Description of nasopharyngeal bacterial pathogens associated with different SARS-CoV-2 variants.

The emergence of the coronavirus pandemic facilitated the acquisition of mutations in the SARS-CoV-2 genome, resulting in the appearance of new variants over the past three years. We previously identified several taxa associated with the clinical outcome of COVID-19 disease in a retrospective study involving 120 patients (infected patients and negative subjects). However, little is known about whether the different variants could influence variations in the composition of the nasopharyngeal microbiota. In this study, we used multiplex pathogen-specific PCR to analyse the presence of nasopharyngeal bacterial pathogens from 400 SARS-CoV-2 positive patients (equally distributed in the four SARS-CoV-2 variants studied: B.1.1.7 (Alpha), B.1 0.617.2 (Delta), B.1.160 (Marseille-4), and B.1.1.529 (omicron)). We then compared them to 400 patients who tested negative for all respiratory viruses tested in this study, including SARS-CoV-2. We first observed an enrichment of Staphylococcus aureus (P ≤ .05) and Corynebacterium propinquum (P ≤ .05) in COVID-19-positive patients, regardless of the variant, compared to negative subjects. We specifically highlighted a significantly higher frequency of S. aureus (P ≤ .0001), C. propinquum (P ≤ .0001), and Klebsiella pneumoniae (P ≤ .0001), in patients infected with the omicron variant, whereas that of Haemophilus influenzae was higher in patients infected with Marseille-4 (P ≤ .001) and Alpha (P ≤ .01) variants. Our results suggest that the nasopharyngeal bacterial pathogens have their own specificity according to the SARS-CoV-2 variant and independently of the season. Additional studies are needed to determine the role of these pathogens in the evolution of the clinical outcome of patients.

Staphylococcal Resistance Patterns, blaZ and SCCmec Cassette Genes in the Nasopharyngeal Microbiota of Pregnant Women.

Antimicrobial resistance in Staphylococcus spp. colonising the nasopharynx can create risk factors of therapeutic treatment failure or prophylaxis in pregnant women. Resistance is mostly encoded on plasmids (e.g., blaZ gene for penicillinase synthesis) or chromosomes (e.g., mecA and mecC for methicillin resistance). The mecA gene is part of the chromosomal mec gene cassette (SCCmec), which is also located on the plasmid. The disc diffusion method for the selected drugs (beta-lactams, fluoroquinolones, streptogramins, aminoglicosides, macrolides, oxasolidinones, tetracyclines and other groups) was used. PCR for blaZ, mecA and mecC genes and SCCmec cassette detection and typing were performed. S. aureus (54.4%) and S. epidermidis (27.9%) were the most prevalent and showed the highest diversity of resistance profiles. The blaZ, mecA and mecC genes were reported in 95.6%, 20.6% and 1.5% of isolates, respectively. The highest resistance was found to beta-lactams, commonly used during pregnancy. Resistance to a variety of antimicrobials, including benzylpenicillin resistance in blaZ-positive isolates, and the existence of a very high diversity of SCCmec cassette structures in all staphylococci selected from the nasopharyngeal microbiota of pregnant women were observed for the first time. Knowledge of the prevalence of antimicrobial-resistant staphylococci in the nasopharynx of pregnant women may be important for the appropriate treatment or prophylaxis of this group of patients.

The nasopharyngeal microbiota of preweaned dairy calves with and without ultrasonographic lung lesions.

The primary objective of this cross-sectional study was to identify associations between the diversity and composition of the nasopharyngeal (NP) microbiota and pneumonia status, as diagnosed by ultrasonography (US), in preweaned dairy calves. Characteristics of the NP microbiota were compared between calves with and without pneumonia, as diagnosed by US. Secondary objectives were to compare the composition of the NP microbiota between calves by age, clinical respiratory score (CRS), and previous antibiotic therapy. Holstein heifer calves (n = 50) from a southern Wisconsin dairy were enrolled at either 3 or 6 wk of age; 4 calves were sampled at both time points. Antibiotic treatment history was also collected for the 30 d before enrollment. For the purpose of this study, pneumonia was defined as having lobar pneumonia, as diagnosed by US, in at least 1 lung lobe. Following examination by CRS and US, a deep nasopharyngeal swab was obtained for 16S rRNA amplicon sequencing. Alpha diversity was reduced in calves that were CRS positive, and beta diversity tended to be different in calves previously treated with antibiotics and in calves that were CRS positive. Microbial diversity was not different between calves with and without pneumonia. The most dominant genus identified was Mycoplasma spp.; however, there was no association between relative abundance (RA) and pneumonia status. The median RA of Mycoplasma spp. was increased by 25 (95% confidence interval, CI: 3, 40) in calves at 3 wk of age compared with 6 wk of age. The median RA of Pasteurella spp. was increased by 1.5 (95% CI: 0.1, 3) in calves with pneumonia, as diagnosed by US, compared with calves without pneumonia. Additionally, Pasteurella spp. was increased by 2.3 (95% CI: 0, 9) in CRS-positive calves compared with CRS-negative calves. The median RA of Psychrobacter spp. was increased by 2 (95% CI: 0, 12) and median RA of Chryseobacterium spp. was increased by 0.15 (95% CI: 0, 2) in calves that were not treated previously with antibiotics compared with calves previously treated with antibiotics.

The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.

BACKGROUND: The nasopharyngeal (NP) microbiota plays an important role in bovine health, comprising a rich and diverse microbial community. The nasopharynx is also the niche for potentially pathogenic agents which are associated with bovine respiratory disease (BRD), a serious and costly illness in feedlot cattle. We used 14 beef heifers from a closed and disease-free herd to assess the dynamics of the NP microbiota of cattle that are transported to a feedlot. Cattle were sampled prior to transport to the feedlot (day 0) and at days 2, 7, and 14. RESULTS: The structure of the NP microbiota changed significantly over the course of the study, with the largest shift occurring between day 0 (prior to transport) and day 2 (P < 0.001). Phylogenetic diversity and richness increased following feedlot placement (day 2; P < 0.05). The genera Pasteurella, Bacillus, and Proteus were enriched at day 0, Streptococcus and Acinetobacter at day 2, Bifidobacterium at day 7, and Mycoplasma at day 14. The functional potential of the NP microbiota was assessed using PICRUSt, revealing that replication and repair, as well as translation pathways, were more relatively abundant in day 14 samples. These differences were driven mostly by Mycoplasma. Although eight cattle were culture-positive for the BRD-associated bacterium Pasteurella multocida at one or more sampling times, none were culture-positive for Mannheimia haemolytica or Histophilus somni. CONCLUSIONS: This study investigated the effect that feedlot placement has on the NP microbiota of beef cattle over a 14-d period. Within two days of transport to the feedlot, the NP microbiota changed significantly, increasing in both phylogenetic diversity and richness. These results demonstrate that there is an abrupt shift in the NP microbiota of cattle after transportation to a feedlot. This may have importance for understanding why cattle are most susceptible to BRD after feedlot placement.

Seven-valent pneumococcal conjugate vaccine and nasopharyngeal microbiota in healthy children.

Seven-valent pneumococcal conjugate vaccine (PCV-7) is effective against vaccine serotype disease and carriage. Nevertheless, shifts in colonization and disease toward nonvaccine serotypes and other potential pathogens have been described. To understand the extent of these shifts, we analyzed nasopharyngeal microbial profiles of 97 PCV-7-vaccinated infants and 103 control infants participating in a randomized controlled trial in the Netherlands. PCV-7 immunization resulted in a temporary shift in microbial community composition and increased bacterial diversity. Immunization also resulted in decreased presence of the pneumococcal vaccine serotype and an increase in the relative abundance and presence of nonpneumococcal streptococci and anaerobic bacteria. Furthermore, the abundance of Haemophilus and Staphylococcus bacteria in vaccinees was increased over that in controls. This study illustrates the much broader effect of vaccination with PCV-7 on the microbial community than currently assumed, and highlights the need for careful monitoring when implementing vaccines directed against common colonizers.

Gut and respiratory tract microbiota in children younger than 12 months hospitalized for bronchiolitis compared with healthy children: can we predict the severity and medium-term respiratory outcome?

Growing evidence indicates that gut and respiratory microbiota have a potential key effect on bronchiolitis, mainly caused by respiratory syncytial virus (RSV). This was a prospective study of 96 infants comparing infants with bronchiolitis (n = 57, both RSV and non-RSV associated) to a control group (n = 39). Gut (feces) and respiratory [nasopharyngeal aspirate (NPA)] microbial profiles were analyzed by 16S rRNA amplicon sequencing, and respiratory viruses were identified by PCR. Clinical data of the acute episode and follow-up during the first year after infection were recorded. Pairwise comparisons showed significant differences in the gut (R2 = 0.0639, P = 0.006) and NPA (R2 = 0.0803, P = 0.006) microbiota between cases and controls. A significantly lower gut microbial richness and an increase in the NPA microbial diversity (mainly due to an increase in Haemophilus, Streptococcus, and Neisseria) were observed in the infants with bronchiolitis, in those with the most severe symptoms, and in those who subsequently developed recurrent wheezing episodes after discharge. In NPA, the higher microbial richness differed significantly between the control group and the non-RSV bronchiolitis group (P = 0.01) and between the control group and the RSV bronchiolitis group (P = 0.001). In the gut, the richness differed significantly between the control group and the non-RSV group (P = 0.01) and between the control group and the RSV bronchiolitis group (P = 0.001), with higher diversity in the RSV group. A distinct respiratory and intestinal microbial pattern was observed in infants with bronchiolitis compared with controls. The presence of RSV was a main factor for dysbiosis. Lower gut microbial richness and increased respiratory microbial diversity were associated with respiratory morbidity during follow-up. IMPORTANCE: Both the intestinal and respiratory microbiota of children with bronchiolitis, especially those with respiratory syncytial virus infection, are altered and differ from that of healthy children. The microbiota pattern in the acute episode could identify those children who will later have other respiratory episodes in the first year of life. Preventive measures could be adopted for this group of infants.

Molecular surveillance of influenza A virus in Saudi Arabia: whole-genome sequencing and metagenomic approaches.

Outbreaks of influenza A viruses are generally seasonal and cause annual epidemics worldwide. Due to their frequent reassortment and evolution, annual surveillance is of paramount importance to guide vaccine strategies. The aim of this study was to explore the molecular epidemiology of influenza A virus and nasopharyngeal microbiota composition in infected patients in Saudi Arabia. A total of 103 nasopharyngeal samples from 2015 and 12 samples from 2022 were collected from patients positive for influenza A. Sequencing of influenza A as well as metatranscriptomic analysis of the nasopharyngeal microbiota was conducted using Oxford Nanopore sequencing. Phylogenetic analysis of hemagglutinin, neuraminidase segments, and concatenated influenza A genomes was performed using MEGA7. Whole-genome sequencing analysis revealed changing clades of influenza A virus: from 6B.1 in 2015 to 5a.2a in 2022. One sample containing the antiviral resistance-mediating mutation S247N toward oseltamivir and zanamivir was found. Phylogenetic analysis showed the clustering of influenza A strains with the corresponding vaccine strains in each period, thus suggesting vaccine effectiveness. Principal component analysis and alpha diversity revealed the absence of a relationship between hospital admission status, age, or gender of infected patients and the nasopharyngeal microbial composition, except for the infecting clade 5a.2a. The opportunistic pathogens Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were the most common species detected. The molecular epidemiology appears to be changing in Saudi Arabia after the COVID-19 pandemic. Antiviral resistance should be carefully monitored in future studies. In addition, the disease severity of patients as well as the composition of the nasopharyngeal microbiota in patients infected with different clades should also be assessed.IMPORTANCEIn this work, we have found that the clade of influenza A virus circulating in Riyadh, KSA, has changed over the last few years from 6B.1 to 5a.2a. Influenza strains clustered with the corresponding vaccine strains in our population, thus emphasizing vaccine effectiveness. Metatranscriptomic analysis showed no correlation between the nasopharyngeal microbiome and the clinical and/or demographic characteristics of infected patients. This is except for the 5a.2a strains isolated post-COVID-19 pandemic. The influenza virus is among the continuously evolving viruses that can cause severe respiratory infections. Continuous surveillance of its molecular diversity and the monitoring of anti-viral-resistant strains are thus of vital importance. Furthermore, exploring potential microbial markers and/or dysbiosis of the nasopharyngeal microbiota during infection could assist in the better management of patients in severe cases.

Exploring nasopharyngeal microbiota profile in children affected by SARS-CoV-2 infection.

The relationship between COVID-19 and nasopharyngeal (NP) microbiota has been investigated mainly in the adult population. We explored the NP profile of children affected by COVID-19, compared to healthy controls (CTRLs). NP swabs of children with COVID-19, collected between March and September 2020, were investigated at the admission (T0), 72 h to 7 days (T1), and at the discharge (T2) of the patients. NP microbiota was analyzed by 16S rRNA targeted-metagenomics. Data from sequencing were investigated by QIIME 2.0 and PICRUSt 2. Multiple machine learning (ML) models were exploited to classify patients compared to CTRLs. The NP microbiota of COVID-19 patients (N = 71) was characterized by reduction of α-diversity compared to CTRLs (N = 59). The NP microbiota of COVID-19 cohort appeared significantly enriched in Streptococcus, Haemophilus, Staphylococcus, Veillonella, Enterococcus, Neisseria, Moraxella, Enterobacteriaceae, Gemella, Bacillus, and reduced in Faecalibacterium, Akkermansia, Blautia, Bifidobacterium, Ruminococcus, and Bacteroides, compared to CTRLs (FDR < 0.001). Exploiting ML models, Enterococcus, Pseudomonas, Streptococcus, Capnocytopagha, Tepidiphilus, Porphyromonas, Staphylococcus, and Veillonella resulted as NP microbiota biomarkers, in COVID-19 patients. No statistically significant differences were found comparing the NP microbiota profile of COVID-19 patients during the time-points or grouping patients on the basis of high, medium, and low viral load (VL). This evidence provides specific pathobiont signatures of the NP microbiota in pediatric COVID-19 patients, and the reduction of anaerobic protective commensals. Our data suggest that the NP microbiota may have a specific disease-related signature since infection onset without changes during disease progression, regardless of the SARS-CoV-2 VL. IMPORTANCE: Since the beginning of pandemic, we know that children are less susceptible to severe COVID-19 disease. A potential role of the nasopharyngeal (NP) microbiota has been hypothesized but to date, most of the studies have been focused on adults. We studied the NP microbiota modifications in children affected by SARS-CoV-2 infection showing a specific NP microbiome profile, mainly composed by pathobionts and almost missing protective anaerobic commensals. Moreover, in our study, specific microbial signatures appear since the first days of infection independently from SARS-CoV-2 viral load.

Screening and selection of essential oils for an intranasal spray against bovine respiratory pathogens based on antimicrobial, antiviral, immunomodulatory, and antibiofilm activities.

Introduction: The rise in antibiotic resistant pathogens associated with bovine respiratory disease (BRD) poses a serious challenge, particularly to the beef feedlot industry, as they currently depend on antibiotics to prevent BRD to mitigate the financial burden (approx. $1 billion annual loss) inflicted by BRD-associated high mortality and morbidity in feedlot cattle. Thus, there is an impetus need for the development of antimicrobial alternative strategies against BRD. This study aimed to screen and select candidate essential oils (EOs) for the development of an intranasal EO spray that can inhibit BRD pathogens and promote microbiota-mediated respiratory health. Methods: The effects of selected EOs (ajowan, cinnamon leaf, citronella, grapefruit, fennel, and thyme) on a bovine nasopharyngeal microbiota culture were evaluated using 16S rRNA gene sequencing. The microbiota culture was enriched by incubating nasopharyngeal swabs obtained from finishing beef heifers in brain heart infusion broth with and without EOs (0.025%, v/v). These EOs were then also evaluated for their immunomodulatory effects on bovine turbinate (BT) cells by analyzing the concentrations of 15 cytokines and chemokines in cell culture after 24 h incubation. The crystal violet assay was done to assess the antibiofilm activity of EOs against Escherichia coli UMN026 strain. Finally, 15 EOs were screened for their antiviral activity against the bovine viral diarrhea virus 1 (BVDV-1) using BT cells and a fluorescence-based method. Results: Ajowan, fennel, and thyme resulted in a moderate reduction of overall nasopharyngeal microbiota growth with significant alterations of both alpha and beta diversity, and the relative abundance of predominant bacterial families (e.g., increasing Enterobacteriaceae and decreasing Moraxellaceae) compared to the control (p < 0.05). Co-incubation of BT cells with selected EOs resulted in minimal alterations in cytokine and chemokine levels (p > 0.05). Ajowan, thyme, fennel, and cinnamon leaf exhibited antibiofilm activity at concentrations of 0.025 and 0.05%. Reduction of BVDV-1 replication in BT cells was observed with thyme (strong), and ajowan and citronella (moderate) at 0.0125% concentration. Discussion: Accordingly, ajowan, thyme, fennel, cinnamon leaf, and citronella EOs were selected for further development as an intranasal EO spray to prevent and control of BRD pathogens in feedlot cattle.

Analysis of Intestinal and Nasopharyngeal Microbiota of Children with Meningococcemia in Pediatric Intensive Care Unit: INMACS-PICU Study.

Microbiota composition might play a role in the pathophysiology and course of sepsis, and understanding its dynamics is of clinical interest. Invasive meningococcal disease (IMD) is an important cause of community-acquired serious infection, and there is no information regarding microbiota composition in children with meningococcemia. In this study, we aimed to evaluate the intestinal and nasopharyngeal microbiota composition of children with IMD. Materials and Methods: In this prospective, multi-center study, 10 children with meningococcemia and 10 age-matched healthy controls were included. Nasopharyngeal and fecal samples were obtained at admission to the intensive care unit and on the tenth day of their hospital stay. The V3 and V4 regions of the 16S rRNA gene were amplified following the 16S Metagenomic Sequencing Library Preparation. Results: Regarding the alpha diversity on the day of admission and on the tenth day at the PICU, the Shannon index was significantly lower in the IMD group compared to the control group (p = 0.002 at admission and p = 0.001, on the tenth day of PICU). A statistical difference in the stool samples was found between the IMD group at Day 0 vs. the controls in the results of the Bray-Curtis and Jaccard analyses (p = 0.005 and p = 0.001, respectively). There were differences in the intestinal microbiota composition between the children with IMD at admission and Day 10 and the healthy controls. Regarding the nasopharyngeal microbiota analysis, in the children with IMD at admission, at the genus level, Neisseria was significantly more abundant compared to the healthy children (p < 0.001). In the children with IMD at Day 10, genera Moraxella and Neisseria were decreased compared to the healthy children. In the children with IMD on Day 0, for paired samples, Moraxella, Neisseria, and Haemophilus were significantly more abundant compared to the children with IMD at Day 10. In the children with IMD at Day 10, the Moraxella and Neisseria genera were decreased, and 20 different genera were more abundant compared to Day 0. Conclusions: We first found alterations in the intestinal and nasopharyngeal microbiota composition in the children with IMD. The infection itself or the other care interventions also caused changes to the microbiota composition during the follow-up period. Understanding the interaction of microbiota with pathogens, e.g., N. meningitidis, could give us the opportunity to understand the disease's dynamics.

Disentangling the interactions between nasopharyngeal and gut microbiome and their involvement in the modulation of COVID-19 infection.

The human organism is inhabited by trillions of microorganisms, known as microbiota, which are considered to exploit a pivotal role in the regulation of host health and immunity. Recent investigations have suggested a relationship between the composition of the human microbiota and COVID-19 infection, highlighting a possible role of bacterial communities in the modulation of the disease severity. In this study, we performed a shotgun metagenomics analysis to explore and compare the nasopharyngeal microbiota of 38 hospitalized Italian patients with and without COVID-19 infection during the third and fourth pandemic waves. In detail, the metagenomic analysis combined with specific correlation analyses suggested a positive association of several microbial species, such as S. parasanguinis and P. melaninogenica, with the severity of COVID-19 infection. Furthermore, the comparison of the microbiota composition between the nasopharyngeal and their respective fecal samples highlighted an association between these different compartments represented by a sharing of several bacterial species. Additionally, lipidomic and deep-shotgun functional analyses of the fecal samples suggested a metabolic impact of the microbiome on the host's immune response, indicating the presence of key metabolic compounds in COVID-19 patients, such as lipid oxidation end products, potentially related to the inflammatory state. Conversely, the patients without COVID-19 displayed enzymatic patterns associated with the biosynthesis and degradation of specific compounds like lysine (synthesis) and phenylalanine (degradation) that could positively impact disease severity and contribute to modulating COVID-19 infection. IMPORTANCE The human microbiota is reported to play a major role in the regulation of host health and immunity, suggesting a possible impact on the severity of COVID-19 disease. This preliminary study investigated the possible correlation between nasopharyngeal microbiota and COVID-19 infection. In detail, the analysis of the nasopharyngeal microbiota of hospitalized Italian patients with and without COVID-19 infection suggested a positive association of several microbial species with the severity of the disease and highlighted a sharing of several bacteria species with the respective fecal samples. Moreover, the metabolic analyses suggested a possible impact of the microbiome on the host's immune response and the disease severity.

Nasopharyngeal Bacterial Prevalence and Microbial Diversity at First Treatment for Bovine Respiratory Disease (BRD) and Its Associations with Health and Mortality Outcomes in Feedyard Cattle.

Bovine respiratory disease (BRD) is an economically important disease in feedyards influencing both animal welfare and antimicrobial utilization. Major pathogens associated with BRD have been identified in previous research, but little information is available on the relationship between nasopharyngeal microbiota and health outcomes. The objective of this study was to identify potential associations between nasopharyngeal microbiota and antimicrobial resistance patterns of clinical cases that lived or died compared to non-diseased controls. Enrolled animals were subdivided based on clinical disease status and case outcome (subsequent mortality). Deep nasopharyngeal swabs were collected on enrolled animals and submitted for bacterial isolation, antimicrobial susceptibility determination, and metagenomics analysis. Enrolled cattle were represented in three groups: animals at first treatment for BRD that subsequently died (BRDM, n = 9), animals at first treatment for BRD that subsequently lived (BRDL, n = 15), and animals that were never treated for BRD during the feeding phase (CONT, n = 11). Antimicrobial resistance patterns for Pasteurella multocida illustrated cattle in each outcome category had isolates that were pan-susceptible or only showed resistance to oxytetracycline. The relative abundance of species and genera illustrated few differences among the three outcomes. Higher alpha diversity was identified in BRDL compared to CONT at the species level, and both BRDL and BRDM showed increased alpha diversity compared to CONT at the general level. Overall, this work illustrated nasopharyngeal microbiota showed relatively few differences among BRD cases that lived or died compared to animals without BRD.

The nasopharyngeal microbiome in COVID-19.

The development of novel culture-independent techniques of microbial identification has allowed a rapid progress in the knowledge of the nasopharyngeal microbiota and its role in health and disease. Thus, it has been demonstrated that the nasopharyngeal microbiota defends the host from invading pathogens that enter the body through the upper airways by participating in the modulation of innate and adaptive immune responses. The current COVID-19 pandemic has created an urgent need for fast-track research, especially to identify and characterize biomarkers to predict the disease severity and outcome. Since the nasopharyngeal microbiota diversity and composition could potentially be used as a prognosis biomarker for COVID-19 patients, which would pave the way for strategies aiming to reduce the disease severity by modifying such microbiota, dozens of research articles have already explored the possible associations between changes in the nasopharyngeal microbiota and the severity or outcome of COVID-19 patients. Unfortunately, results are controversial, as many studies with apparently similar experimental designs have reported contradictory data. Herein we put together, compare, and discuss all the relevant results on this issue reported to date. Even more interesting, we discuss in detail which are the limitations of these studies, that probably are the main sources of the high variability observed. Therefore, this work is useful not only for people interested in current knowledge about the relationship between the nasopharyngeal microbiota and COVID-19, but also for researchers who want to go further in this field while avoiding the limitations and variability of previous works.

Gut, oral, and nasopharyngeal microbiota dynamics in the clinical course of hospitalized infants with respiratory syncytial virus bronchiolitis.

Introduction: Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and hospitalization in infants worldwide. The nasopharyngeal microbiota has been suggested to play a role in influencing the clinical course of RSV bronchiolitis, and some evidence has been provided regarding oral and gut microbiota. However, most studies have focused on a single timepoint, and none has investigated all three ecosystems at once. Methods: Here, we simultaneously reconstructed the gut, oral and nasopharyngeal microbiota dynamics of 19 infants with RSV bronchiolitis in relation to the duration of hospitalization (more or less than 5 days). Fecal samples, oral swabs, and nasopharyngeal aspirates were collected at three timepoints (emergency room admission, discharge and six-month follow-up) and profiled by 16S rRNA amplicon sequencing. Results: Interestingly, all ecosystems underwent rearrangements over time but with distinct configurations depending on the clinical course of bronchiolitis. In particular, infants hospitalized for longer showed early and persistent signatures of unhealthy microbiota in all ecosystems, i.e., an increased representation of pathobionts and a depletion of typical age-predicted commensals. Discussion: Monitoring infant microbiota during RSV bronchiolitis and promptly reversing any dysbiotic features could be important for prognosis and long-term health.

Prevalence of Culturable Bacteria and Yeasts in the Nasopharynx Microbiota during the Physiological Course of Pregnancy.

The aim of the study was to compare the prevalence of the nasopharyngeal carriage of culturable microorganisms in the microbiota of asymptomatic women with a physiological pregnancy (PW) and nonpregnant women (NPW). Nasopharyngeal swabs were collected from 53 PW and 30 NPW to detect bacterial and fungal colonization. Isolates were identified using the culture method and the MALDI-TOF MS technique. The nasopharyngeal microbiota (NPM) partially differed between PW and NPW. These differences in the frequency of nasopharyngeal colonization between the PW and NPW groups were not statistically significant (p > 0.05); all cases were colonized by bacteria and only two cases in the PW group were colonized by yeasts, namely, Rhodotorula spp. High levels of staphylococcal colonization, including predominantly coagulase-negative staphylococci and S. aureus in the nasopharyngeal sample, were present in both groups. The reduced number of Gram-negative rods colonized in the cases studied was seen in samples from the NPW group, particularly with Enterobacterales, and anaerobic Cutibacterium spp. were isolated only in the PW group (p < 0.05). Moreover, a higher carriage rate of Enterobacter aerogenes colonization was statistically significant (p < 0.05) and correlated with the NPW group. Pregnancy may disturb the composition of the NPM represented by commensals and opportunistic bacteria and promote yeast colonization as compared to nonpregnant women.

Impact of COVID-19 Lockdown on the Nasopharyngeal Microbiota of Children and Adults Self-Confined at Home.

The increased incidence of COVID-19 cases and deaths in Spain in March 2020 led to the declaration by the Spanish government of a state of emergency imposing strict confinement measures on the population. The objective of this study was to characterize the nasopharyngeal microbiota of children and adults and its relation to SARS-CoV-2 infection and COVID-19 severity during the pandemic lockdown in Spain. This cross-sectional study included family households located in metropolitan Barcelona, Spain, with one adult with a previous confirmed COVID-19 episode and one or more exposed co-habiting child contacts. Nasopharyngeal swabs were used to determine SARS-CoV-2 infection status, characterize the nasopharyngeal microbiota and determine common respiratory DNA/RNA viral co-infections. A total of 173 adult cases and 470 exposed children were included. Overall, a predominance of Corynebacterium and Dolosigranulum and a limited abundance of common pathobionts including Haemophilus and Streptococcus were found both among adults and children. Children with current SARS-CoV-2 infection presented higher bacterial richness and increased Fusobacterium, Streptococcus and Prevotella abundance than non-infected children. Among adults, persistent SARS-CoV-2 RNA was associated with an increased abundance of an unclassified member of the Actinomycetales order. COVID-19 severity was associated with increased Staphylococcus and reduced Dolosigranulum abundance. The stringent COVID-19 lockdown in Spain had a significant impact on the nasopharyngeal microbiota of children, reflected in the limited abundance of common respiratory pathobionts and the predominance of Corynebacterium, regardless of SARS-CoV-2 detection. COVID-19 severity in adults was associated with decreased nasopharynx levels of healthy commensal bacteria.

Effect of intra-partum azithromycin on the development of the infant nasopharyngeal microbiota: A post hoc analysis of a double-blind randomized trial.

BACKGROUND: Sepsis is a leading cause of neonatal death. Intrapartum azithromycin reduces neonatal nasopharyngeal carriage of potentially pathogenic bacteria, a prerequisite for sepsis. Early antibiotic exposure has been associated with microbiota perturbations with varying effects. This study aims to understand the effect of intrapartum azithromycin intervention on the developing nasopharyngeal microbiota of the child. METHODS: Using 16S rRNA gene sequencing, we analysed the microbiota of 343 nasopharyngeal samples collected from birth to 12 months from 109 healthy infants selected from a double-blind randomized placebo-controlled clinical trial conducted in the Gambia (PregnAnZI-1). In the trial, 829 women were given 2g oral azithromycin or placebo (1:1) during labour with the objective of reducing bacterial carriage in mother and child during the neonatal period. The post-hoc analysis presented here assessed the effect of the intervention on the child nasopharyngeal microbiota development. FINDINGS: 55 children were from mothers given azithromycin and 54 from mothers given placebo. Comparing arms, we found an increase in alpha-diversity at day-6 (p = 0·018), and a significant effect on overall microbiota composition at days 6 and 28 (R2 = 4.4%, q = 0·007 and R2 = 2.3%, q = 0·018 respectively). At genus level, we found lower representation of Staphylococcus at day-6 (q = 0·0303) and higher representation of Moraxella at 12 months (q = 0·0443). Unsupervised clustering of samples by microbial community similarity showed different community dynamics between the intervention and placebo arms during the neonatal period. INTERPRETATION: These results indicate that intrapartum azithromycin caused short-term alterations in the nasopharyngeal microbiota with modest overall effect at 12 months of age. Further exploration of the effects of these variations on microbiome function will give more insight on the potential risks and benefits, for the child, associated with this intervention. FUNDING: This work was jointly funded by the Medical Research Council (UK) (MC_EX_MR/J010391/1/MRC), Bill & Melinda Gates Foundation (OPP1196513), and MRCG@LSHTM Doctoral Training Program.

Super Dominant Pathobiontic Bacteria in the Nasopharyngeal Microbiota Cause Secondary Bacterial Infection in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease responsible for many infections worldwide. Differences in respiratory microbiota may correlate with disease severity. Samples were collected from 20 severe and 51 mild COVID-19 patients. High-throughput sequencing of the 16S rRNA gene was used to analyze the bacterial community composition of the upper and lower respiratory tracts. The indices of diversity were analyzed. When one genus accounted for >50% of reads from a sample, it was defined as a super dominant pathobiontic bacterial genus (SDPG). In the upper respiratory tract, uniformity indices were significantly higher in the mild group than in the severe group (P < 0.001). In the lower respiratory tract, uniformity indices, richness indices, and the abundance-based coverage estimator were significantly higher in the mild group than in the severe group (P < 0.001). In patients with severe COVID-19, SDPGs were detected in 40.7% of upper and 63.2% of lower respiratory tract samples. In patients with mild COVID-19, only 10.8% of upper and 8.5% of lower respiratory tract samples yielded SDPGs. SDPGs were present in both upper and lower tracts in seven patients (35.0%), among which six (30.0%) patients possessed the same SDPG in the upper and lower tracts. However, no patients with mild infections had an SDPG in both tracts. Staphylococcus, Corynebacterium, and Acinetobacter were the main SDPGs. The number of SDPGs identified differed significantly between patients with mild and severe COVID-19 (P < 0.001). SDPGs in nasopharyngeal microbiota cause secondary bacterial infection in COVID-19 patients and aggravate pneumonia. IMPORTANCE The nasopharyngeal microbiota is composed of a variety of not only the true commensal bacterial species but also the two-face pathobionts, which are one a harmless commensal bacterial species and the other a highly invasive and deadly pathogen. In a previous study, we found that the diversity of nasopharyngeal microbiota was lost in severe influenza patients. We named the genus that accounted for over 50% of microbiota abundance as super dominant pathobiontic genus, which could invade to cause severe pneumonia, leading to high fatality. Similar phenomena were found here for SARS-CoV-2 infection. The diversity of nasopharyngeal microbiota was lost in severe COVID-19 infection patients. SDPGs in nasopharyngeal microbiota were frequently detected in severe COVID-19 patients. Therefore, the SDPGs in nasopharynx microbiota might invade into low respiratory and be responsible for secondary bacterial pneumonia in patients with SARS-CoV-2 infection.

Alloiococcus otitidis-Cause of Nonspecific Acute Sinusitis: First Case Report and Review of Literature.

Although most sinus infections are viral, potential bacterial pathogens such as Streptococcus pneumoniae, Haemophilus influenza and Moraxella catarrhalis can migrate during a viral respiratory infection from the nasopharynx into the sinus cavity causing sinusitis. Alloiococcus otitidis is a commensal of the external auditory canal and is considered one of the potential middle ear pathogens. Unlike most otopathogens, A. otitidis is rarely found in the nasopharynx of healthy individuals. This difficult-to-culture organism has not previously been described as a causative agent of sinusitis. Here we describe one case of acute sinusitis due to A. otitidis and review previous knowledge of this controversial organism based on recent literature.

Impact of Pneumococcal Vaccination on Nasopharyngeal Carriage of Streptococcus pneumoniae and Microbiota Profiles in Preschool Children in South East Poland.

In 2017, Poland introduced the 10-valent pneumococcal conjugate vaccine (PCV) into its national immunization schedule. This prospective study was conducted between March and June 2020 to determine the impact of vaccination on prevalence of the nasopharyngeal carriage of S. pneumoniae in 176 healthy children and to determine how conjugate vaccines indirectly affect colonization of nasopharyngeal microbiota. Pneumococcal isolates were analyzed by serotyping and antimicrobial resistance tests. Nasopharyngeal microbiota were detected and identified using the culture method and real-time PCR amplification primers and hydrolysis-probe detection with the 16S rRNA gene as the target. In the vaccinated group of children, colonization was in 24.2% of children, compared to 21.4% in the unvaccinated group. Serotypes 23A and 23B constituted 41.5% of the isolates. Serotypes belonging to PCV10 and PCV13 constituted 4.9% and 17.1% of the isolates, respectively. S. pneumoniae isolates were resistant to penicillin (34.1%), erythromycin (31.7%), and co-trimoxazole (26.8%). Microbial DNA qPCR array correlated to increased amounts of Streptococcus mitis and S. sanguinis in vaccinated children, with reduced amounts of C. pseudodiphtericum, S. aureus, and M. catarrhalis. Introduction of PCV for routine infant immunization was associated with significant reductions in nasopharyngeal carriage of PCV serotypes and resistant strains amongst vaccine serotypes, yet carriage of non-PCV serotypes increased modestly, particularly serotype 23B.