Succession and determinants of the early life nasopharyngeal microbiota in a South African birth cohort

Background: Bacteria colonizing the nasopharynx play a key role as gatekeepers of respiratory health. Yet, dynamics of early life nasopharyngeal (NP) bacterial profiles remain understudied in low- and middle-income countries (LMICs), where children have a high prevalence of risk factors for lower respiratory tract infection. We investigated longitudinal changes in NP bacterial profiles, and associated exposures, among healthy infants from low-income households in South Africa. Methods: We used short fragment (V4 region) 16S rRNA gene amplicon sequencing to characterize NP bacterial profiles from 103 infants in a South African birth cohort, at monthly intervals from birth through the first 12 months of life and six monthly thereafter until 30 months. Results: Corynebacterium and Staphylococcus were dominant colonizers at 1 month of life; however, these were rapidly replaced by Moraxella- or Haemophilus-dominated profiles by 4 months. This succession was almost universal and largely independent of a broad range of exposures. Warm weather (summer), lower gestational age, maternal smoking, no day-care attendance, antibiotic exposure, or low height-for-age z score at 12 months were associated with higher alpha and beta diversity. Summer was also associated with higher relative abundances of Staphylococcus, Streptococcus, Neisseria, or anaerobic gram-negative bacteria, whilst spring and winter were associated with higher relative abundances of Haemophilus or Corynebacterium, respectively. Maternal smoking was associated with higher relative abundances of Porphyromonas. Antibiotic therapy (or isoniazid prophylaxis for tuberculosis) was associated with higher relative abundance of anerobic taxa (Porphyromonas, Fusobacterium, and Prevotella) and with lower relative abundances of health associated-taxa Corynebacterium and Dolosigranulum. HIV-exposure was associated with higher relative abundances of Klebsiella or Veillonella and lower relative abundances of an unclassified genus within the family Lachnospiraceae. Conclusions: In this intensively sampled cohort, there was rapid and predictable replacement of early profiles dominated by health-associated Corynebacterium and Dolosigranulum with those dominated by Moraxella and Haemophilus, independent of exposures. Season and antibiotic exposure were key determinants of NP bacterial profiles. Understudied but highly prevalent exposures prevalent in LMICs, including maternal smoking and HIV-exposure, were associated with NP bacterial profiles

Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens

Careful consideration of experimental artefacts is required in order to successfully apply high-throughput 16S ribosomal ribonucleic acid (rRNA) gene sequencing technology. Here we introduce experimental design, quality control and “denoising” approaches for sequencing low biomass specimens. Results We found that bacterial biomass is a key driver of 16S rRNA gene sequencing profiles generated from bacterial mock communities and that the use of different deoxyribonucleic acid (DNA) extraction methods [DSP Virus/Pathogen Mini Kit® (Kit-QS) and ZymoBIOMICS DNA Miniprep Kit (Kit-ZB)] and storage buffers [PrimeStore® Molecular Transport medium (Primestore) and Skim-milk, Tryptone, Glucose and Glycerol (STGG)] further influence these profiles. Kit-QS better represented hard-to-lyse bacteria from bacterial mock communities compared to Kit-ZB. Primestore storage buffer yielded lower levels of background operational taxonomic units (OTUs) from low biomass bacterial mock community controls compared to STGG. In addition to bacterial mock community controls, we used technical repeats (nasopharyngeal and induced sputum processed in duplicate, triplicate or quadruplicate) to further evaluate the effect of specimen biomass and participant age at specimen collection on resultant sequencing profiles. We observed a positive correlation (r = 0.16) between specimen biomass and participant age at specimen collection: low biomass technical repeats (represented by < 500 16S rRNA gene copies/μl) were primarily collected at < 14 days of age. We found that low biomass technical repeats also produced higher alpha diversities (r = − 0.28); 16S rRNA gene profiles similar to no template controls (Primestore); and reduced sequencing reproducibility. Finally, we show that the use of statistical tools for in silico contaminant identification, as implemented through the decontam package in R, provides better representations of indigenous bacteria following decontamination. Conclusions We provide insight into experimental design, quality control steps and “denoising” approaches for 16S rRNA gene high-throughput sequencing of low biomass specimens. We highlight the need for careful assessment of DNA extraction methods and storage buffers; sequence quality and reproducibility; and in silico identification of contaminant profiles in order to avoid spurious results

Using a multiple-delivery-mode training approach to develop local capacity and infrastructure for advanced bioinformatics in Africa

With more microbiome studies being conducted by African-based research groups, there is an increasing demand for knowledge and skills in the design and analysis of microbiome studies and data. However, high-quality bioinformatics courses are often impeded by differences in computational environments, complicated software stacks, numerous dependencies, and versions of bioinformatics tools along with a lack of local computational infrastructure and expertise. To address this, H3ABioNet developed a 16S rRNA Microbiome Intermediate Bioinformatics Training course, extending its remote classroom model. The course was developed alongside experienced microbiome researchers, bioinformaticians, and systems administrators, who identified key topics to address. Development of containerised workflows has previously been undertaken by H3ABioNet, and Singularity containers were used here to enable the deployment of a standard replicable software stack across different hosting sites. The pilot ran successfully in 2019 across 23 sites registered in 11 African countries, with more than 200 participants formally enrolled and 106 volunteer staff for onsite support

Current knowledge and future research directions on fecal bacterial patterns and their association with asthma

CITATION: Claassen-Weitz, S., et al. 2016. Current knowledge and future research directions on fecal bacterial patterns and their association with asthma. Frontiers in Microbiology, 7:838, doi:10.3389/fmicb.2016.00838.The original publication is available at http://journal.frontiersin.orgENGLISH SUMMARY : Asthma is a complex respiratory condition that involves interplay between genetic predisposition, environmental, and immunological factors (Edwards et al., 2012). It is considered to be one of the most common chronic diseases, affecting ~300 million people (Masoli et al., 2004), and causing an estimated 250,000 deaths annually (Bateman et al., 2008). Furthermore, because of an increased Westernized lifestyle and urbanization in developing countries, it is estimated that by 2025 the global burden of asthma will increase by 100 million people (Masoli et al., 2004).http://journal.frontiersin.org/article/10.3389/fmicb.2016.00838/fullPublisher's versio

HIV-exposure, early life feeding practices and delivery mode impacts on faecal bacterial profiles in a South African birth cohort

CITATION: Claassen-Weitz, S., et al. 2018. HIV-exposure, early life feeding practices and delivery mode impacts on faecal bacterial profiles in a South African birth cohort. Scientific Reports, 8:5078, doi:10.1038/s41598-018-22244-6.The original publication is available at https://www.nature.comThere are limited data on meconium and faecal bacterial profiles from African infants and their mothers. We characterized faecal bacterial communities of infants and mothers participating in a South African birth cohort. Stool and meconium specimens were collected from 90 mothers and 107 infants at birth, and from a subset of 72 and 36 infants at 4–12 and 20–28 weeks of age, respectively. HIV-unexposed infants were primarily exclusively breastfed at 4–12 (49%, 26/53) and 20–28 weeks (62%, 16/26). In contrast, HIV-exposed infants were primarily exclusively formula fed at 4–12 (53%; 10/19) and 20–28 weeks (70%, 7/10). Analysis (of the bacterial 16S rRNA gene sequences of the V4 hypervariable region) of the 90 mother-infant pairs showed that meconium bacterial profiles [dominated by Proteobacteria (89%)] were distinct from those of maternal faeces [dominated by Firmicutes (66%) and Actinobacteria (15%)]. Actinobacteria predominated at 4–12 (65%) and 20–28 (50%) weeks. HIV-exposed infants had significantly higher faecal bacterial diversities at both 4–12 (p = 0.026) and 20–28 weeks (p = 0.002). HIV-exposed infants had lower proportions of Bifidobacterium (p = 0.010) at 4–12 weeks. Maternal faecal bacterial profiles were influenced by HIV status, feeding practices and mode of delivery. Further longitudinal studies are required to better understand how these variables influence infant and maternal faecal bacterial composition.https://www.nature.com/articles/s41598-018-22244-6Publisher's versio

Influence of socio-economic and psychosocial profiles on the human breast milk bacteriome of South African women

CITATION: Ojo-Okunola, A. et al. 2019. Influence of Socio-Economic and Psychosocial Profiles on the Human Breast Milk Bacteriome of South African Women. Nutrients, 11(6). doi:10.3390/nu11061390The original publication is available at https://www.mdpi.com/journal/nutrientsThe human breast milk (HBM) bacteriome is an important, continuous source of microbes to the neonate in early life, playing an important role in shaping the infant’s intestinal bacteriome. Study of the composition of the HBM bacteriome is an emerging area of research, with little information available, particularly from low- and middle-income countries. The aim of this study was to characterize the diversity of bacterial communities in HBM samples collected between 6–10 weeks postpartum from lactating South African women and to study potential influencing factors of the bacteriome. Using 16S rRNA gene sequencing of samples from 554 women, we demonstrated that the HBM bacteriome was largely dominated by the phyla Firmicutes (mean relative abundance: 71.1%) and Actinobacteria (mean relative abundance: 16.4%). The most abundant genera identified from the HBM bacteriome were Streptococcus (mean relative abundance: 48.6%), Staphylococcus (mean relative abundance: 17.8%), Rothia (mean relative abundance: 5.8%), and Corynebacterium (mean relative abundance: 4.3%). “Core” bacterial genera including Corynebacterium, Streptococcus, Staphylococcus, Rothia, Veillonella, Gemella, Acinetobacter, Micrococcus and a genus belonging to the Enterobacteriaceae family were present in 80% of samples. HBM samples were classified, according to their bacteriome, into three major clusters, dominated by the genera Staphylococcus (cluster 1), a combination of Staphylococcus and Streptococcus (cluster 2), and Streptococcus (cluster 3). The cluster groups differed significantly for Shannon and chao1 richness indices. Bacterial interactions were studied using co-occurrence networks with positive associations observed between the abundances of Staphylococcus and Corynebacteria (members of the skin microflora) and between Streptococcus, Rothia, Veillonella, and Gemella (members of the oral microflora). HBM from older mothers had a higher Shannon diversity index. The study site was associated with differences in HBM bacteriome composition (permutational multivariate analysis of variance using distance matrices (PERMANOVA), p < 0.05). No other tested socio-demographic or psychosocial factors were associated with HBM bacterial composition.https://www.mdpi.com/2072-6643/11/6/1390Publisher’s versio

Genital tract infections, the vaginal microbiome and gestational age at birth among pregnant women in South Africa: a cohort study protocol.

IntroductionPreterm birth complications are the most common cause of death in children under 5 years. The presence of multiple microorganisms and genital tract inflammation could be the common mechanism driving early onset of labour. South Africa has high levels of preterm birth, genital tract infections and HIV infection among pregnant women. We plan to investigate associations between the presence of multiple lower genital tract microorganisms in pregnancy and gestational age at birth.Methods and analysisThis cohort study enrols around 600 pregnant women at one public healthcare facility in East London, South Africa. Eligible women are ≥18 years and at Chlamydia trachomatis and Neisseria gonorrhoeae, with treatment if test results are positive. At these visits, additional vaginal specimens are taken for: PCR detection and quantification of Trichomonas vaginalis, Candida spp., Mycoplasma genitalium, M. hominis, Ureaplasma urealyticum and U. parvum; microscopy and Nugent scoring; and for 16S ribosomal RNA gene sequencing and quantification. Pregnancy outcomes are collected from a postnatal visit and birth registers. The primary outcome is gestational age at birth. Statistical analyses will explore associations between specific microorganisms and gestational age at birth. To explore the association with the quantity of microorganisms, we will construct an index of microorganism load and use mixed-effects regression models and classification and regression tree analysis to examine which combinations of microorganisms contribute to earlier gestational age at birth.Ethics and disseminationThis protocol has approvals from the University of Cape Town Research Ethics Committee and the Canton of Bern Ethics Committee. Results from this study will be uploaded to preprint servers, submitted to open access peer-reviewed journals and presented at regional and international conferences.Trial registration numberNCT06131749; Pre-results

Composition of gut microbiota of children and adolescents with perinatal HIV infection taking antiretroviral therapy in Zimbabwe

Background - Human immunodeficiency virus (HIV) infection causes impairment of the gastrointestinal barrier, with substantial depletion of CD4+ T cells in the gut. Antiretroviral therapy (ART) restores CD4+ counts and may have beneficial effects on gut microbiota in adults. Little is known about effect of long-term ART on gut microbiome in HIV-infected children. We investigated composition of gut microbiota in HIV-infected and -uninfected children and assessed associations between gut microbiota and patient characteristics. Methods - In a cross-sectional study, rectal swabs were collected from 177 HIV-infected and 103 HIV-uninfected controls. Gut microbial composition was explored using 16S ribosomal ribonucleic acid sequencing. Results - Human immunodeficiency virus-infected children had significantly lower alpha-diversity and higher beta-diversity compared to HIV-uninfected. No association was observed between microbiome diversity and CD4+ T-cell count, HIV viral load, or HIV-associated chronic lung disease. We found enriched levels of Corynebacterium (P Finegoldia (P Anaerococcus (P Enterobacteriaceae (P = .02) in participants with low CD4+ counts (3). Prolonged ART-treatment (≥10 years) was significantly associated with a richer gut microbiota by alpha diversity. Conclusions - Human immunodeficiency virus-infected children have altered gut microbiota. Prolonged ART may restore the richness of the microbiota closer to that of HIV-uninfected children

Sputum bacterial load and bacterial composition correlate with lung function and are altered by long-term azithromycin treatment in children with HIV-associated chronic lung disease

Background: Long-term azithromycin (AZM) treatment reduces the frequency of acute respiratory exacerbation in children and adolescents with HIV-associated chronic lung disease (HCLD). However, the impact of this treatment on the respiratory bacteriome is unknown. Method: African children with HCLD (defined as forced expiratory volume in 1 s z-score (FEV1z) less than − 1.0 with no reversibility) were enrolled in a placebo-controlled trial of once-weekly AZM given for 48-weeks (BREATHE trial). Sputum samples were collected at baseline, 48 weeks (end of treatment) and 72 weeks (6 months post-intervention in participants who reached this timepoint before trial conclusion). Sputum bacterial load and bacteriome profiles were determined using 16S rRNA gene qPCR and V4 region amplicon sequencing, respectively. The primary outcomes were within-participant and within-arm (AZM vs placebo) changes in the sputum bacteriome measured across baseline, 48 weeks and 72 weeks. Associations between clinical or socio-demographic factors and bacteriome profiles were also assessed using linear regression. Results: In total, 347 participants (median age: 15.3 years, interquartile range [12.7–17.7]) were enrolled and randomised to AZM (173) or placebo (174). After 48 weeks, participants in the AZM arm had reduced sputum bacterial load vs placebo arm (16S rRNA copies/µl in log10, mean difference and 95% confidence interval [CI] of AZM vs placebo − 0.54 [− 0.71; − 0.36]). Shannon alpha diversity remained stable in the AZM arm but declined in the placebo arm between baseline and 48 weeks (3.03 vs. 2.80, p = 0.04, Wilcoxon paired test). Bacterial community structure changed in the AZM arm at 48 weeks compared with baseline (PERMANOVA test p = 0.003) but resolved at 72 weeks. The relative abundances of genera previously associated with HCLD decreased in the AZM arm at 48 weeks compared with baseline, including Haemophilus (17.9% vs. 25.8%, p < 0.05, ANCOM ω = 32) and Moraxella (1% vs. 1.9%, p < 0.05, ANCOM ω = 47). This reduction was sustained at 72 weeks relative to baseline. Lung function (FEV1z) was negatively associated with bacterial load (coefficient, [CI]: − 0.09 [− 0.16; − 0.02]) and positively associated with Shannon diversity (0.19 [0.12; 0.27]). The relative abundance of Neisseria (coefficient, [standard error]: (2.85, [0.7], q = 0.01), and Haemophilus (− 6.1, [1.2], q < 0.001) were positively and negatively associated with FEV1z, respectively. An increase in the relative abundance of Streptococcus from baseline to 48 weeks was associated with improvement in FEV1z (3.2 [1.11], q = 0.01) whilst an increase in Moraxella was associated with decline in FEV1z (-2.74 [0.74], q = 0.002). Conclusions: AZM treatment preserved sputum bacterial diversity and reduced the relative abundances of the HCLD-associated genera Haemophilus and Moraxella. These bacteriological effects were associated with improvement in lung function and may account for reduced respiratory exacerbations associated with AZM treatment of children with HCLD