Informed interpretation of metagenomic data by StrainPhlAn enables strain retention analyses of the upper airway microbiome.

IMPORTANCE: The usage of 16S rRNA gene sequencing has become the state-of-the-art method for the characterization of the microbiota in health and respiratory disease. The method is reliable for low biomass samples due to prior amplification of the 16S rRNA gene but has limitations as species and certainly strain identification is not possible. However, the usage of metagenomic tools for the analyses of microbiome data from low biomass samples is not straight forward, and careful optimization is needed. In this work, we show that by validating StrainPhlAn 3 results with the data from bacterial cultures, the strain-level tracking of the respiratory microbiome is feasible despite the high content of host DNA being present when parameters are carefully optimized to fit low biomass microbiomes. This work further proposes that strain retention analyses are feasible, at least for more abundant species. This will help to better understand the longitudinal dynamics of the upper respiratory microbiome during health and disease.

A distinct clade of Bifidobacterium longum in the gut of Bangladeshi children thrives during weaning.

The gut microbiome has an important role in infant health and development. We characterized the fecal microbiome and metabolome of 222 young children in Dhaka, Bangladesh during the first two years of life. A distinct Bifidobacterium longum clade expanded with introduction of solid foods and harbored enzymes for utilizing both breast milk and solid food substrates. The clade was highly prevalent in Bangladesh, present globally (at lower prevalence), and correlated with many other gut taxa and metabolites, indicating an important role in gut ecology. We also found that the B. longum clades and associated metabolites were implicated in childhood diarrhea and early growth, including positive associations between growth measures and B. longum subsp. infantis, indolelactate and N-acetylglutamate. Our data demonstrate geographic, cultural, seasonal, and ecological heterogeneity that should be accounted for when identifying microbiome factors implicated in and potentially benefiting infant development.

The Influence of FUT2 and FUT3 Polymorphisms and Nasopharyngeal Microbiome on Respiratory Infections in Breastfed Bangladeshi Infants from the Microbiota and Health Study.

Acute respiratory infections (ARIs) are one of the most common causes of morbidity and mortality in young children. The aim of our study was to examine whether variation in maternal FUT2 (α1,2-fucosyltransferase 2) and FUT3 (α1,3/4-fucosyltransferase 3) genes, which shape fucosylated human milk oligosaccharides (HMOs) in breast milk, are associated with the occurrence of ARIs in breastfed infants as well as the influence of the nasopharyngeal microbiome on ARI risk. Occurrences of ARIs were prospectively recorded in a cohort of 240 breastfed Bangladeshi infants from birth to 2 years. Secretor and Lewis status was established by sequencing of FUT2/3 genes. The nasopharyngeal microbiome was characterized by shotgun metagenomics, complemented by specific detection of respiratory pathogens; 88.6% of mothers and 91% of infants were identified as secretors. Maternal secretor status was associated with reduced ARI incidence among these infants in the period from birth to 6 months (incidence rate ratio [IRR], 0.66; 95% confidence interval [CI], 0.47 to 0.94; P = 0.020), but not at later time periods. The nasopharyngeal microbiome, despite precise characterization to the species level, was not predictive of subsequent ARIs. The observed risk reduction of ARIs among infants of secretor mothers during the predominant breastfeeding period is consistent with the hypothesis that fucosylated oligosaccharides in human milk contribute to protection against respiratory infections. However, we found no evidence that modulation of the nasopharyngeal microbiome influenced ARI risk. IMPORTANCE The observed risk reduction of acute respiratory infections (ARIs) among infants of secretor mothers during the predominant breastfeeding period is consistent with the hypothesis that fucosylated oligosaccharides in human milk contribute to protection against respiratory infections. Respiratory pathogens were only weak modulators of risk, and the nasopharyngeal microbiome did not influence ARI risk, suggesting that the associated protective effects of human milk oligosaccharides (HMOs) are not conveyed via changes in the nasopharyngeal microbiome. Our observations add to the evidence for a role of fucosylated HMOs in protection against respiratory infections in exclusively or predominantly breastfed infants in low-resource settings. There is no indication that the nasopharyngeal microbiome substantially modulates the risk of subsequent mild ARIs. Larger studies are needed to provide mechanistic insights on links between secretor status, HMOs, and risk of respiratory infections.

Nurturing the Early Life Gut Microbiome and Immune Maturation for Long Term Health.

Early life is characterized by developmental milestones such as holding up the head, turning over, sitting up and walking that are typically achieved sequentially in specific time windows. Similarly, the early gut microbiome maturation can be characterized by specific temporal microorganism acquisition, colonization and selection with differential functional features over time. This orchestrated microbial sequence occurs from birth during the first years of age before the microbiome reaches an adult-like composition and function between 3 and 5 years of age. Increasingly, these different steps of microbiome development are recognized as crucial windows of opportunity for long term health, primarily linked to appropriate immune and metabolic development. For instance, microbiome disruptors such as preterm and Cesarean-section birth, malnutrition and antibiotic use are associated with increased risk to negatively affect long-term immune and metabolic health. Different age discriminant microbiome taxa and functionalities are used to describe age-appropriate microbiome development, and advanced modelling techniques enable an understanding and visualization of an optimal microbiome maturation trajectory. Specific microbiome features can be related to later health conditions, however, whether such features have a causal relationship is the topic of intense research. Early life nutrition is an important microbiome modulator, and 'Mother Nature' provides the model with breast milk as the sole source of nutrition for the early postnatal period, while dietary choices during the prenatal and weaning period are to a large extent guided by tradition and culture. Increasing evidence suggests prenatal maternal diet and infant and child nutrition impact the infant microbiome trajectory and immune competence development. The lack of a universal feeding reference for such phases represents a knowledge gap, but also a great opportunity to provide adequate nutritional guidance to maintain an age-appropriate microbiome for long term health. Here, we provide a narrative review and perspective on our current understanding of age-appropriate microbiome maturation, its relation to long term health and how nutrition shapes and influences this relationship.

Bacterial Signatures of Paediatric Respiratory Disease: An Individual Participant Data Meta-Analysis.

Introduction: The airway microbiota has been linked to specific paediatric respiratory diseases, but studies are often small. It remains unclear whether particular bacteria are associated with a given disease, or if a more general, non-specific microbiota association with disease exists, as suggested for the gut. We investigated overarching patterns of bacterial association with acute and chronic paediatric respiratory disease in an individual participant data (IPD) meta-analysis of 16S rRNA gene sequences from published respiratory microbiota studies. Methods: We obtained raw microbiota data from public repositories or via communication with corresponding authors. Cross-sectional analyses of the paediatric (<18 years) microbiota in acute and chronic respiratory conditions, with >10 case subjects were included. Sequence data were processed using a uniform bioinformatics pipeline, removing a potentially substantial source of variation. Microbiota differences across diagnoses were assessed using alpha- and beta-diversity approaches, machine learning, and biomarker analyses. Results: We ultimately included 20 studies containing individual data from 2624 children. Disease was associated with lower bacterial diversity in nasal and lower airway samples and higher relative abundances of specific nasal taxa including Streptococcus and Haemophilus. Machine learning success in assigning samples to diagnostic groupings varied with anatomical site, with positive predictive value and sensitivity ranging from 43 to 100 and 8 to 99%, respectively. Conclusion: IPD meta-analysis of the respiratory microbiota across multiple diseases allowed identification of a non-specific disease association which cannot be recognised by studying a single disease. Whilst imperfect, machine learning offers promise as a potential additional tool to aid clinical diagnosis.

Changing Epidemiology of Acute Respiratory Infections in Under-Two Children in Dhaka, Bangladesh.

Objectives: Risk factors for acute respiratory infections (ARIs) in community settings are not fully understood, especially in low-income countries. We examined the incidence and risk factors associated with ARIs in under-two children from the Microbiota and Health study. Methods: Children from a peri-urban area of Dhaka (Bangladesh) were followed from birth to 2 years of age by both active surveillance of ARIs and regular scheduled visits. Nasopharyngeal samples were collected during scheduled visits for detection of bacterial facultative respiratory pathogens. Information on socioeconomic, environmental, and household conditions, and mother and child characteristics were collected. A hierarchical modeling approach was used to identify proximate determinants of ARIs. Results: Of 267 infants, 87.3% experienced at least one ARI episode during the first 2 years of life. The peak incidence of ARIs was 330 infections per 100 infant-years and occurred between 2 and 4 months of age. Season was the main risk factor (rainy monsoon season, incidence rate ratio [IRR] 2.43 [1.92-3.07]; cool dry winter, IRR 2.10 [1.65-2.67] compared with hot dry summer) in the first 2 years of life. In addition, during the first 6 months of life, young maternal age (<22 years; IRR 1.34 [1.01-1.77]) and low birth weight (<2,500 g; IRR 1.39 [1.03-1.89]) were associated with higher ARI incidence. Conclusions: Reminiscent of industrialized settings, cool rainy season rather than socioeconomic and hygiene conditions was a major risk factor for ARIs in peri-urban Bangladesh. Understanding the causal links between seasonally variable factors such as temperature, humidity, crowding, diet, and ARIs will inform prevention measures.

Preterm Birth: A Narrative Review of the Current Evidence on Nutritional and Bioactive Solutions for Risk Reduction.

Preterm birth (PTB) (<37 weeks of gestation) is the leading cause of newborn death and a risk factor for short and long-term adverse health outcomes. Most cases are of unknown cause. Although the mechanisms triggering PTB remain unclear, an inappropriate increase in net inflammatory load seems to be key. To date, interventions that reduce the risk of PTB are effective only in specific groups of women, probably due to the heterogeneity of its etiopathogenesis. Use of progesterone is the most effective, but only in singleton pregnancies with history of PTB. Thus, primary prevention is greatly needed and nutritional and bioactive solutions are a promising alternative. Among these, docosahexaenoic acid (DHA) is the most promising to reduce the risk for early PTB. Other potential nutrient interventions include the administration of zinc (possibly limited to populations with low nutritional status or poor zinc status) and vitamin D; additional preliminary evidence exists for vitamin A, calcium, iron, folic acid, combined iron-folate, magnesium, multiple micronutrients, and probiotics. Considering the public health relevance of PTB, promising interventions should be studied in large and well-designed clinical trials. The objective of this review is to describe, summarize, and discuss the existing evidence on nutritional and bioactive solutions for reducing the risk of PTB.

Host Microbe Interactions in the Lactating Mammary Gland.

The bacteria present in human milk constitute the human milk microbiome (hMM). Both the older culture-based work and the more recent studies using molecular detection of bacterial DNA have reached similar conclusions: the hMM mostly consists of commensal staphylococci such as Staphylococcus epidermidis, and streptococci. The prevalence of other bacterial groups such lactobacilli varies widely, while the abundance and prevalence of bifidobacteria is generally low. Recently, the hMM became accepted as a part of a physiologically normal state with suggested potential health benefits. Most research on the hMM has focused on its composition and potential effect on the breastfed infant. A major role as a microbiome inoculum for the infant gut has been proposed, but remains to be clearly demonstrated. Herein, we also discuss the emerging connection between the hMM and mammary gland physiology and lactation. Similarities between the mammary gland and mucosal interfaces are considerable, and in particular mucosal-like immune attributes of mammary gland. The potential role of hMM-host interactions in the mammary gland in maternal health is explored with a primary focus on lactational mastitis.

Bangladeshi children with acute diarrhoea show faecal microbiomes with increased Streptococcus abundance, irrespective of diarrhoea aetiology.

We report streptococcal dysbiosis in acute diarrhoea irrespective of aetiology. Compared with 20 healthy local controls, 71 Bangladeshi children hospitalized with acute diarrhoea (AD) of viral, mixed viral/bacterial, bacterial and unknown aetiology showed a significantly decreased bacterial diversity with loss of pathways characteristic for the healthy distal colon microbiome (mannan degradation, methylerythritol phosphate and thiamin biosynthesis), an increased proportion of faecal streptococci belonging to the Streptococcus bovis and Streptococcus salivarius species complexes, and an increased level of E. coli-associated virulence genes. No enteropathogens could be attributed to a subgroup of patients. Elevated lytic coliphage DNA was detected in 2 out of 5 investigated enteroaggregative E. coli (EAEC)-infected patients. Streptococcal outgrowth in AD is discussed as a potential nutrient-driven consequence of glucose provided with oral rehydration solution.

Microbiota in Breast Milk of Chinese Lactating Mothers.

The microbiota of breast milk from Chinese lactating mothers at different stages of lactation was examined in the framework of a Maternal Infant Nutrition Growth (MING) study investigating the dietary habits and breast milk composition in Chinese urban mothers. We used microbiota profiling based on the sequencing of fragments of 16S rRNA gene and specific qPCR for bifidobacteria, lactobacilli and total bacteria to study microbiota of the entire breast milk collected using standard protocol without aseptic cleansing (n = 60), and the microbiota of the milk collected aseptically (n = 30). We have also investigated the impact of the delivery mode and the stage of lactation on the microbiota composition. The microbiota of breast milk was dominated by streptococci and staphylococci for both collection protocols and, in the case of standard collection protocol, Acinetobacter sp. While the predominance of streptococci and staphylococci was consistently reported previously for other populations, the abundance of Acinetobacter sp. was reported only once before in a study where milk collection was done without aseptic cleansing of the breast and rejection of foremilk. Higher bacterial counts were found in the milk collected using standard protocol. Bifidobacteria and lactobacilli were present in few samples with low abundance. We observed no effect of the stage of lactation or the delivery mode on microbiota composition. Methodological and geographical differences likely explain the variability in microbiota composition reported to date.

Rate of establishing the gut microbiota in infancy has consequences for future health.

The gut of the human neonate is colonized rapidly after birth from an early sparse and highly distinct microbiota to a more adult-like and convergent state, within 1 to 3 years. The progression of colonizing bacterial species is non-random. During the first months of life several shifts commonly occur in the species prevalent in our guts. Although the sequential progression of these species is remarkably consistent across individuals and geographies, there is inter-individual variation in the rate of progression. Our study and others suggest that the rate is influenced by environmental factors, and influences our future health. In this article, we review our recent contribution to cataloging the developing infant gut microbiota alongside other important recent studies. We suggest testable hypotheses that arise from this synthesis.

Dynamics of infant gut microbiota are influenced by delivery mode and gestational duration and are associated with subsequent adiposity.

UNLABELLED: We found that the relatively simple microbiota of young infants shifts predictably to a more mature anaerobic microbiota during infancy and the dynamics of this shift are influenced by environmental factors. In this longitudinal study of 75 infants, we demonstrate high interindividual variability within the normal range of birth outcomes, especially in the rate of microbiota progression. Most had acquired a microbiota profile high in Bifidobacterium and Collinsella by 6 months of age, but the time point of this acquisition was later in infants delivered by caesarean section and those born after a shorter duration of gestation. Independently of the delivery mode and gestation duration, infants who acquired a profile high in Bifidobacterium and Collinsella at a later age had lower adiposity at 18 months of age. IMPORTANCE: This study shows that the acquisition of the early microbiota is strongly influenced by environmental factors such as the delivery mode and duration of gestation, even in healthy neonates. The composition of the early microbiota has been linked with long-lasting effects on health and disease. Here we show that the rate of acquisition of certain microbiota predicts adiposity at 18 months of age and so potentially the risk of later obesity.

Nasopharyngeal microbiota in healthy children and pneumonia patients.

Our study is the first to compare the nasopharyngeal microbiota of pediatric pneumonia patients and control children by 454 pyrosequencing. A distinct microbiota was associated with different pneumonia etiologies. Viral pneumonia was associated with a high abundance of the operational taxonomic unit (OTU) corresponding to Moraxella lacunata. Patients with nonviral pneumonia showed high abundances of OTUs of three typical bacterial pathogens, Streptococcus pneumoniae complex, Haemophilus influenzae complex, and Moraxella catarrhalis. Patients classified as having no definitive etiology harbored microbiota particularly enriched in the H. influenzae complex. We did not observe a commensal taxon specifically associated with health. The microbiota of the healthy nasopharynx was more diverse and contained a wider range of less abundant taxa.

Oral health in older patients with oropharyngeal dysphagia.

BACKGROUND: oropharyngeal dysphagia (OD), aspiration and poor oral health status are potential risk factors in elderly patients with aspiration pneumonia (AP). AIM: to assess the oral hygiene status and the prevalence of periodontal disease and dental caries in elderly patients with OD. PATIENTS AND METHODS: fifty elderly patients (79.7 ± 6.64 years) with OD associated with ageing or neurological diseases and 15 elderly patients without OD (77.01 ± 4.51 years) were enrolled in this observational-transversal study. OD and aspiration were evaluated by videofluoroscopy (VFS). Oral health was assessed by: (i) the Simplified Oral Hygiene Index (OHI-S); (ii) a complete periodontal examination, assessing the periodontal pocket depth, clinical attachment loss and bleeding on probing to study periodontal diseases (periodontitis, gingivitis); and (iii) the presence of dental caries. RESULTS: 8/50 elderly patients with OD presented VFS signs of aspiration, half of them silent; 40/50, signs of penetration into laryngeal vestibule and 16/50, oropharyngeal residue. Prevalence of edentulism and caries was higher in patients with OD. Dentate older patients with OD (30/50) presented the following complications (i) poor oral hygiene in 18 patients (OHI-S 3.1-6), (ii) gingivitis in 2 and periodontitis in 28 and (iii) caries in 16. CONCLUSIONS: older patients with OD presented polymorbidity and impaired health status, high prevalence of VFS signs of impaired safety of swallow and poor oral health status with high prevalence of periodontal diseases and caries. These patients are at great risk of developing AP. We recommend a policy of systematic oral health assessment in elderly patients with OD.

Human-to-bovine jump of Staphylococcus aureus CC8 is associated with the loss of a ß-hemolysin converting prophage and the acquisition of a new staphylococcal cassette chromosome.

Staphylococcus aureus can colonize and infect both humans and animals, but isolates from both hosts tend to belong to different lineages. Our recent finding of bovine-adapted S. aureus showing close genetic relationship to the human S. aureus clonal complex 8 (CC8) allowed us to examine the genetic basis of host adaptation in this particular CC. Using total chromosome microarrays, we compared the genetic makeup of 14 CC8 isolates obtained from cows suffering subclinical mastitis, with nine CC8 isolates from colonized or infected human patients, and nine S. aureus isolates belonging to typical bovine CCs. CC8 isolates were found to segregate in a unique group, different from the typical bovine CCs. Within this CC8 group, human and bovine isolates further segregated into three subgroups, among which two contained a mix of human and bovine isolates, and one contained only bovine isolates. This distribution into specific clusters and subclusters reflected major differences in the S. aureus content of mobile genetic elements (MGEs). Indeed, while the mixed human-bovine clusters carried commonly human-associated ß-hemolysin converting prophages, the bovine-only isolates were devoid of such prophages but harbored an additional new non-mec staphylococcal cassette chromosome (SCC) unique to bovine CC8 isolates. This composite cassette carried a gene coding for a new LPXTG-surface protein sharing homologies with a protein found in the environmental bacterium Geobacillus thermoglucosidans. Thus, in contrast to human CC8 isolates, the bovine-only CC8 group was associated with the combined loss of ß-hemolysin converting prophages and gain of a new SCC probably acquired in the animal environment. Remaining questions are whether the new LPXTG-protein plays a role in bovine colonization or infection, and whether the new SCC could further acquire antibiotic-resistance genes and carry them back to human.

Concentration of airborne Staphylococcus aureus (MRSA and MSSA), total bacteria, and endotoxins in pig farms.

Pigs are very often colonized by Staphylococcus aureus and transmission of such pig-associated S. aureus to humans can cause serious medical, hygiene, and economic problems. The transmission route of zoonotic pathogens colonizing farm animals to humans is not well established and bioaerosols could play an important role. The aim of this study was to assess the potential occupational risk of working with S. aureus-colonized pigs in Switzerland. We estimated the airborne contamination by S. aureus in 37 pig farms (20 nursery and 17 fattening units; 25 in summer, 12 in winter). Quantification of total airborne bacterial DNA, airborne Staphylococcus sp. DNA, fungi, and airborne endotoxins was also performed. In this experiment, the presence of cultivable airborne methicillin-resistant S. aureus (MRSA) CC398 in a pig farm in Switzerland was reported for the first time. Airborne methicillin-sensitive S. aureus (MSSA) was found in ~30% of farms. The average airborne concentration of DNA copy number of total bacteria and Staphylococcus sp. measured by quantitative polymerase chain reaction was very high, respectively reaching values of 75 (± 28) × 10(7) and 35 (± 9.8) × 10(5) copy numbers m(-3) in summer and 96 (± 19) × 10(8) and 40 (± 12) × 10(6) copy numbers m(-3) in winter. Total mean airborne concentrations of endotoxins (1298 units of endotoxin m(-3)) and fungi (5707 colony-forming units m(-3)) exceeded the Swiss recommended values and were higher in winter than in summer. In conclusion, Swiss pig farmers will have to tackle a new emerging occupational risk, which could also have a strong impact on public health. The need to inform pig farmers about biological occupational risks is therefore crucial.

Antimicrobial resistance of Staphylococcus aureus strains acquired by pig farmers from pigs.

Carriage of animal-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) is common among pig farmers. This study was conducted (i) to investigate whether pig farmers are colonized with pig-specific S. aureus genotypes other than CC398 and (ii) to survey antimicrobial resistance of S. aureus isolates from pigs and pig farmers. Forty-eight S. aureus isolates from pig farmers and veterinarians and 130 isolates from pigs collected in Western Switzerland were genotyped by spa typing and amplified fragment length polymorphism (AFLP). Antimicrobial resistance profiles were determined for representative sample of the isolates. The data obtained earlier on healthy S. aureus carriers without exposure to agriculture were used for comparison. The genotype composition of S. aureus isolates from pig farmers and veterinarians was similar to isolates from pigs with predominant AFLP clusters CC398, CC9, and CC49. The resistance to tetracycline and macrolides (clarithromycin) was common among the isolates from farmers and veterinarians (52 and 21%, respectively) and similar to resistance levels in isolates from pigs (39 and 23%, respectively). This was in contrast to isolates from persons without contact with agriculture, where no (0/128) isolates were resistant to tetracycline and 3% of the isolates were resistant to clarithromycin. MRSA CC398 was isolated from pigs (n = 11) and pig farmers (n = 5). These data imply that zoonotic transmission of multidrug-resistant S. aureus from pigs to farmers is frequent, and well-known MRSA transmission merely represents the tip of the iceberg for this phenomenon. We speculate that the relatively low frequency of MRSA isolation is related to lower antimicrobial use in Switzerland compared to, for example, the Netherlands.