Strain-level resolution and pneumococcal carriage dynamics by single-molecule real-time (SMRT) sequencing of the plyNCR marker: a longitudinal study in Swiss infants

BACKGROUND Pneumococcal carriage has often been studied from a serotype perspective; however, little is known about the strain-specific carriage and inter-strain interactions. Here, we examined the strain-level carriage and co-colonization dynamics of Streptococcus pneumoniae in a Swiss birth cohort by PacBio single-molecule real-time (SMRT) sequencing of the plyNCR marker. METHODS A total of 872 nasal swab (NS) samples were included from 47 healthy infants during the first year of life. Pneumococcal carriage was determined based on the quantitative real-time polymerase chain reaction (qPCR) targeting the lytA gene. The plyNCR marker was amplified from 214 samples having lytA-based carriage for pneumococcal strain resolution. Amplicons were sequenced using SMRT technology, and sequences were analyzed with the DADA2 pipeline. In addition, pneumococcal serotypes were determined using conventional, multiplex PCR (cPCR). RESULTS PCR-based plyNCR amplification demonstrated a 94.2% sensitivity and 100% specificity for Streptococcus pneumoniae if compared to lytA qPCR. The overall carriage prevalence was 63.8%, and pneumococcal co-colonization (≥ 2 plyNCR amplicon sequence variants (ASVs)) was detected in 38/213 (17.8%) sequenced samples with the relative proportion of the least abundant strain(s) ranging from 1.1 to 48.8% (median, 17.2%; IQR, 5.8-33.4%). The median age to first acquisition was 147 days, and having ≥ 2 siblings increased the risk of acquisition. CONCLUSION The plyNCR amplicon sequencing is species-specific and enables pneumococcal strain resolution. We therefore recommend its application for longitudinal strain-level carriage studies of Streptococcus pneumoniae. Video Abstract

Strain-level resolution and pneumococcal carriage dynamics by single-molecule real-time (SMRT) sequencing of the plyNCR marker: a longitudinal study in Swiss infants.

BACKGROUND Pneumococcal carriage has often been studied from a serotype perspective; however, little is known about the strain-specific carriage and inter-strain interactions. Here, we examined the strain-level carriage and co-colonization dynamics of Streptococcus pneumoniae in a Swiss birth cohort by PacBio single-molecule real-time (SMRT) sequencing of the plyNCR marker. METHODS A total of 872 nasal swab (NS) samples were included from 47 healthy infants during the first year of life. Pneumococcal carriage was determined based on the quantitative real-time polymerase chain reaction (qPCR) targeting the lytA gene. The plyNCR marker was amplified from 214 samples having lytA-based carriage for pneumococcal strain resolution. Amplicons were sequenced using SMRT technology, and sequences were analyzed with the DADA2 pipeline. In addition, pneumococcal serotypes were determined using conventional, multiplex PCR (cPCR). RESULTS PCR-based plyNCR amplification demonstrated a 94.2% sensitivity and 100% specificity for Streptococcus pneumoniae if compared to lytA qPCR. The overall carriage prevalence was 63.8%, and pneumococcal co-colonization (≥ 2 plyNCR amplicon sequence variants (ASVs)) was detected in 38/213 (17.8%) sequenced samples with the relative proportion of the least abundant strain(s) ranging from 1.1 to 48.8% (median, 17.2%; IQR, 5.8-33.4%). The median age to first acquisition was 147 days, and having ≥ 2 siblings increased the risk of acquisition. CONCLUSION The plyNCR amplicon sequencing is species-specific and enables pneumococcal strain resolution. We therefore recommend its application for longitudinal strain-level carriage studies of Streptococcus pneumoniae. Video Abstract

Pneumococcal carriage and serotype variation before and after introduction of pneumococcal conjugate vaccines in patients with acute otitis media in Switzerland.

BACKGROUND Acute otitis media (AOM) is an important cause for antibiotic prescription within the paediatric population and Streptococcus pneumoniae is a major pathogen associated with AOM episodes. This study aimed at analysing the influence of the heptavalent and 13-valent pneumococcal conjugate vaccines (PCV7 and PCV13) on pneumococcal carriage and serotype distribution in AOM. METHODS Nasopharyngeal swabs (NPS) and middle ear fluid (MEF) were collected within a Swiss surveillance study of outpatients from all ages with AOM between 2004 and 2015, covering three vaccination eras (pre-PCV7, PCV7 and PCV13). Samples were cultured for pneumococcal identification, and the association of vaccine era with pneumococcal carriage was investigated by logistic regression analysis adjusting for sociodemographic factors. FINDINGS In total, 3300 NPS and 620 MEF were included in this study. The number of samples from patients with AOM dropped over vaccination eras and S. pneumoniae was less frequently isolated in the PCV13 era as compared to the other two eras. The latest (PCV13) vaccination era was independently associated with a reduced pneumococcal carriage within NPS (adjusted odds ratio 0.65, 95%-CI 0.45-0.94). Investigating serotype epidemiology, vaccine serotypes decreased significantly after the conjugate vaccine introductions with the exception of serotype 3. Within the non-PCV13 serotypes, a particular increase of serogroups 11, 15 and 23 was observed in both NPS and MEF. CONCLUSION A substantial change in pneumococcal carriage and serotype epidemiology suggests an impact of the conjugate vaccines on pneumococcal AOM in Switzerland

Nasopharyngeal Microbiota in Infants With Acute Otitis Media

Background. Interspecies interactions of the nasopharyngeal microbiota are likely to be involved in the pathogenesis of acute otitis media (AOM). Capturing the breadth of microbial interactions requires a detailed description of the microbiota during health and AOM. Methods. The nasopharyngeal microbiota of 163 infants with (n = 153) or without (n = 10) AOM was characterized using nasopharyngeal swabs and multiplexed pyrosequencing of 16S rRNA. Nasopharyngeal swab specimens were collected during 4 winter seasons from 2004 through 2010 for infants with AOM and during 2010 for controls. Results. Fifty-eight bacterial families were identified, of which Moraxellaceae, Streptococcaceae, and Pasteurellaceae were the most frequent. Commensal families were less prevalent in infants with AOM than in controls. In infants with AOM, prior exposure to antimicrobials and administration of the heptavalent conjugated pneumococcal polysaccharide vaccine (PCV7) were also associated with reduced prevalence of distinct commensal families (Streptococcaceae and Corynebacteriaceae). In addition, antimicrobial exposure increased the prevalence of Enterobacteriaceae and the abundance of Pasteurellaceae. Other factors, such as age, sex, day care, and a history of recurrent AOM, did not influence the microbiota. Conclusions. Infants' nasopharyngeal microbiota undergoes significant changes during AOM and after exposure to antimicrobials and PCV7, which is mainly attributable to reduced prevalence of commensal bacterial familie

Bacterial contamination of air and surfaces during dental procedures—An experimental pilot study using Staphylococcus aureus

Objective: The oral cavity contains numerous microorganisms, including antimicrobial-resistant bacteria. These microorganisms can be transmitted via respiratory particles from patients to healthcare providers and vice versa during dental care. We evaluated the spread of Staphylococcus aureus during standardized dental procedures using different scaling devices and rinsing solutions. Methods: During systematic therapy for dental biofilm removal (guided biofilm therapy), using an airflow or ultrasound device to a model simulation head. Staphylococcus aureus suspension was injected into the mouth of the model to mimic saliva. Different suction devices (conventional saliva ejector or a prototype) and rising solutions (water or chlorhexidine) were used. To assess contamination with S. aureus, an air-sampling device was placed near the oral cavity and samples of surface areas were collected. Results: S. aureus was only detected by air sampling when the conventional saliva ejector with airflow was used. No growth was observed during treatments with the ultrasonic piezo instrument or the prototype suction device. Notably, a rinsing solution of chlorhexidine digluconate decreased the bacterial load compared to water. Surface contamination was rarely detected (1 of 120 samples). Conclusions: Although our findings indicate potential airborne bacterial transmission during routine prophylactic procedures, specific treatment options during biofilm removal appear to reduce air contamination. These options include ultrasonic piezo devices or the prototype suction device. The use of chlorhexidine reduced the CFU counts of S. aureus detected by air sampling. Surface contamination during dental procedures was a rare occurrence

Understanding short-term transmission dynamics of methicillin-resistant Staphylococcus aureus in the patient room.

OBJECTIVE Little is known about the short-term dynamics of methicillin-resistant Staphylococcus aureus (MRSA) transmission between patients and their immediate environment. We conducted a real-life microbiological evaluation of environmental MRSA contamination in hospital rooms in relation to recent patient activity. DESIGN Observational pilot study. SETTING Two hospitals, hospital 1 in Zurich, Switzerland, and hospital 2 in Ann Arbor, Michigan, United States. PATIENTS Inpatients with MRSA colonization or infection. METHODS At baseline, the groin, axilla, nares, dominant hands of 10 patients and 6 environmental high-touch surfaces in their rooms were sampled. Cultures were then taken of the patient hand and high-touch surfaces 3 more times at 90-minute intervals. After each swabbing, patients' hands and surfaces were disinfected. Patient activity was assessed by interviews at hospital 1 and analysis of video footage at hospital 2. A contamination pressure score was created by multiplying the number of colonized body sites with the activity level of the patient. RESULTS In total, 10 patients colonized and/or infected with MRSA were enrolled; 40 hand samples and 240 environmental samples were collected. At baseline, 30% of hands and 20% of high-touch surfaces yielded MRSA. At follow-up intervals, 8 (27%) of 30 patient hands, and 10 (6%) of 180 of environmental sites were positive. Activity of the patient explained 7 of 10 environmental contaminations. Patients with higher contamination pressure score showed a trend toward higher environmental contamination. CONCLUSION Environmental MRSA contamination in patient rooms was highly dynamic and was likely driven by the patient's MRSA body colonization pattern and the patient activity

Rapid detection of Staphylococcus aureus and Streptococcus pneumoniae by real-time analysis of volatile metabolites

Early detection of pathogenic bacteria is needed for rapid diagnostics allowing adequate and timely treatment of infections. In this study, we show that secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) can be used as a diagnostic tool for rapid detection of bacterial infections as a supportive system for current state-of-the-art diagnostics. Volatile organic compounds (VOCs) produced by growing S. aureus or S. pneumoniae cultures on blood agar plates were detected within minutes and allowed for the distinction of these two bacteria on a species and even strain level within hours. Furthermore, we obtained a fingerprint of clinical patient samples within minutes of measurement and predominantly observed a separation of samples containing live bacteria compared to samples with no bacterial growth. Further development of this technique may reduce the time required for microbiological diagnosis and should help to improve patient's tailored treatment. Keywords: Applied microbiology; Biological sciences tools; Diagnostics; Microbiology

Pulmonary Surfactant Proteins are Inhibited by IgA Autoantibodies in Severe COVID-19

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 bronchoalveolar lavage fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on bronchoalveolar lavage fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and main results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19, but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA against pulmonary surfactant proteins B and C and that these antibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Long-Term Persisting SARS-CoV-2 RNA and Pathological Findings: Lessons Learnt From a Series of 35 COVID-19 Autopsies

BackgroundLong-term sequelae of coronavirus disease 2019 (COVID-19), including the interaction between persisting viral-RNA and specific tissue involvement, pose a challenging issue. In this study, we addressed the chronological correlation (after first clinical diagnosis and postmortem) between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and organ involvement.MethodsThe presence of postmortem SARS-CoV-2 RNA from 35 complete COVID-19 autopsies was correlated with the time interval between the first diagnosis of COVID-19 and death and with its relationship to morphologic findings.ResultsSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be evident up to 40 days after the first diagnosis and can persist to 94 hours after death. Postmortem SARS-CoV-2 RNA was mostly positive in lungs (70%) and trachea (69%), but all investigated organs were positive with variable frequency. Late-stage tissue damage was evident up to 65 days after initial diagnosis in several organs. Positivity for SARS-CoV-2 RNA in pulmonary swabs correlated with diffuse alveolar damage (p = 0.0009). No correlation between positive swabs and other morphologic findings was present. Cerebral (p = 0.0003) and systemic hemorrhages (p = 0.009), cardiac thrombi (p = 0.04), and ischemic events (p = 0.03) were more frequent in the first wave, whereas bacterial pneumonia (p = 0.03) was more prevalent in the second wave. No differences in biometric data, clinical comorbidities, and other autopsy findings were found.ConclusionsOur data provide evidence not only of long-term postmortem persisting SARS-CoV-2 RNA but also of tissue damage several weeks after the first diagnosis of SARS-CoV-2 infection. Additional conditions, such as concomitant bacterial pulmonary superinfection, lung aspergillosis, thromboembolic phenomena, and hemorrhages can further worsen tissue damage

A retrospective analysis of blood culture-negative endocarditis at a tertiary care centre in Switzerland

AIMS OF THE STUDY Numerous studies from different countries have contributed to an improved understanding of blood culture-negative infective endocarditis. However, little is known about its epidemiology and microbiology in Switzerland. We aimed to assess the epidemiology and microbiology of blood culture-negative endocarditis at the University Hospital Zurich, Switzerland. METHODS We screened all patients hospitalised between 1997 and 2020 with possible or definite endocarditis at our institution. Thereof, we identified all cases with blood culture-negative endocarditis and retrospectively retrieved patient characteristics, microbiological, histopathological, radiographic and surgical data from medical records. RESULTS Among 861 patients screened, 66 (7.7%) cases of blood culture-negative endocarditis were identified. Thereof, 31 cases could be microbiologically documented or not documented (n = 30), and in five cases a non-infectious aetiology was confirmed. Endocarditis predominantly affected men (77%) and the left heart (79%); predisposing factors were prosthetic valves (42%), congenital heart disease (35%) and prior endocarditis (14%). The most common reasons for negative blood cultures were antibiotic treatment prior to blood culture sampling (35%), fastidious and slow growing microorganisms (30%) and definite non-infective endocarditis (8%). Coxiella burnetii and Bartonella spp. were the most common fastidious bacteria identified. In addition to serology, identification of causative microorganisms was possible by microbiological and/or histopathological analysis of tissue samples, of which polymerase chain reaction testing (PCR) of the 16S ribosomal RNA proved to be most successful. CONCLUSIONS The present study provides a detailed analysis of blood culture-negative endocarditis over a time span of more than 20 years in Zurich, Switzerland. Antibiotic treatment prior to blood collection, and fastidious and slow growing organisms were identified as main reasons for sterile blood cultures. Typical culture-negative bacteria were mainly found by PCR and/or culture of tissue samples