Bacterial Respiratory Pathogens Research Articles

2223. Real-time Prediction of Respiratory Pathogen Infection Based on Machine Learning Decision Support Tool

BackgroundRespiratory pathogens are a common cause of disease. Currently there is not a practical tool to predict the putative etiology of each case with an inexpensive, fast point-of-care assay. Here, we describe a decision support tool that enables the prediction of both bacterial and viral respiratory pathogen infections in a single patient, using a Machine Learning model.MethodsThe data were obtained from the Hadassah-Hebrew University Medical Center during a period of 10 years beginning from 2007 and contained more than 40,000 patients from a 1,000,000-population community for whom specimens were tested by either PCR or culture. The pathogens included were, H. influenzae; M. catarrhalis; S. pneumoniae; M. pneumoniae; Adenovirus; Human metapneumovirus; Influenza H1N1, A, B; parainfluenza 1,2 and 3; and RSV. We then created a Machine-Learning algorithm to simulate the spread of infection in the entire Jerusalem area. We defined transmission areas based on geographical distances of patients’ home-addresses. Then we prospectively tested the tool accuracy over a 4-month period, in addition to real-time improvement of the model.ResultsInitial model was created based on gender, age, home addresses and the diagnostics test results. We then reconstructed a putative spread pattern for each of the pathogens that can be correlated to potential “transmission routes.” The initial prediction tool had an AUC for most pathogens around 0.85. It ranged from 0.75 to 0.8 for the bacterial and 0.82 to 0.89 for the viral pathogens. In almost all pathogens the NPV was 0.98–0.99. We then tested the decision support tool prospectively over four consecutive months (January to April 2019—1,700 patients with respiratory complaints from whom samples were sent to the lab). While the AUC in the prospective cohort was 0.81 on average, the NPV remained high on 0.98.ConclusionThe implementation of the decision support tool on respiratory pathogen diagnostics enables better prediction of patients not infected with either viral or bacterial pathogens. The use of such a tool can save more than 50% of diagnostic tests expenses as well as real-time mapping of disease spread. Improvement of the Machine Learning protocol may further promote the optimization of positive predictive values.Disclosures All authors: No reported disclosures.

703. In Vitro Activity of Lefamulin Against Bacterial Pathogens Causing Community-Acquired Bacterial Pneumonia (CABP): SENTRY Surveillance 2017–2018 Results From the United States (US)

BackgroundLefamulin (LEF), a novel pleuromutilin protein synthesis inhibitor in development for use as an empiric IV and oral monotherapy for CABP, recently demonstrated safety and efficacy in two phase 3 trials in adults with CABP (PORT II–V). LEF IV or IV/oral (5–7 days; 10 days for methicillin-resistant Staphylococcus aureus [MRSA]) and LEF oral (5 days) were noninferior to MOX IV or IV/oral (7 days; 10 days for MRSA) and MOX oral (7 days) in patients with CABP caused by the most prevalent typical and atypical bacterial pathogens. This study investigated the in vitro activity of LEF and comparators against bacterial respiratory pathogens collected in the United States in 2017 and 2018.MethodsAs part of the SENTRY Surveillance Programme, isolates (n = 2299, 1/patient) were collected from 39 medical centers in the United States from patients with community-acquired respiratory tract infections (1812/2299 [78.8%]) and pneumonia in hospitalized patients (487/2299 [21.2%]). LEF and comparators were tested by broth microdilution and CLSI (2019) breakpoints were applied.ResultsLEF demonstrated potent antibacterial activity against all pathogens tested and was unaffected by resistance to other antibiotic classes (table). Streptococcus pneumoniae isolates were largely susceptible ( >80%) to most comparators; however, 45.6% and 20.4% were resistant (R) to macrolides and tetracycline, respectively. LEF exhibited a MIC50/90 of 0.12/0.25 mg/L for S. pneumoniae, including all R subsets. Among S. aureus isolates, and particularly MRSA, resistance to macrolides was high (48.5% and 81.2% R, respectively). LEF showed a MIC50/90 of 0.06/0.12 mg/L for S. aureus, including all R subsets. Haemophilus influenzae isolates were susceptible to all comparators except for ampicillin (31.4% R) and trimethoprim-sulfamethoxazole (35.3% R). LEF displayed a MIC50/90 of 0.5/2 mg/L for H. influenzae isolates. Moraxella catarrhalis isolates, which were largely β-lactamase positive (98%), were susceptible to all comparators.ConclusionLEF displayed potent in vitro activity against contemporary CABP pathogens collected in the United States. LEF activity was unaffected by resistance to other antibiotic classes, including fluoroquinolones, macrolides, β-lactams, and tetracyclines. Disclosures All authors: No reported disclosures.

Verification of a Novel Multiplex PCR Respiratory Virus Panel in a US Biocontainment Unit

Emerging infectious diseases carry unique logistical, financial, and clinical ramifications. Rapid diagnostic testing methods can alleviate some of these challenges by providing definitive diagnoses earlier in the clinical course, leading to appropriate targeted therapy, cost savings, and improved patient outcomes. The BioFire FilmArray Respiratory Panel 2 plus (RP2plus; bioMérieux, Marcy l’Etoile, France) is a multiplexed nucleic acid test for detection of Middle East respiratory syndrome coronavirus (MERS-CoV) and 14 common viral and 4 bacterial respiratory pathogens in nasopharyngeal swabs obtained from those meeting MERS-CoV epidemiological criteria. The aim of this study was to verify the FilmArray RP2plus for use in our biocontainment unit. Of note, the RP2plus is FDA approved but not currently available for sale in the United States. Eight patient samples were tested with known results (GenMark Respiratory Virus Panel [RVP] or Cepheid Xpert Flu/RSV). We had concordant results between the platforms for samples containing influenza A, respiratory syncytial virus (RSV), parainfluenza virus 2, rhinovirus, and a negative sample. We evaluated two influenza B samples from two different patients. The FilmArray RP2plus did not detect influenza B in one of the patient samples. The sample was exhausted and repeat testing could not be performed. A second rhinovirus sample was not detected by the RP2plus, but Coronavirus 229E was detected in this sample, a virus not detected by the RVP. The sample was repeated and again did not detect rhinovirus. Further investigation into this discrepancy revealed that rhinovirus was originally detected by RVP at a signal of 34.4 nA (repeat of 46.9 nA). The concordant rhinovirus sample had a signal of 226.7 nA by RVP, which was much higher than the discrepant sample. Because of the low signal by RVP in the discrepant sample, perhaps the viral load was below the limit of detection of the RP2plus. All other quality control sample pools passed verification testing, including day-to-day and operator variance. It is not uncommon for a person under investigation (PUI) for a highly communicable disease to be evaluated in our facility. The performance of the RP2plus test on clinical samples showed acceptable concordance with our current means of testing for respiratory pathogens. The RP2plus will eliminate challenges implicated in storing and transporting specimens to an off-site lab, facilitate quicker turnaround time, and streamline the often cumbersome, complex protocols and practices required to work up a serious communicable disease.

Identification of genes regulated by the two-component system response regulator NarP of Actinobacillus pleuropneumoniae via DNA-affinity-purified sequencing

Identifying the direct target genes of response regulators (RRs) of a bacterial two-component system (TCS) is critical to understand the roles of TCS in bacterial environmental adaption and pathogenesis. Actinobacillus pleuropneumoniae is an important respiratory bacterial pathogen that causes considerable economic losses to swine industry worldwide. The targets of A. pleuropneumoniae NarP (nitrate/nitrite RR), which is the cognate RR of the nitrate/nitrite sensor histidine kinase NarQ, are still unknown. In the present study, a DNA-affinity-purified sequencing (DAP-Seq) approach was established. The upstream regions of a total of 131 candidate genes from the genome of A. pleuropneumoniae were co-purified with the activated NarP protein. Electrophoretic mobility shift assay (EMSA) results confirmed the interactions of NarP with the promoter regions of five selected target genes, including dmsA, pgaA, ftpA, cstA and ushA. The EMSA-confirmed target genes were significantly up-regulated in the narP-deleted mutant in the presence of additional nitrate, whilst the transcriptional changes were restored in the complemented strain. The NarP binding motif in the upstream regions of the target genes dmsA and ftpA were further identified and confirmed by EMSA using the truncated binding motif. The NarP binding sites were present in a total of 25.2% of the DNA fragments captured by DAP-Seq. These results demonstrated that the established DAP-Seq method is effective for exploring the direct targets of RRs of bacterial TCSs and that the A. pleuropneumoniae NarP could be a repressor in response to nitrate.

A randomized, placebo-controlled, double-blinded, single-centre, phase IV trial to assess the efficacy and safety of OM-85 in children suffering from recurrent respiratory tract infections

BackgroundOver many years, OM-85, a lysate of 21 common bacterial respiratory pathogens, has been demonstrated to prevent respiratory recurrences in children. However, further studies are needed to explore the true importance of OM-85 in the prevention of respiratory tract infections (RTIs) in children. This study was planned to further contribute to the evaluation of the role played by OM-85 in prevention of recurrent RTIs in children.MethodsThis study was a randomized (3:3:1), placebo-controlled, double-blind, single-centre, phase IV trial carried out in Italy to assess the efficacy of OM-85 (Broncho-Vaxom®; Vifor Pharma; Meyrin 2/Geneva, Switzerland) in reducing the number of new RTI episodes in 288 children aged 1 to 6 years with a history of recurrent RTIs and to compare the efficacy of the standard 3-month regimen with that of administration of OM-85 for 6 months during a 6-month study period.ResultsThe number of RTIs and of children who experienced at least one RTI were significantly lower among patients receiving OM-85 for 3 months than among those given placebo (33% vs 65.1%, p < 0.0001). Differences were statistically significant for upper RTIs (i.e., common cold/viral pharyngitis and acute otitis media; p < 0.0001 and p = 0.006, respectively). Days of absence from day-care for children and working days lost by parents were significantly lower in the group with children treated with OM-85 for 3 months than in the placebo group (p = 0.007 and p = 0.004, respectively). No difference was seen between children who received OM-85 for 3 and those who received OM-85 for 6 months. The prevalence of atopy as well as the history of recurrent wheezing and age of the study child did not influence the results. Benefit was maximally evident among children with a history of frequent recurrences. OM-85 was well tolerated and safe, even in children who received an influenza vaccination.ConclusionsThe use of OM-85 for 3 months in 3 series of 10 consecutive days each time reduces the risk of recurrent RTIs in children, with a favourable safety profile. The greater effect observed in children prone to several respiratory episodes than in non-prone children seems to indicate that this lysate should be administered especially to children with a proven high susceptibility to RTIs.

Seasonality of respiratory viruses and bacterial pathogens

BackgroundSeasonal variation has been observed for various bacterial and viral infections. We aimed to further study seasonality of respiratory viruses and bacterial pathogens in relation to antibiotic use, as well as meteorological parameters.MethodsAn ecologic study of antibiotic exposure, meteorological parameters, detection of respiratory viruses and clinical isolates of Clostridioides difficile, Methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, and Escherichia coli and Klebsiella pneumoniae (grouped together as gram-negative bacteria; GNB) in Rhode Island from 2012 to 2016.ResultsPeak detection of C. difficile occurred 3 months after the peak in antibiotic prescriptions filled (OR = 1.24, 95% CI, 1.07–1.43; P = 0.006). Peak MRSA detection was noted 7 months after the peak in antibiotic prescriptions filled (OR = 1.69, 95% CI, 1.21–2.35; P = 0.003) and 10 months after the peak in respiratory virus detection (OR = 1.04, 95% CI, 1.01–1.06; P = 0.003). Peak GNB detection was noted 2 months after the peak mean monthly ambient temperature (OR = 1.69, 95% C.I., 1.20–2.39; P = 0.004). Peak detection of S. pneumoniae was noted at the same time as the peak in detection of respiratory viruses (OR = 1.01, 95% C.I., 1.00–1.01; P = 0.015).ConclusionsWe identified distinct seasonal variation in detection of respiratory viruses and bacterial pathogens. C. difficile seasonality may, in part, be related to antibiotic prescriptions filled; GNB seasonality may be related to ambient temperature and S. pneumoniae may be related to concurrent respiratory viral infections.

Exploitation of Host Epithelial Signaling Networks by Respiratory Bacterial Pathogens

Although tremendous effort has been put towards identifying the surface molecules of nontypeable Haemophilus influenzae (NTHi) for vaccine development over the past decades, it is only recently that we have begun to appreciate the intricate host epithelial signaling networks activated by NTHi, an important human pathogen causing respiratory infections. From what has been reported, it is evident that NTHi activates multiple signaling pathways in host epithelial cells that, in turn, inadvertently contribute to the pathogenesis. Among those signaling pathways, activation of NF-κB leads to up-regulation of IL-1β, IL-8 and TNF-α, mucin MUC2 and Toll-like receptor 2 (TLR2), whereas activation of p38 MAP kinase mediates not only up-regulation of inflammatory mediators and mucin MUC5AC but also down-regulation of TLR2. Interestingly, NTHi-induced activation of the PI3K-Akt pathway, however, leads to inhibition of p38 mitogen-activated protein (MAP) kinase. Moreover, the TGF-β-Smad signaling pathway cooperates with NF-κB to mediate up-regulation of mucin MUC2. Finally, glucocorticoids synergistically enhance NTHi-induced TLR2 expression via specific up-regulation of the MAP kinase phosphatase-1 that, in turn, leads to inactivation of p38 MAP kinase, the negative regulator for TLR2 expression. These studies may bring new insights into the molecular pathogenesis of NTHi-induced infections and open up novel therapeutic targets for these diseases.

Nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for clinical microbiology in 2014: General view of the pathogens' antibacterial susceptibility

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from the patients in Japan was conducted by Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2014.The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period between January 2014 and April 2015 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical Laboratory Standards Institute.Susceptibility testing was evaluated in 1534 strains (335 Staphylococcus aureus, 264 Streptococcus pneumoniae, 29 Streptococcus pyogenes, 281 Haemophilus influenzae, 164 Moraxella catarrhalis, 207 Klebsiella pneumoniae, and 254 Pseudomonas aeruginosa). Ratio of methicillin-resistant S. aureus was 43.6%, and those of penicillin-susceptible S. pneumoniae was 100%. Among H. influenzae, 8.2% of them were found to be β-lactamase-producing ampicillin-resistant strains, and 49.1% 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 9.2% and 0.4%, respectively.

Serotype-Independent Protection Against Invasive Pneumococcal Infections Conferred by Live Vaccine With lgt Deletion.

Streptococcus pneumoniae is the most common respiratory bacterial pathogen among cases of community-acquired infection in young children, older adults, and individuals with underlying medical conditions. Although capsular polysaccharide-based pneumococcal vaccines have contributed to significant decrease in invasive pneumococcal infections, these vaccines have some limitations, including limited serotype coverage, lack of effective mucosal antibody responses, and high costs. In this study, we investigated the safety and immunogenicity of a live, whole-cell pneumococcal vaccine constructed by deleting the gene for prolipoprotein diacylglyceryl transferase (lgt) from the encapsulated pneumococcal strain TIGR4 (TIGR4Δlgt) for protection against heterologous pneumococcal strains. Pneumococcal strain TIGR4 was successfully attenuated by deletion of lgt, resulting in the loss of inflammatory activity and virulence. TIGR4Δlgt colonized the nasopharynx long enough to induce strong mucosal IgA and IgG2b-dominant systemic antibody responses that were cross-reactive to heterologous pneumococcal serotypes. Finally, intranasal immunization with TIGR4Δlgt provided serotype-independent protection against pneumococcal challenge in mice. Taken together, our results suggest that TIGR4Δlgt is an avirulent and attractive broad-spectrum pneumococcal vaccine candidate. More broadly, we assert that modulation of such “master” metabolic genes represents an emerging strategy for developing more effective vaccines against numerous infectious agents.

What is the role of Ewingella americana in humans? A case report in a healthy 4-year-old girl

BackgroundEwingella americana (Ea) is a Gram-negative, lactose-fermenting, oxidase-negative and catalase-positive bacterium that was first described in 1983 as a new genus and species in the family Enterobacteriaceae. It is not known whether Ea is a true pathogen or simply an opportunistic infectious agent, as most of the cases have been described in patients at risk.Case presentationA 4-year-old girl described here was hospitalized due to a productive cough over the previous 3 weeks and a fever > 38 °C associated with tachypnea over the previous 2 days. Her familial and personal medical histories were negative for relevant diseases, including respiratory infections. At admission, she was febrile (axillary temperature 39.2 °C) and had dyspnea with retractions, grunting and nasal flaring. A chest examination revealed fine crackling rales in the left upper field associated with bilateral wheezing. A chest X-ray revealed segmental consolidation of the lingula of the left lung. Laboratory tests revealed leukocytosis (15.,800 white blood cells/mm3 with 50.3% neutrophils), a slight increase in serum C-reactive protein (11.9 mg/L) and normal procalcitonin values (< 0.12 ng/mL). A nasopharyngeal swab culture did not reveal viral or bacterial respiratory pathogens, including atypical bacteria. A blood culture revealed the presence of a Gram-negative, lactose-fermenting rod that was oxidase negative and catalase positive. The isolate was identified by means of the VITEK®2 identification system (bioMérieux, Firenze, Italy) as Ea. This identification was confirmed by sequencing the 16 s ribosomal deoxyribonucleic acid (rDNA). The pathogen was sensitive to aminoglycoside, fluoroquinolones, carbapenems, cefotaxime, and ceftazidime but was intermediate against sulfametoxazole/trimethoprim and resistant to amoxicillin-clavulanic acid, fosfomycin, and oxacillin. The child was immediately treated orally with amoxicillin-clavulanic acid and erythromycin. Based on the results of a blood culture and sensitivity tests, the amoxicillin-clavulanic acid medication was stopped after 3 days. Erythromycin was continued for a total of 10 days, and the child was discharged after 3 days in the hospital. Follow-up visit 1 month later did not reveal any respiratory problems.ConclusionThis case shows that Ea infections in healthy subjects are mild even in pediatric age, and the need for antibiotic therapy is debated. Cases occurring in subjects with underlying chronic disease can be significantly more complicated and require appropriate antibiotic therapy.

Protective role of TH17 cells in an FcγR targeting immunization strategy against the respiratory bacterial pathogen, Francisella tularensis

Abstract Francisella tularensis is a category A intracellular bacterial pathogen with no FDA approved vaccine. Understanding the immunological mechanisms involved in host defense can improve vaccine efficacy. Previously we have shown using an F. tularensis murine infection model that targets the inactivated bacterium (iFt) to Fcγ receptors on antigen presenting cells (APCs) using anti-LPS monoclonal antibodies (mAb-iFt) provides full protection against lethal pulmonary challenge with F. tularensis live vaccine strain (LVS). The protection provided by this mucosal vaccine strategy involves both innate and adaptive immune components, with a strong inflammatory response being critical for protection. This study focuses on elucidating the role of Th 17 cells, a T helper cell subpopulation involved in pathology and host defenses against bacterial pathogens. C57BL/6 mice were immunized with iFt and mAb-iFt on day 0 and 21 and then challenged i.n. with a lethal dose of LVS on day 35. Flow cytometry data revealed a significant population of Th17 cells (characterized as CD3+/CD4+/IL- 17a+/RORγ-T+ cells) in the lungs and spleen post-infection in the mAb-iFt immunized group when compared to PBS and iFt alone. Additionally, cytokine levels of IL-6 and TGF-β1 (two known synergistic Th17 polarizing cytokines) were elevated in the lungs and spleens of mAb-iFt immunized mice on days 2 and 5 post-infection when compared to iFt alone. Lastly, cytokine levels of IL-17a, an effector cytokine secreted predominantly by active Th17 cells, was shown to be significantly increased in mAb-iFt immunized mice when compared to iFt alone. Collectively, our data suggests that Th17 cells possess a protective role in the immune response to pulmonary F. tularensis infection.

PIN92 PEDIATRIC BACTERIAL PNEUMONIA CLASSIFICATION THROUGH CHEST X-RAYS USING TRANSFER LEARNING

Chest X-ray (CXR)-confirmed pneumonia in children is more specific for bacterial pneumonia than symptom-based diagnosis, and it may be more responsive for assessing the impact of vaccines that target respiratory bacterial pathogens. However, the variability of CXR interpretation by radiologists makes it costly and difficult to use CXR-confirmed pneumonia as an endpoint in large-scale epidemiology research. We aim to develop an automated system to interpret pediatric CXR. We applied a deep neural network (DNN) to classify suspected bacterial pneumonia from pediatric CXRs. A DNN model was first trained using the largest publicly available CXR dataset, ChestX-ray14 (N=112,120). A transfer learning technique was applied to train this pre-trained model on two smaller pediatric datasets, Guangzhou Women and Children’s Hospital CXR (GZ-CXR, N=5,856, 47% bacterial pneumonia) and World Health Organization pediatric training CXR (WHO-CXR, N=410, 39% primary endpoint consolidation (PEP)), separately. Data augmentation techniques (i.e., horizonal flipping, minor rotation, translation and shear) were applied to boost model robustness. We assessed eight DNN models using the above framework, and we evaluated results using the average area under the curve (AUC) from 10-fold cross-validation. While the classification accuracy across the eight DNN models was similar in GZ-CXR, there was more variation in WHO-CXR due to the small sample size. DenseNet-121 had the best trade-off between speed and accuracy. It achieved AUC 0.91 and 0.92 for classifying bacterial pneumonia on GZ-CXR and for PEP on WHO-CXR, respectively. DenseNet-121 was also computationally efficient, with average training time about twenty and four minutes in GZ-CXR and WHO-CXR, respectively. It was two-to-four times faster than NASNetLarge and InceptionResNetV2, with similar accuracy. We developed a CXR-based pneumonia classification framework which achieved high classification accuracy. With substantial reduction in human time required, computer-aided reading of CXR-confirmed pneumonia may facilitate future disease burden and vaccine impact studies of pneumonia in children.

Etiology of Pulmonary Infections in Human Immunodeficiency Virus-infected Inpatients Using Sputum Multiplex Real-time Polymerase Chain Reaction.

BackgroundThere are limited data on the etiology of respiratory infections in human immunodeficiency virus (HIV)–infected patients in resource-limited settings.MethodsWe performed quantitative multiplex real-time polymerase chain reaction (PCR) for Pneumocystis jirovecii and common bacterial and viral respiratory pathogens on sputum samples (spontaneous or induced) from a prospective cohort study of HIV-infected inpatients with World Health Organization danger signs and cough. Mycobacterial culture was done on 2 sputum samples, blood cultures, and relevant extrapulmonary samples.ResultsWe enrolled 284 participants from 2 secondary-level hospitals in Cape Town, South Africa: median CD4 count was 97 cells/μL, 64% were women, and 38% were on antiretroviral therapy. One hundred forty-eight had culture-positive tuberculosis, 100 had community-acquired pneumonia (CAP), 26 had P. jirovecii pneumonia (PJP), and 64 had other diagnoses. Probable bacterial infection (>105 copies/mL) was detected in 133 participants; the prevalence was highest in those with CAP (52%). Haemophilus influenzae and Streptococcus pneumoniae were the commonest bacterial pathogens detected; atypical bacteria were uncommon. Viruses were detected in 203 participants; the prevalence was highest in those with PJP (85%). Human metapneumovirus was the commonest virus detected. Multiple coinfections were commonly detected.ConclusionsSputum multiplex PCR could become a useful diagnostic tool for bacterial respiratory infections in HIV-infected inpatients, but its value is limited as quantitative cutoffs have only been established for a few bacterial pathogens and validation has not been done in this patient population. We found a high prevalence of respiratory viruses, but it is unclear whether these viruses were causing infection as there are no accepted quantitative PCR cutoffs for diagnosing respiratory viral infections.

Do combined upper airway cultures identify lower airway infections in children with chronic cough?

Obtaining lower airway specimens is important for guiding therapy in chronic lung infection but is difficult in young children unable to expectorate. While culture-based studies have assessed the diagnostic accuracy of nasopharyngeal or oropharyngeal specimens for identifying lower airway infection, none have used both together. We compared respiratory bacterial pathogens cultured from nasopharyngeal and oropharyngeal swabs with bronchoalveolar lavage (BAL) cultures as the "gold standard" to better inform the diagnosis of lower airway infection in children with chronic wet cough. Nasopharyngeal and oropharyngeal swabs and BAL fluid specimens were collected concurrently from consecutive children undergoing flexible bronchoscopy for chronic cough and cultured for bacterial pathogens. In cultures from 309 children (median age, 2.3 years) with chronic endobronchial suppuration, all main pathogens detected (Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis) were more prevalent in nasopharyngeal than oropharyngeal swabs (37%, 34%, and 23% vs 21%, 6.2%, and 3.2%, respectively). Positive and negative predictive values for lower airway infection by any of these three pathogens were 63% (95% confidence interval [95% CI] 55, 70) and 85% (95% CI, 78, 91) for nasopharyngeal swabs, 65% (95% CI, 54, 75), and 66% (95% CI, 59, 72) for oropharyngeal swabs, and 61% (95% CI, 54,68), and 88% (95% CI, 81, 93) for both swabs, respectively. Neither nasopharyngeal nor oropharyngeal swabs, alone or in combination, reliably predicted lower airway infection in children with chronic wet cough. Although upper airway specimens may be useful for bacterial carriage studies and monitoring antimicrobial resistance, their clinical utility in pediatric chronic lung disorders of endobronchial suppuration is limited.

Antibacterial activity against porcine respiratory bacterial pathogens and in vitro biocompatibility of essential oils.

Bacterial respiratory infections affecting pigs such as pneumonia, pleuropneumonia, and pleurisy, are a major health concern in the swine industry and are associated with important economic losses. This study aimed to investigate the antibacterial activities of essential oils against major swine respiratory pathogens with a view to developing a potential alternative to antibiotics. Their synergistic interactions with the bacteriocin nisin was also examined. Lastly, we assessed the in vitro biocompatibility of the most efficient essential oils using a pig tracheal epithelial cell line. Of the nine essential oils tested, those from cinnamon, thyme, and winter savory were the most active against Streptococcus suis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Bordetella bronchiseptica, Haemophilus parasuis, and Pasteurella multocida, with minimum inhibitory concentrations and minimum bactericidal concentrations ranging from 0.01 to 0.156% (v/v). The main component found in cinnamon, thyme, and winter savory oils were cinnamaldehyde, thymol, and carvacrol, respectively. Treating pre-formed S. suis and A. pleuropneumoniae biofilms with thyme or winter savory oils significantly decreased biofilm viability. We also observed a synergistic growth inhibition of S. suis with mixtures of nisin and essential oils from thyme and winter savory. Concentrations of nisin and cinnamon, thyme and winter savory essential oils that were effective against bacterial pathogens had no effect on the viability of pig tracheal epithelial cells. The present study brought evidence that essential oils are potential antimicrobial agents against bacteria associated with porcine respiratory infections.

Comparative Emergence of Resistance to Clofoctol, Erythromycin, and Amoxicillin against Community-Acquired Bacterial Respiratory Tract Pathogens in Italy

Due to increasing bacterial resistance and poor availability of new antibiotics, physicians need to use old, still active antibiotics more frequently. In this study, we focused on clo­foctol and aimed to verify the emergence of clofoctol resistance over time. Additionally, the ability of clofoctol to induce resistance under static and dynamic conditions was evaluated. The minimum inhibitory concentration (MIC) values measured in pathogens isolated from 1990 to 1995 were compared to those isolated from 2017 to 2018. The behaviour of clofoctol is similar to that of amoxicillin, while erythromycin shows a different behaviour with an increase in MIC. A rapid decline in CFUs with complete eradication at 96 and 120 h in the case of clofoctol and amoxicillin, respectively, was observed in a dynamic in vitro model of a pharmacokinetic simulation. Erythromycin provides a reduction in CFUs of approximately one order of magnitude for up to 72 h, and then re-growth is observed. The MIC trend was observed during 5 days of kinetic simulation. The clofoctol MICs remain almost stable up to 96 h, after which the colonies are no longer detectable. The MICs of amoxicillin show a 2-fold increase starting from 36 h; however, at 120 h the colonies are no longer detectable. The MICs of erythromycin show a progressive increase starting from 72 h and reaching 32-fold. Clofoctol maintains its activity towards the common pathogens of respiratory tract infections and, similarly to amoxicillin, does not induce resistance in a strain of Streptococcus pneumoniae, resulting in complete eradication, while erythromycin was able to select resistant mutants.

Impact of Respiratory Syncytial Virus Infection on Bronchiolitis Obliterans Syndrome in Lung Transplant Recipients

Purpose Respiratory syncytial virus infection (RSVI) in lung transplant recipients (LTRs) has been associated with bronchiolitis obliterans syndrome (BOS) development and progression, but data have been derived from small studies. Methods This is a retrospective study of RSVI in LTRs at Duke University from Jan 2013 - May 2017. Decrement in FEV1 was determined by comparison of the FEV1 at 90 days post-RSVI to pre-RSVI baseline. Results Of 114 LTRs with RSVI, 15 had preexisting BOS and 99 did not have preexisting BOS. Those with preexisting BOS had a longer duration from transplant surgery to RSVI, more concomitant respiratory bacterial pathogens and less acute cellular rejection (ACR) within 90 days preceding RSVI compared to those without. Within 1-year of RSVI, 7/15 (46.7%) patients with preexisting BOS had BOS progression and 25/99 (25.3%) patients developed new BOS. In patients with documented FEV1 measured pre- and post-RSVI, 6/12 (50%) with preexisting BOS and 23/90 (25.5%) without preexisting BOS had FEV1 decline ≥ 5%. Kaplan-Meier analysis showed patients with decline in FEV1 ≥ 5% at 90 days post-RSVI had a higher 1-year mortality (p=0.003). Conclusion Our experience supports an increase in BOS development post-RSVI. While BOS progression post-RSVI was common in patients with pre-existing BOS, rates of progression were not greater than those described for the natural history of BOS post-lung transplant. FEV1 decline ≥ 5% at 90 days post-RSVI was associated with higher mortality regardless of BOS status prior to RSVI.

Identifying and inhibiting co-aggregation occurring between bacterial respiratory pathogens and commensal species associated with the altered microbiota of chronic pulmonary disease to identify the effect on the development of multispecies biofilms in vitro

Comparisons of pulmonary microbiotas associated with healthy individuals and cystic fibrosis (CF) patients identified a less diverse microbiota among CF patients comprising numerous bacterial species not found among healthy controls. This research aimed to identify if coadhesion occurs between respiratory pathogens P. aeruginosa and S. aureus and some commensal species associated with the microbiota of CF. This was conducted in vitro by spectrophotometric co-aggregation analysis and multi-species biofilm assays in the presence and absence of an inhibitor of lectin-mediated coaggregation, testing the ability of eleven commensal representative species of the CF pulmonary microbiota. Results show that all commensal species selected to represent genera that are associated with the CF pulmonary microbiota are capable of coaggregation in vitro with respiratory pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. S. aureus co-aggregated with a higher affinity to commensal species than P. aeruginosa with a median coaggregation percentage of 62.9 % after 24 h incubation, compared to 41.9 % in P. aeruginosa. When commensal and pathogenic species were cultured in the presence of lactose, biofilm inhibition was observed in 5 of 12 cultures with P. aeruginosa and 7 of 12 with S. aureus. These results demonstrate that some commensals associated with CF participate in coaggregation with respiratory pathogens in vitro which has the ability to alter biofilm production. Identifying coadhesion between commensal and pathogenic species in vitro suggests that coadhesion could be occurring in vivo potentially causing increased susceptibility of the host by enhancing adhesion to the epithelial surface of the respiratory tract, aiding colonization and therefore increased susceptibility to infections.

TNF-α disrupts the integrity of the porcine respiratory epithelial barrier

The airway epithelium plays an important role in protecting the host against environmental microbial pathogens. The disruption of the epithelial barrier may lead to inflammation and invasion by bacterial pathogens. The aim of the present study was to determine whether TNF-α can damage the integrity of the epithelial barrier using a tracheal epithelial cell model. TNF-α (10 and 100 ng/mL) significantly and time-dependently decreased the transepithelial electrical resistance of the epithelial barrier. The ability of TNF-α to disrupt epithelial barrier integrity was confirmed by quantifying the passage of fluorescein isothiocyanate-conjugated dextran across an epithelial monolayer. Immunofluorescence microscopy showed that TNF-α has a disruptive effect on both occludin and zonula occludens-1, which are important tight junction proteins. In addition to disrupting the epithelial barrier, TNF-α induced the secretion of IL-6 and IL-8 by tracheal epithelial cells. Lastly, incubating Gram-negative respiratory bacterial pathogens with the tracheal epithelial cells caused the cells to secrete TNF-α. In summary, the present study showed that TNF-α, which was secreted by the tracheal epithelial cells when they were stimulated with several swine pathogens, damaged tight junction proteins and disrupted the epithelial barrier of a pig tracheal epithelial cell model. TNF-α can also cause inflammation through its ability to induce IL-6 and IL-8 secretion by epithelial cells.

Laboratory diagnostics of selected feline respiratory pathogens and their prevalence in the Czech Republic

Respiratory problems in cats have a multifactorial character. Therapy without the detection of pathogen is often ineffective. Our study was therefore focused on the detection of important feline respiratory bacterial pathogens such as Mycoplasma felis, Chlamydia felis and Bordetella bronchiseptica and viral pathogens such as Felid alphaherpesvirus-1 and feline calicivirus. The goal of this study was to map the occurrence of these pathogens in cat populations in the Czech Republic with the aim of introducing rapid and highly sensitive methods into routine diagnostics and to provide consulting services to animal health professionals based on the acquired data. A total of 218 cats were investigated in the study: 69 were outdoor and 149 were indoor cats. Three groups of animals were compared: up to one year of age (60 cats), one to three years of age (68 cats) and more than three years of age (90 cats). Samples were taken from conjunctiva and/or the oropharynx. Samples originated from cats with various forms of respiratory disease or from healthy cats from different parts of the Czech Republic. Real-Time RT-PCR, multiplex Real-Time PCR, nested PCR and sequencing analyses were performed. Outdoor cats were infected more often (84 detected pathogens in 69 cats) than indoor cats (110 detected infections in 149 cats). More than one pathogen was detected in a total of 38 cats, and six cats were infected with more than two pathogens. The difference was statistically significant in the case of co-infections, but not for mono-infections (P &amp;lt; 0.05). Kittens and young adults up to the age of one year were the most common reservoirs of respiratory infections (only 19 cats out of 60 were negative and positive cats often harboured coinfections). The difference in age groups were not statistically significant (P &amp;gt; 0.05). Concerning the site of the sampling, feline calicivirus, M. felis and B. bronchiseptica were detected more often from oropharynx than from conjunctival swabs. M. felis was slightly more common in clinically diseased animals (39.6%) than in healthy ones (26.1%). The obtained results reveal the frequency of individual pathogens and their co-infections in cats kept on the territory of the Czech Republic, data which can be used to make the treatment of respiratory infections and breeding measures more effective. Therefore, the diagnostic methods are now available to veterinary surgeons with the possibility of consultation and discussion of the results.