DNA extraction approaches substantially influence the assessment of the human breast milk microbiome

In addition to providing nutritional and bioactive factors necessary for infant development, human breast milk contains bacteria that contribute to the establishment of commensal microbiota in the infant. However, the composition of this bacterial community differs considerably between studies. We hypothesised that bacterial DNA extraction methodology from breast milk samples are a substantial contributor to these inter-study differences. We tested this hypothesis by applying five widely employed methodologies to a mock breast milk sample and four individual human breast milk samples. Significant differences in DNA yield and purity were observed between methods (P < 0.05). Microbiota composition, assessed by 16S rRNA gene amplicon sequencing, also differed significantly with extraction methodology (P < 0.05), including in the contribution of contaminant signal. Concerningly, many of the bacterial taxa identified here as contaminants have been reported as components of the breast milk microbiome in other studies. These findings highlight the importance of using stringent, well-validated, DNA extraction methodologies for analysis of the breast milk microbiome, and exercising caution interpreting microbiota data from low-biomass contexts.Chloe A. Douglas, Kerry L. Ivey, Lito E. Papanicolas, Karen P. Best, Beverly S. Muhlhausler and Geraint B. Roger

Total bacterial load, inflammation, and structural lung disease in paediatric cystic fibrosis

Background: Cystic fibrosis (CF) is characterised by reduced airway clearance, microbial accumulation, inflammation, and lung function decline. Certain bacterial species may contribute disproportionately to worsening lung disease. However, the relative importance of these microorganisms compared to the ab- solute abundance of all bacteria is uncertain. We aimed to identify the characteristics of lower airway microbiology that best reflect CF airway inflammation and disease in children. Methods: Analysis was performed on bronchoalveolar lavage (BAL) fluid from 78 participants of the Australasian CF Bronchoalveolar Lavage (ACFBAL) clinical trial, aged 4.5–5.5 years. Universal bacterial quantitative PCR (qPCR), species-specific qPCR, and 16S rRNA gene sequencing were performed on DNA ex- tracts to determine total bacterial load, species-specific load and taxa relative abundance. Quantification of prespecified pathogens was performed by culture-based methods. Bacteriological data were related to neutrophil counts, interleukin-8, lung function, and two computed-tomography based measures, CF-CT (as the primary measure) and PRAGMA. Results: Of all bacteriological measures assessed, total bacterial load determined by qPCR correlated most strongly with structural disease (CF-CT total score, r s = 0.30, P = 0.0095). Specifically, total bacterial load correlated with bronchiectasis, airway wall thickening, mucus plugging and parenchymal disease sub-scores. In contrast, culture-based quantification , microbiota-derived measures, and pathogen-specific qPCR-based quantification were weakly associated with total CF-CT. Regression analyses supported cor- relation findings, with total bacterial load explaining the greatest variance in total CF-CT (R 2 = 0.097, P = 0.0061). Correlations with PRAGMA score were comparable to CF-CT total score. Conclusions: Within the ACFBAL trial, culture-independent quantification of total bacteria provided the most clinically-informative bacteriological measure in 5-year-old CF patients.Steven L. Taylor, Lex E.X. Leong, Kerry L. Ivey, Steve Wesselingh, Keith Grimwood, Claire E. Wainwright, Geraint B. Rogers, On behalf of the Australasian Cystic Fibrosis Bronchoalveolar Lavage, (ACFBAL), study grou

Sarcopenia: its assessment, etiology, pathogenesis, consequences and future

Sarcopenia is a loss of muscle protein mass and loss of muscle function. It occurs with increasing age, being a major component in the development of frailty. Current knowledge on its assessment, etiology, pathogenesis, consequences and future perspectives are reported in the present review. On-going and future clinical trials on sarcopenia may radically change our preventive and therapeutic approaches of mobility disability in older peopleY. Rolland, S. Czerwinski, G. Abellan Van Kan, J.E. Morley, M. Cesari, G. Onder, J. Woo, R. Baumgartner, F. Pillard, Y. Boirie, W.M.C. Chumlea, B. Vella