to show that Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis were present in the LRT of Spanish children with suspected chronic LRTI • Concordance between isolates from bronchoalveolar lavage fluid and nasopharyngeal swabs was low, suggesting that samples from the upper respiratory tract could not reliably predict the bacterial etiology of suspected chronic LRTI Electronic supplementary material The online version of this article (10.1007/s00431-017-3044-3) contains supplementary material, which is available to authorized users.

Background Delafloxacin (DLX) is an anionic fluoroquinolone (FQ) antimicrobial that was approved in 2017 by the United States (US) Food and Drug Administration for the treatment of acute bacterial skin and skin structure infections. DLX recently successfully completed a clinical trial for the treatment of community-acquired bacterial pneumonia (CABP). In the present study, in vitro susceptibility (S) results for DLX and comparator agents were determined for CABP pathogens including Streptococcus pneumoniae (SPN), Haemophilus influenzae (HI), H. parainfluenzae (HP) and Moraxella catarrhalis (MC) clinical isolates from US hospitals participating in the SENTRY Program during 2014–2018. Methods A total of 1,975 SPN, 1,128 HI, 684 MC, and 43 HP isolates were collected from community-acquired respiratory tract infections (CARTI) during 2014–2018 from US hospitals. Sites included only 1 isolate/patient/infection episode. Isolate identifications were confirmed at JMI Laboratories. Susceptibility testing was performed according to CLSI broth microdilution methodology, and CLSI (2019) breakpoints were applied where applicable. Other antimicrobials tested included levofloxacin (LEV) and moxifloxacin (MOX; not tested in 2015). Multidrug-resistant (MDR) SPN isolates were categorized as being nonsusceptible (NS) to amoxicillin-clavulanate, erythromycin, and tetracycline; other SPN phenotypes were LEV-NS or penicillin (PEN)-NS. β-Lactamase (BL) presence was determined for HI, HP, and MC. Results The activities of the 3 FQs are shown in the table. The most active agent against SPN was DLX, with the lowest MIC50/90 values of 0.015/0.03 mg/L. DLX activities were similar when tested against the MDR or PEN-NS for SPN phenotypes. LEV-NS isolates had DLX MIC50/90 results of 0.12/0.25 mg/L. DLX was the most active FQ against HI, HP, and MC. BL presence did not affect FQ MIC values for HI or MC; only 2 HP isolates were BL-positive. Conclusion DLX demonstrated potent in vitro antibacterial activity against SPN, HI, HP, and MC. DLX was active against MDR SPN that were NS to the agents commonly used as treatments for CABP. DLX had excellent activity against LEV-NS SPN. These data support the continued study of DLX as a potential treatment for CABP. Disclosures All authors: No reported disclosures.

There is no licensed vaccine for Moraxella catarrhalis (Mcat), which is a prominent bacterium causing acute otitis media (AOM) in children and lower respiratory tract infections in adults. Nasopharyngeal (NP) colonization caused by respiratory bacteria results in natural immunization of the host. To identify Mcat antigens as vaccine candidates, we evaluated the development of naturally induced antibodies to 5 Mcat surface proteins in children 6-30 months of age during Mcat NP colonization and AOM.Human serum IgG against the recombinant Mcat proteins, outer membrane protein (OMP) CD, oligopeptide permease (Opp)A, hemagglutinin (Hag), Moraxella surface protein (Msp)22, and PilA clade 2 (PilA2) was quantitated by using an ELISA assay.There were 223 Mcat NP colonization episodes documented in 111 (60%) of 184 children in the study. Thirty five Mcat AOM episodes occurred in 30 (16%) of 184 children. All 5 Mcat candidate vaccine antigens evaluated stimulated a significant rise in serum IgG levles over time from 6 to 36 months of age (P0.001), with a rank order as follows: Msp22=OppAOMP CD=Hag=PilA2. Children with no detectable Mcat NP colonization showed a higher serum IgG level against OppA, Hag, and Msp22 compared to those with Mcat NP colonization (P0.05). Individual data showed that some children responded to AOM with an antibody increase to one or more of the studied Mcat proteins but some children failed to respond.Serum antibody to Mcat candidate vaccine proteins OMP CD, OppA, Msp22, Hag, and PilA2 increased with age in naturally immunized children age 6-30 months following Mcat NP colonization and AOM. High antibody levels against OppA, Msp22, and Hag correlated with reduced carriage. The results support further investigation of these vaccine candidates in protecting against Mcat colonization and infection.

Moraxella catarrhalis is a prominent pathogen that causes acute otitis media in children and lower respiratory tract infections in adults, resulting in a significant socioeconomic burden on healthcare systems globally. No vaccine is currently available for M. catarrhalis. Promising M. catarrhalis target antigens have been characterized in animal models and should soon enter human clinical trials.This review discusses the detailed features and research status of current candidate target antigens for an M. catarrhalis vaccine. The approaches for assessing M. catarrhalis vaccine efficacy are also discussed.Targeting the key molecules contributing to serum resistance may be a viable strategy to identify effective vaccine targets among M. catarrhalis antigens. Elucidating the role and mechanisms of the serum and mucosal immune responses to M. catarrhalis is significant for vaccine target selection, testing and evaluation. Developing animal models closely simulating M. catarrhalis-caused human respiratory diseases is of great benefit in better understanding pathogenesis and evaluating vaccine efficacy. Carrying out clinical trials will be a landmark in the progress of M. catarrhalis vaccine research. Combined multicomponent vaccines will be a focus of future M. catarrhalis vaccine studies.

Background Virus is detected in about 80% of upper respiratory tract infections (URTIs) in children and is also detectable in the nasopharynx of 30% of asymptomatic children. The effect of asymptomatic viral infection on the dynamics of bacterial density and colonization of the nasopharynx has not been reported. The current study was performed to assess the presence and density of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the nasopharynx of 4-7-year-old children during URTI and when well. Methods Nasal samples were obtained during 4 surveillance periods when children were asymptomatic and whenever they had symptoms of URTI. Respiratory viruses and bacterial pathogens were identified and quantified using polymerase chain reaction. Results The proportion of children colonized with all 3 bacteria was higher during visits for acute URTI than during asymptomatic surveillance visits. Mean bacterial densities were significantly higher at all visits for all 3 pathogens when a virus was detected. The differences between the means were 1.0, 0.4, and 0.7 log10 colony-forming unit equivalents per milliliter for S. pneumoniae, H. influenzae, and M. catarrhalis, respectively, compared with visits in which virus was not detected. The percentage of children colonized and density were also higher at asymptomatic visits in which virus was detected than at visits in which virus was not detected. Conclusion The density and frequency of colonization with S. pneumoniae, H. influenzae, and M. catarrhalis in nasal wash samples increase during periods of both symptomatic and asymptomatic viral infection. Increases in bacterial colonization observed during asymptomatic viral infection were nearly the same magnitude as when children were symptomatic.

This work revealed the drug resistance and population structure of Moraxella catarrhalis strains isolated from children less than three years old with pneumonia. Forty-four independent M. catarrhalis strains were analyzed using broth dilution antimicrobial susceptibility testing and multilocus sequence typing (MLST). The highest non-susceptibility rate was observed for amoxicillin (AMX), which reached 95.5%, followed by clindamycin (CLI) (n=33; 75.0%), azithromycin (AZM) (61.4%), cefaclor (CEC) (25.0%), trimethoprim-sulfamethoxazole (SXT) (15.9%), cefuroxime (CXM) (4.5%), tetracycline (TE) (2.3%), and doxycycline (DOX) (2.3%). There was no strain showing non-susceptibility to other six antimicrobials. Using MLST, the 44 M. catarrhalis strains were divided into 33 sequence types (STs). Based on their allelic profiles, the 33 STs were divided into one CC (CC363) and 28 singletons. CC363 contained five STs and ST363 was the founder ST. CC363 contained 63.6%, 33.3%, and 40.7% of CEC non-susceptible, CLI non-susceptible and AZM non-susceptible strains, respectively. The proportions of CEC non-susceptible, CLI non-susceptible and AZM non-susceptible strains in CC363 were higher than that of singletons; these differences were significant for CEC (p=0.002) and AZM (p=0.011). Furthermore, CC363 contained more AMX-CLI-AZM co-non-susceptible and AMX-CEC-CLI-AZM co-non-susceptible strains than the singletons (p=0.007 and p

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhalis.

Submitted by Flávia Ferreira (flaviaccf@yahoo.com.br) on 2019-11-29T14:14:48Z No. of bitstreams: 1 Tese ICS Igor Carmo Borges.pdf: 10586455 bytes, checksum: ad4f34b5698fa37cf5e70b829ab6f4d2 (MD5) Made available in DSpace on 2019-11-29T14:14:48Z (GMT). No. of bitstreams: 1 Tese ICS Igor Carmo Borges.pdf: 10586455 bytes, checksum: ad4f34b5698fa37cf5e70b829ab6f4d2 (MD5) Pneumonia está entre as principais causas de mortalidade infantil no mundo. Contudo, o diagnóstico etiológico de pneumonia ainda é um desafio. Nós avaliamos diversos fatores que podem influenciar o diagnóstico de infecção por Streptococcus pneumoniae, Haemophilus influenzae e Moraxella catarrhalis em crianças com pneumonia utilizando testes sorológicos com antígenos proteicos. Utilizando diversas combinações de proteínas pneumocócicas, mostramos que testes sorológicos combinando diferentes proteínas pneumocócicas detectam uma frequência maior de resposta anticórpica contra S. pneumoniae, comparado a testes que utilizam uma única proteína. Também demonstramos que o nível de anticorpos preexistentes e o intervalo entre a coleta das amostras pareadas de soro influenciam a detecção de respostas anticórpicas contra proteínas de pneumococo e H. influenzae por testes sorológicos. Comparando testes sorológicos utilizando proteínas e polissacarídeos pneumocócicos, encontramos que testes sorológicos utilizando proteínas são mais sensíveis para a detecção de infecção pneumocócica em crianças com pneumonia. Adicionalmente, avaliamos o desenvolvimento natural de anticorpos contra proteínas das três bactérias estudadas nos primeiros 13 anos de vida de crianças sadias. Encontramos que há maturação do sistema imune para produção de anticorpos contra antígenos proteicos dessas bactérias antes dos cinco anos de vida. Também avaliamos a distribuição sazonal das infecções causadas por essas bactérias em crianças com pneumonia em uma região tropical. Demonstramos que pneumonia causada por M. catarrhalis é mais frequente durante o outono-inverno. Concluindo, testes sorológicos utilizando antígenos proteicos são métodos diagnósticos adequados para a investigação de infecção causa por S. pneumoniae, H. influenzae ou M. catarrhalis em crianças com pneumonia.

Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon ( opp ) consisting of five genes ( oppB , oppC , oppD , oppF , and oppA ), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l -arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.

Aim This study aims to describe the microbiology of middle ear fluid (MEF) in a cohort of children vaccinated with Streptococcus pneumoniae conjugate vaccine (PCV7) having ventilation tube insertion. Nasopharyngeal (NP) carriage of otopathogens in these children is compared with children without history of otitis media. Methods Between May and November 2011, MEF and NP samples from 325 children aged

Moraxella catarrhalis is a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeable Haemophilus influenzae , M. catarrhalis has become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, of M. catarrhalis . Among 30 clinical isolates tested, the sbp2 gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not the sbp2 mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance of M. catarrhalis from the lung compared to that in the control group at both 25-μg and 50-μg doses ( P < 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen against M. catarrhalis .

To assess the differential response of the secretory gel forming mucins (GFM) to the most common bacterial pathogens causing otitis media, Streptococcus pneumoniae (SP), nontypeable Haemophilus influenza (NTHi), and Moraxella catarrhalis (Mcat), in a culture model of human middle ear epithelium (HMEEC).In vitro cultured HMEEC was exposed to 5 μg/ml of bacterial whole cell lysate (WCL). RNA was extracted to generate cDNA. The expression levels of each of the targeted mucin transcripts, MUC2, MUC5AC, MUC5B and MUC19, were detected by quantitative PCR.The submerged HMEEC exposed to NTHi-86028NP WCL demonstrated a significant increase of MUC2, MUC5AC and MUC5B as compared to the control non-treated cells while MUC19 transcript level remained unchanged. WCL of additional major OM pathogens significantly increase the transcription of these three mucin genes as well. A combination of NTHi and SP further synergistically induced MUC2 and MUC5AC gene expression however, not all NTHi strains synergized with SP in the induction. Addition of Mcat WCL to the synergized combination of NTHi and SP did not participate in the synergistic response of mucins.The specific pathogen combinations were important in determining the degree of synergistic effects to GFM expression. The current data are substantive in guiding future work to extend our understanding of OM pathogens and GFMs.

戦前・戦中を通じて、我が国における国民の死亡原因はそのほとんどが感染症、特に呼吸器感染症即ち肺炎と肺結核であった。それが戦後、ペニシリンに始まる一連の抗生剤の開発・進展と国民一般の生活水準の向上によって、感染症による死亡者数は激減し、代って癌・心疾患・脳血管障害が主たる死亡原因疾患になった。しかし肺炎による死亡者数は激減したとはいえ、国民全般の長寿化に伴い、依然として高い水準にある。そこでその、我が国における肺炎の臨床像変遷の概要を、病原体とその対策の観点から、年代順に記述した。(1)肺炎球菌、(2)インフルエンザ・ウイルス、(3)マイコプラズマ、(4)レジオネラ菌、(5)クラミディア、(6)インフルエンザ菌とモラクセラ菌、(7)黄色ブドウ球菌と緑膿菌、そして、これからの問題として誤嚥性肺炎と肺炎球菌ワクチンについて論じた。(著者抄録)

Background Chronic lower airway inflammation is considered to be a major cause of pathogenesis and disease progression in chronic obstructive pulmonary disease (COPD). Moraxella catarrhalis is a COPD-associated pathogen causing exacerbations and bacterial colonization in the lower airways of patients, which may contribute to chronic inflammation. Increasing evidence suggests that the epidermal growth factor receptor (EGFR) modulates inflammatory processes in the human airways. The goal of this study was to investigate the role of EGFR in the M. catarrhalis- induced pro-inflammatory immune response in airway epithelial cells. Methods The effects of inhibition and gene silencing of EGFR on M. catarrhalis- dependent pro-inflammatory cytokine expression in human primary bronchial epithelial cells (NHBEs), as well as the pulmonary epithelial cell lines BEAS-2B and A549 were analyzed. We also assessed the involvement of EGFR-dependent ERK and NF-κB signaling pathways. Results The M. catarrhalis- induced pro-inflammatory immune response depends, at least in part, on the phosphorylation and activation of the EGF receptor. Interaction of M. catarrhalis with EGFR increases the secretion of pro-inflammatory cytokines, which is mediated via ERK and NF-κB activation. Conclusion The interaction between M. catarrhalis and EGFR increases airway inflammation caused by this pathogen. Our data suggest that the inhibition of EGFR signaling in COPD could be an interesting target for reducing M. catarrhalis -induced airway inflammation.

Otitis media is an extremely common pediatric ailment caused by opportunists that reside within the nasopharynx. Inflammation within the upper airway can promote ascension of these opportunists into the middle ear chamber. Otitis media can be chronic/recurrent in nature, and a wealth of data indicates that in these cases the bacteria persist within biofilms. Epidemiological data demonstrates most cases of otitis media are polymicrobial, which may have significant impact on antibiotic resistance. In this study, we used in vitro biofilm assays and rodent infection models to examine the impact of polymicrobial infection with Moraxella catarrhalis and Streptococcus pneumoniae (pneumococcus) on biofilm resistance to antibiotic treatment and persistence in vivo. Consistent with prior work, M. catarrhalis conferred beta-lactamase dependent passive protection from beta-lactam killing to pneumococci within polymicrobial biofilms. Moreover, pneumococci increased resistance of M. catarrhalis to macrolide killing in polymicrobial biofilms. However, pneumococci increased colonization in vivo by M. catarrhalis in a quorum signal-dependent manner. We also found that co-infection with M. catarrhalis affects middle ear ascension of pneumococci in both mice and chinchillas. Therefore, we conclude that residence of M. catarrhalis and pneumococci within the same biofilm community significantly impacts resistance to antibiotic treatment and bacterial persistence in vivo.

Background Upper respiratory tract colonization with a number of bacterial pathogens has been associated with significant respiratory disease and asthma in children. As part of a larger study to evaluate microbial contributions from animals to children with asthma, we tested the hypothesis that mammalian pets could harbor respiratory pathogens of relevance to disease exacerbation among inner-city children with asthma. Methods We tested nasal and pharyngeal biospecimens from subset of 5–17 years old primarily African-American children with asthma enrolled in an ongoing cohort (ECATCh, NCT02251379) prior to trial randomization. At a home visit within three weeks prior to the clinic visit at which children were swabbed, mammalian pets whose owners consented to participate were sampled at nares, mouth, and perineum, depending on animal access and temperament. Aliquots (400 µL) of medium from Copan e-swabs from children and mammalian pets were cultured for multiple respiratory pathogens at the clinical microbiology laboratory at Johns Hopkins Hospital. Results We evaluated 95 children with asthma and 60 mammalian pets at the baseline clinic and home visits, respectively. In children, carriage of respiratory pathogens was: Staphylococcus aureus, 36.8%; Moraxella catarrhalis, 8.4%; Group A Strep, 7.4%; Streptococcus pneumoniae, 1%. In mammalian pets, carriage of respiratory pathogens was: Moraxella catarrhalis, 11.7% (1 dog, 6 cats where 5 of the cats were in the same household); Streptococcus pneumoniae, 1.7% (1 dog). In the home where the dog carried Moraxella catarrhalis (perineum site), the child also carried Moraxella catarrhalis (nares site). Children with dogs had 8-fold higher odds of detection of Moraxella catarrhalis (95% Confidence Interval: 1.4, 46.9, P = 0.02), controlling for other pet ownership and demographic variables. Dogs had higher contact with child participants than cats (contact score higher by 0.7 points on average, P < 0.05). Conclusion Mammalian pets may harbor respiratory pathogens, including Moraxella catarrhalis. Future studies are needed to determine the direction of transmission and whether mammalian pets can serve as a vehicle or reservoir of pathogens of relevance to respiratory disease in children. Disclosures All authors: No reported disclosures.

Background Previous studies reported that Moraxella catarrhalis was one of the main pathogen in community-acquired pneumonia (CAP); however, only a few investigations describing CAP due to M. catarrhalis (MCAP) have been published until now. Therefore, the data regarding clinical features of MCAP is still unknown. Methods The primary objective of the present study was to determine the clinical characteristics of patients with MCAP. Pneumonia patients aged over 20 hospitalized between April 2014 and March 2018 were enrolled. Differences on patients’ background and clinical parameters between MCAP and CAP caused by S. pneumoniae (SCAP) were compared with elucidate the clinical characteristics of MCAP. Patients with bed-ridden status, residents in nursing home, more than two microorganisms were detected from sputum, were excluded. Results During the study period, 114 MCAP and 107 SCAP were identified. In two groups, general status was mild (score ≤2 was 65.7% vs. 64.4%) according to Japanese pneumonia severity scoring system (A-DROP), and the qSOFA score was also relatively low (score ≤2 was 95.6% vs. 91.5%). Although there was no difference in the ratio of sex in these groups, the age was significantly higher in MCAP cohort (the mean age; 77 vs. 68 years old, P < 0.01). Compared with SCAP, MCAP had significantly higher pulmonary underling diseases such as bronchiectasis (P < 0.01), asthma (P < 0.05), interstitial pneumonia (P < 0.05), and lung cancer (P < 0.05), home oxygen therapy (P < 0.01), and systemic disease (P < 0.05). Diagnostic concordance rate between sputum smear on Gram-stain and bacterial cultivation was lower in MCAP patients (78% vs. 87.8%; P = 0.05). In radiological findings, bronchopneumonia pattern was predominant in MCAP group than PCAP group (95.6% vs. 62.6%; P < 0.01). On the other hand, developing a chill and co-infection with Flu were common in PCAP patients (P < 0.01). There was no statistical significant difference on length of treatment and hospital stay in two groups (P = 0.66 and 0.55, respectively). All patients in both groups recovered. Conclusion In the present study, the characteristics of MCAP were as follows; (i) mainly occurred in elderly patients under pulmonary and systemic diseases, (ii) presented with relatively mild symptoms, (iii) bronchopneumonia pattern was predominant, and (iv) benign prognosis. Disclosures All authors: No reported disclosures.

We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.

The otopathogen distribution colonizing the nasopharynx (NP) and causing acute otitis media (AOM) is in flux following the introduction of pneumococcal conjugate vaccine 7 (PCV7) and will continue to change.Two hundred seventy-seven children were followed prospectively; tympanocentesis was performed during AOM and 208 NP samples were collected to compare with middle ear fluid (MEF) isolates. Eight hundred sixty-three NP samples were collected at 7 healthy visits between 6 and 30 months of age. All children received PCV7 until April 2010 when it was substituted by PCV13. Multilocus sequence typing was used to speciate Streptococcus pneumoniae.The distribution of otopathogens in the MEF during the study time frame was stable. PCV7 serotypes of pneumococci were virtually absent. The frequency of isolation of S. pneumoniae was 26-36% compared with 28-34% for nontypeable Haemophilus influenzae. Moraxella catarrhalis isolation was less common, 7-18%. The proportion of S. pneumoniae that were penicillin nonsusceptible was stable during the 3 years, 40-52%. All M. catarrhalis and 34% of nontypeable H. influenzae were β-lactamase producing. NP isolates of otopathogens at onset of AOM included the isolate from the MEF and was dissimilar from the distribution at times of health. Sequence types 320 and 199 of S. pneumoniae expressing serotypes 19A and 15 most often caused AOM.The otopathogen distribution, antibiotic susceptibility and the diversity of strains within the S. pneumoniae species during 2008 through late 2010 were stable. NP isolation of otopathogens at onset of AOM better reflected, albeit incompletely, likely MEF isolates compared with NP isolates at times of health.

The human respiratory tract pathogen Moraxella catarrhalis expresses lipooligosaccharides (LOS), glycolipid surface moieties that are associated with enhanced colonization and virulence. Recent studies have delineated the major steps required for the biosynthesis and assembly of the M. catarrhalis LOS molecule. We previously demonstrated that the glucosyltransferase enzyme Lgt3 is responsible for the addition of at least one glucose (Glc) molecule, at the β-(1-4) position, to the inner core of the LOS molecule. Our data further suggested a potential multifunctional role for Lgt3 in LOS biosynthesis. The studies reported here demonstrate that the Lgt3 enzyme possesses two glycosyltransferase domains (A1 and A2) similar to that of other bifunctional glycosyltransferase enzymes involved in surface polysaccharide biosynthesis in Escherichia coli, Pasteurella multocida and Streptococcus pyogenes. Each Lgt3 domain contains a conserved DXD motif, shown to be involved in the catalytic activity of other glycosyltransferases. To determine the function of each domain, A1 (N-terminal), A2 (C-terminal) and double A1A2 site-directed DAD to AAA mutants were constructed and the resulting LOS phenotypes of these modified strains were analyzed. Our studies indicate that the Lgt3 N-terminal A1 catalytic domain is responsible for the addition of the first β-(1-3) Glc to the first Glc on the inner core. The C-terminal catalytic domain A2 then adds the β-(1-4) Glc and the β-(1-6) Glc, confirming the bifunctional nature of this domain. The results from these experiments demonstrate that Lgt3 is a novel, multifunctional transferase responsible for the addition of three Glcs with differing linkages onto the inner core of M. catarrhalis LOS. © 2013 The Author 2013.

Bacterial colonization of neonatal airways with the pathogenic bacterial species, Moraxella catarrhalis, Streptococcus pneumoniae, and Haemophilus influenzae, is associated with later development of childhood asthma.To study a possible association between colonization with pathogenic bacterial strains and the immune signature of the upper airways in healthy neonates.A total of 20 cytokines and chemokines were quantified in vivo in the airway mucosal lining fluid of 662 neonates from the Copenhagen Prospective Study of Asthma in Childhood 2010 birth cohort. Colonization of the hypopharynx with M. catarrhalis, S. pneumoniae, H. influenzae, and Staphylococcus aureus was assessed simultaneously. The association between immune signatures and bacterial colonization or noncolonized controls was analyzed using conventional statistical methods supplemented by a multivariate approach for pattern identification.Colonization with M. catarrhalis and H. influenzae induced a mixed T helper cell (Th) type 1/Th2/Th17 response with high levels of IL-1β (M. catarrhalis, P = 2.2 × 10(-12); H. influenzae, P = 7.1 × 10(-10)), TNF-α (M. catarrhalis, P = 1.5 × 10(-9); H. influenzae, P = 5.9 × 10(-7)), and macrophage inflammatory protein-1β (M. catarrhalis, P = 1.6 × 10(-11); H. influenzae, P = 2.7 × 10(-7)). S. aureus colonization demonstrated a Th17-promoting profile with elevated IL-17 levels (P = 1.6 × 10(-24)). S. pneumoniae colonization was not significantly associated with any of the mediators.M. catarrhalis and H. influenzae colonization of the airways of asymptomatic neonates is associated with an inflammatory immune response of the airway mucosa, which may result in chronic inflammation.

The lack of a transcriptional reporter system for use in Moraxella catarrhalis has hindered studies of gene regulation in this pathogen. PCR and recombinant DNA methods were used to insert a multicloning site (MCS) and promoterless full-length Escherichia coli lacZ gene, flanked by transcriptional terminators both immediately upstream and downstream, into the M. catarrhalis recombinant plasmid pWW115. Insertion into the MCS in the newly constructed plasmid pASE222 of M. catarrhalis promoter regions controlled by either a repressor (i.e., NsrR) or activator (i.e., PhoB) yielded transcriptional fusion constructs that were appropriately responsive to signal inputs dependent on the host strain genotype, as measured quantitatively by means of a Miller β-galactosidase assay. The transcriptional reporter plasmid pASE222 should prove to be a useful tool for rapid screening of factors affecting gene expression in M. catarrhalis.

Objectives/Hypothesis Acute otitis media (AOM) involves an inflammatory response to microbes in the middle ear that facilitates clearance of otopathogens. Clinically, Streptococcus pneumoniae (Spn) infections of the respiratory tract are characterized by greater inflammatory responses than nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat). Interleukin 10 (IL-10) plays an important role in down-regulating the inflammatory response. We compared serum IL-10 levels in children before onset, at onset, and after recovery from AOM caused by Spn, NTHi, and Mcat. We sought to determine if IL-10 could serve as a biomarker to distinguish AOM caused by Spn versus NTHi and Mcat. Study Design Prospective, longitudinal study in a primary care pediatric practice in Rochester, NY. Methods Participants were 54 children, 6 to 30 months of age. Outcomes measured were serum IL-10 levels when healthy, at onset of AOM, and after recovery from AOM. Results Serum IL-10 was elevated when children developed AOM (P=0.013) due to infections caused by Spn (P=0.011) but not AOM caused by NTHi or Mcat. Middle ear fluid levels of IL-10 mirrored those seen in serum but were 10-fold higher (P=0.02). Other effector cytokines in serum: IL-4, IFN-γ, and TNF-alpha, did not show the same increases as IL-10 at onset of AOM. Conclusion Our study indicates that AOM caused by Spn elicits a significantly higher IL-10 response compared to NTHi and Mcat and may prove to be a biomarker of AOM infections by Spn. Level of Evidence 4.

Background Moraxella catarrhalis is a common causative agent of acute otitis media and other respiratory infections in children. Toll-like receptor (TLR) 4 is an important protein of human innate immunity. One polymorphic site Asp299Gly of TLR4 is proven to result in an impaired response to lipopolysaccharide from Gram-negative bacteria. We investigated whether Finnish children who carry Asp299Gly had increased risk of M. catarrhalis colonization during their first 2 years of life. Methods This was a prospective cohort study carried out in Turku, Finland. We studied M. catarrhalis colonization in 161 Finnish children, whose nasopharyngeal specimens were taken at 2, 12 and 24 months of age. The semiquantitative culture method was used for identification of different bacterial species and the pyrosequencing-based method for detection of TLR4 Asp299Gly. Results Of 161 subjects, 126 (78%) were TLR4 A/A wild type and 35 (22%) were A/G heterozygote variants. The prevalence of M. catarrhalis increased from 24% at 2 months to 48% at 12 months and to 58% at 24 months of age. Of the 35 subjects with TLR4 variant, 15 (43%) were M. catarrhalis positive at all 3 time points, whereas only 11 (9%) subjects with TLR4 wild type were positive at these time points (relative risk 4.91, 95% confidence interval: 2.482-9.711, P=0.0001). Moreover, subjects with TLR4 variant had significantly higher bacterial load of M. catarrhalis in their nasopharynx than those with TLR4 wild type (P=0.0032). Conclusions Our results indicate that children with TLR4 Asp299Gly polymorphism have an increased risk of repeated M. catarrhalis colonization.

We studied nasopharyngeal (NP) colonization in a cohort of children to determine the impact of viral upper respiratory infections (URIs) on nonpneumococcal α-hemolytic streptococci (AHS) and otopathogen colonization in association with acute otitis media (AOM).NP samples were collected routinely when children were aged 6, 9, 12, 15, 18, 24 and 30 months and during episodes of AOM. NP samples were prospectively obtained from 248 children during a 5-year time span: 1018 during routine visits, 161 at the time of AOM and 59 at follow-up visits 3 weeks after AOM.The overall NP colonization rate of AHS was 50.8% during a non-AOM visit but declined to 38.3% during a viral URI with concurrent AOM (P = 0.0006). Of 56 AOM visits with paired follow-ups, 6 (10.7%) had AHS in the NP at the time of viral URI and concurrent AOM whereas 29 (51.8%) had AHS at the follow-up (P0.001). Lower NP colonization rates with AHS were associated with significant increases in Streptococcus pneumoniae carriage during non-AOM visits (P0.001) and during viral URI and concurrent AOM visits (P = 0.003). AHS NP colonization rates were not different when children had a viral URI without AOM versus when they were URI negative, but NP colonization with nontypeable Haemophilus influenzae rates increased (P0.001) and Moraxella catarrhalis decreased (P0.001) during viral URI.Respiratory viral infections alter NP carriage rates of commensal AHS and otopathogens, including before AOM.

Seven hundred twenty-three isolates of Moraxella catarrhalis obtained from outpatients with a variety of infections in 30 medical centers in the United States between 1 November 1994 and 30 April 1995 were characterized in a central laboratory. The overall rate of beta-lactamase production was 95.3%. When the National Committee for Clinical Laboratory Standards MIC interpretive breakpoints for Haemophilus influenzae were applied, percentages of strains found to be susceptible to selected oral antimicrobial agents were as follows: azithromycin, clarithromycin, and erythromycin, 100%; tetracycline and chloramphenicol, 100%; amoxicillin-clavulanate, 100%; cefixime, 99.3%; cefpodoxime, 99.0%; cefaclor, 99.4%; loracarbef, 99.0%; cefuroxime, 98.5%; cefprozil, 94.3%; and trimethoprim-sulfamethoxazole, 93.5%.

From November 1, 1997 to April 30, 1998, 726 Moraxella catarrhalis isolates and 1529 Haemophilus influenzae isolates were obtained from 34 medical centres throughout the United States. Rates of beta-lactamase production were 94.6% among M. catarrhalis and 31.1% among H. influenzae strains. Susceptibility rates of M. catarrhalis isolates to selected antimicrobial agents were greater than 99% for amoxycillin-clavulanate, cefixime, cefpodoxime, cefuroxime, cefaclor, loracarbef, clarithromycin, azithromycin, chloramphenicol and tetracycline, 97.8% for cefprozil, 50.4% for trimethoprim-sulphamethoxazole and 28.1% for ampicillin. Of the antimicrobials tested against H. influenzae, the only agents with susceptibility rates below 96% were loracarbef (87.6%), cefprozil (83.4%), cefaclor (82.7%), trimethoprim-sulphamethoxazole (67.3%) and ampicillin (64.7%). The clarithromycin susceptibility rate was 67.4% but this agent was not tested in the presence of its 14-OH metabolite.