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See detailPersistence of birth mode-dependent effects on gut microbiome composition, immune system stimulation and antimicrobial resistance during the first year of life
Busi, Susheel Bhanu UL; de Nies, Laura UL; Habier, Janine UL et al

in ISME Communications (2021)

Caesarean section delivery (CSD) disrupts mother-to-neonate transmission of specific microbial strains and functional repertoires as well as linked immune system priming. Here we investigate whether ... [more ▼]

Caesarean section delivery (CSD) disrupts mother-to-neonate transmission of specific microbial strains and functional repertoires as well as linked immune system priming. Here we investigate whether differences in microbiome composition and impacts on host physiology persist at 1 year of age. We perform high-resolution, quantitative metagenomic analyses of the gut microbiomes of infants born by vaginal delivery (VD) or by CSD, from immediately after birth through to 1 year of life. Several microbial populations show distinct enrichments in CSD-born infants at 1 year of age including strains of Bacteroides caccae, Bifidobacterium bifidum and Ruminococcus gnavus, whereas others are present at higher levels in the VD group including Faecalibacterium prausnitizii, Bifidobacterium breve and Bifidobacterium kashiwanohense. The stimulation of healthy donor-derived primary human immune cells with LPS isolated from neonatal stool samples results in higher levels of tumour necrosis factor alpha (TNF-α) in the case of CSD extracts over time, compared to extracts from VD infants for which no such changes were observed during the first year of life. Functional analyses of the VD metagenomes at 1 year of age demonstrate a significant increase in the biosynthesis of the natural antibiotics, carbapenem and phenazine. Concurrently, we find antimicrobial resistance (AMR) genes against several classes of antibiotics in both VD and CSD. The abundance of AMR genes against synthetic (including semi-synthetic) agents such as phenicol, pleuromutilin and diaminopyrimidine are increased in CSD children at day 5 after birth. In addition, we find that mobile genetic elements, including phages, encode AMR genes such as glycopeptide, diaminopyrimidine and multidrug resistance genes. Our results demonstrate persistent effects at 1 year of life resulting from birth mode-dependent differences in earliest gut microbiome colonisation. [less ▲]

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See detailThe RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions
Malabirade, Antoine UL; Habier, Janine UL; Heintz-Buschart, Anna et al

in Frontiers in Microbiology (2018), 9

Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host–pathogen interactions. The presence of larger RNA transcripts in OMVs has been ... [more ▼]

Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host–pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host–pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by Salmonella enterica serovar Typhimurium (S. Typhimurium) cultured under different conditions, which mimic host–pathogen interactions. S. Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium (phosphate-carbon-nitrogen, PCN), as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of Salmonella pathogenicity island 2 (SPI-2) genes. Moreover, Salmonella pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing S. Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts (IsrB-1/2; IsrA; ffs; SsrS; CsrC; pSLT035; 10Sa; rnpB; STM0277; sseB; STM0972; STM2606), whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts (SsrS, CsrC, 10Sa, and rnpB). In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active. [less ▲]

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See detailExtraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles
Habier, Janine UL; May, Patrick UL; Heintz-Buschart, Anna et al

in Arluison, Véronique; Valverde, Claudio Valverde (Eds.) Bacterial Regulatory RNA (2018)

Outer membrane vesicles (OMVs) are released by commensal as well as pathogenic Gram-negative bacteria. These vesicles contain numerous bacterial components, such as proteins, peptidoglycans ... [more ▼]

Outer membrane vesicles (OMVs) are released by commensal as well as pathogenic Gram-negative bacteria. These vesicles contain numerous bacterial components, such as proteins, peptidoglycans, lipopolysaccharides, DNA, and RNA. To examine if OMV-associated RNA molecules are bacterial degradation products and/or are functionally active, it is necessary to extract RNA from pure OMVs for subsequent analysis. Therefore, we describe here an isolation method of ultrapure OMVs and the subsequent extraction of RNA and basic steps of RNA-Seq analysis. Bacterial culture, extracellular supernatant concentration, OMV purification, and the subsequent RNA extraction out of OMVs are described. Specific pitfalls within the protocol and RNA contamination sources are highlighted. [less ▲]

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