The rate of caesarean section delivery (CSD) is increasing worldwide. It remains unclear whether disruption of mother-to-neonate transmission of microbiota through CSD occurs and whether it affects human physiology. Here we perform metagenomic analysis of earliest gut microbial community structures and functions. We identify differences in encoded functions between microbiomes of vaginally delivered (VD) and CSD neonates. Several functional pathways are over-represented in VD neonates, including lipopolysaccharide (LPS) biosynthesis. We link these enriched functions to individual-specific strains, which are transmitted from mothers to neonates in case of VD. The stimulation of primary human immune cells with LPS isolated from early stool samples of VD neonates results in higher levels of tumour necrosis factor (TNF-α) and interleukin 18 (IL-18). Accordingly, the observed levels of TNF-α and IL-18 in neonatal blood plasma are higher after VD. Taken together, our results support that CSD disrupts mother-to-neonate transmission of specific microbial strains, linked functional repertoires and immune-stimulatory potential during a critical window for neonatal immune system priming. The effects of caesarean section delivery on mother-to-neonate transmission of microbiota are unclear. Here the authors show that caesarean section delivery can affect the transmission of specific microbial strains and the immunomodulatory potential of the microbiota. During vaginal birth, specific bacterial strains are transmitted from mothers to infants6 and differences in microbial colonization in neonates born by CSD have been identified10 as early as 3 days postpartum10. However, due to conflicting results, which principally imply a negligible impact of delivery mode on the colonizing neonatal microbiome in the gut11, it remains unclear whether disruption of mother-to-infant transmission of microbiota through CSD occurs and whether it affects human physiology early on, with potentially persistent effects in later life. As the first few days after birth represent a ‘critical window’ in neonatal health and development14, there is growing concern that disruption of microbial transmission from mother to neonate is linked to conditions more frequently observed in CSD-born individuals, including allergies15, chronic immune disorders16 and metabolic disorders17. To address these concerns, it is essential to determine if there are differences in the functional complement conferred by the earliest colonizing microbiota in relation to CSD, if any differences result from changes in the transmission of strains from mothers to neonates, and if these impact neonatal physiology.The past decades have witnessed steadily increasing rates in caesarean section deliveries (CSD) performed largely in the absence of medical necessity and reaching proportions of 19.1% worldwide and 25% in Europe18, these effects are either extenuated or largely absent in other studies19. In this context, it is important to consider that CSD may be performed as a result of underlying maternal or foetal medical conditions 20 and can co-occur with other microbiome-influencing factors. More specifically, CSD is most often accompanied by the administration of antibiotics to mothers due to local health regulations or hospital practices 21. Being born small for gestational age (SGA) frequently coincides with CSD as well 22. SGA neonates have an elevated propensity for developing metabolic disorders during childhood or adulthood, which has been associated with alterations to the gut microbiome23, and may be linked to the elevated rate of CSD in this population.While the majority of studies so far indicate that delivery mode is the strongest factor determining early neonatal gut microbiome colonization6. In addition, although single nucleotide variants (SNVs) have been tracked over time, no such studies have so far covered the earliest time points after delivery (days 0–5) in well-matched mother–neonate pairs in relation to a direct comparison of delivery modes. Consequently, there is a strong need for adequate high-resolution metagenomic analyses capable of resolving the vertical transmission of individual-specific strains and encoded functions from mothers to neonates on an individual basis, while also supplementing observed in silico findings with further in vitro validation experiments.Apart from confounding factors, the methods and study designs employed over the past years may in part explain some of the conflicting results regarding the effect of delivery mode on the early gut microbiome. Notably, taxonomic profiling based on 16S rRNA gene amplicon sequencing does not offer sufficient resolution to assess the direct effect of the delivery mode at the level of strain transmission, which is expected to be a determinant of succession. Although recent studies have focused on mother-to-neonate strain transfer and have shown that maternal strains do colonize the neonatal gut, non-vaginal delivery was not assessed comprehensively24, delivery marks the moment of extensive exposure to microbial communities of faecal, vaginal, skin and environmental origins and this event thereby has a profound impact on the colonization of the neonatal gut25. The initial low microbial biomass in the earliest neonatal stool samples7 makes the sequencing data prone to the over-representation of putative artefactual reads26 and may contribute to the generation of inconsistent results. Therefore, the removal of any artefactual sequences is essential to ensure unambiguous, high-resolution overviews of the earliest microbial colonization of the neonatal gut.Independent of whether prenatal colonization of the foetus takes place or notHere, we performed a detailed analysis of the earliest microbial colonization of the neonatal gut using a combination of 16S rRNA gene amplicon sequencing and high-resolution metagenomics. Our results highlight differences in gut microbiome composition according to delivery mode as well as concurrent differences in the encoded functional potential, which in turn are linked to differences in the transfer of strains from mother to neonate. Based on the enrichment of the LPS biosynthesis pathway in VD neonates, we performed LPS extractions from neonatal stool samples, in vitro immune stimulation assays as well as extensive assessments of LPS purity. The stimulation of primary human immune cells with purified LPS from the faeces of VD neonates collected at day 3 postpartum resulted in the production of higher levels of TNF-α and IL-18. In accordance with these results, the levels of TNF-α and IL-18 in neonatal blood plasma were also higher in VD neonates when compared to CSD. Taken together, we observe a microbiome-driven relationship between delivery mode and endotoxin-induced immune system priming with the potential for lasting effects in later life.7. For a subset of neonates, well-matched neonatal and maternal samples were subjected to high-resolution metagenomic analyses. To differentiate between potential effects of CSD and/or SGA, neonates born by CSD and neonates born by CSD and being SGA were included in the cohort and analysed separately. For each mother–neonate pair, we sampled microbiomes of maternal body sites, which are indicated to be important in relation to neonatal gut colonization  less than 24 h before delivery. Additionally, earliest neonatal stool samples were collected at ≤ 24 h, 3 days and 5 days postpartum . Taking into account the effects of the above-mentioned factors, delivery mode was found to be the dominant driver of neonatal gut microbiome colonization, with other measured factors having considerably less of an effect .To assess whether the apparent taxonomic differences between the gut microbiomes of VD and CSD neonates are reflected at the level of functional potential, we used the metagenomic sequencing data to calculate Jensen-Shannon divergences for all samples  as covariates , and the proportion of reads mapping to genes involved in this pathway was larger in VD neonates compared to both CSD groups neonates . Other important microbial metabolic pathways, which were enriched with differentially abundant genes between VD and CSD (±SGA), included flagellar assembly , bacterial chemotaxis , cationic antimicrobial peptide (CAMP) resistance , two-component system  and ABC transporters . As comparisons between VD and CSD as well as VD and CSD + SGA were largely matching independent of SGA status, we combined both groups (CSD and CSD + SGA) to increase statistical power (CSD ± SGA). Notably, all pathways also showed higher relative gene abundances in VD compared to CSD (±SGA) neonates except for the ABC transporter pathway .We detected a total of 1,697 functional categories from the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) database that were differentially abundant in the comparisons of the gut microbiome of CSD or CSD + SGA neonates to VD neonates. These presented the same directionality of change using the R package DESeq2tes Fig. . Among tP = 1.7 × 10−3 and P = 4.0 × 10−3 for day 3 and 5 respectively; Supplementary Fig. 34 based on the extensive 16S rRNA gene amplicon data (Supplementary Data 32) was performed to compare the functional profiles of CSD (±SGA) to VD neonates and for both generated data sets . Results from the multivariate analyses demonstrated that delivery mode was the strongest determining factor in both data sets  for explaining the differentially abundant genes .To corroborate the apparent higher propensity of the VD microbiome for LPS biosynthesis, we annotated the OTUs resulting from the 16S rRNA gene amplicon sequencing data according to their attributed Gram staining information. Hereby, we observed that the gut microbiomes of VD neonates harboured significantly higher relative abundances of Gram-negative bacteria at days 3 and 5 compared to CSD (±SGA) neonates . This enabled the tracking of specific strains from mothers to neonates (Supplementary Data P = 3 × 10−3). While the reconstructed genomes of 25 taxa belonging to the phyla Proteobacteria and Firmicutes were identified in maternal–neonatal pairs for all birth modes , the reconstructed genomes of 23 enteric taxa belonging to the phyla Bacteroidetes and Actinobacteria (notably Bacteroides and Bifidobacterium spp.) were exclusively observed in VD pairs. Notably, in the case of vaginal delivery, multiple strains of Gram-positive bacteria  were transferred from mother to neonate  and intra-population diversity (π), on the basis of the resolved SNVs. Our results reflected a shift in population structures during the transfer from mothers to neonates. We observed higher fixation indices (Wilcoxon signed-rank test: P < 4 × 10−3) between maternal and neonates’ strains  neonates . Among these strains, Bacteroidetes (B. vulgatus and other Bacteroides species) and Clostridium spp. were common. Most strikingly, a B. vulgatus genome, which shared the majority of SNVs with the corresponding maternal reconstructed genome, was enriched in functions that were significantly more abundant in VD neonates compared to CSD (±SGA) neonates  in functions with higher relative abundances in CSD (±SGA) neonates, and considerably fewer SNVs were shared with the respective mother. These results indicate that vaginal delivery not only favours the vertical transfer of enteric strains from mother to neonate, but also results in the transfer of specific functional traits to the neonate, which are involved in important microbial pathways such as LPS biosynthesis and may be relevant in stimulating the developing immune system during the first days of life.To assess whether the transferred strains conferred specific functional traits to the neonate or not, we assessed the genomic complements of the earliest microbiota. Analysis of reconstructed genomes that were linked to maternal metagenomes showed that vertically transmitted strains were more likely to be enriched in functions that were depleted in CSD neonates (odds ratio (OR) 5.0, Fisher’s exact test dis Fig.  was enriAs LPS forms part of the outer membrane of Gram-negative bacteria, the attributed Gram staining information of microorganisms directly corresponds to their propensity to synthetize LPS. Importantly, LPS is a highly potent innate immune activator that is recognized by the Toll-like receptor (TLR) 4. The earliest VD gut microbiome exhibited an enrichment in the microbial LPS biosynthesis pathway Figs.  and 3h a12. We isolated LPS from faecal samples from day 3 with sufficient biomass from 16 neonates  from 12 adult donors with neonatal LPS extracts (Methods). In order to reflect the in vivo situation as closely as possible, we stimulated the MoDCs with the exact same LPS volume that was obtainable from the same initial amount of faecal material from each neonate sample and subsequently measured levels of the LPS-inducible cytokine TNF-α in the supernatants using an ELISA assay  and their respective neonates  born by distinct delivery modes. All microbiome samples yielded high-resolution, artefact-curated sequencing data, which was analysed at the strain level.Here we employed high-resolution, artefact-curated metagenomic analyses of paired, high-quality samples from mothers and neonates to resolve the neonatal gut microbiome over the first few days of life. While previous studies have used analogous analytical approaches (16S rRNA gene amplicon sequencing and metagenomics) to resolve the early neonatal gut microbiome, these studies did not involve the systematic collection and appropriate preservation of paired mother–neonate samplesBacteroides and Parabacteroides in VD neonates as well as OTUs assigned to Staphylococcus being enriched in CSD neonates39. No fundamental differences in taxonomical compositions nor functional potentials were apparent when comparing CSD and CSD + SGA neonates, indicating that delivery mode is a stronger determinant for neonatal gut microbial colonization than SGA status.As earliest neonatal gut microbiome samples are naturally of low biomass, the accurate identification and curation of potential artefactual sequences is essential. In the absence of appropriate controls, sequences derived from contaminant taxa in reagents may be relatively prominent, thereby masking actual signals and confounding results regarding in particular the transfer of taxa and functions from mothers to neonates. In our study, adequate controls were included and putative artefactual reads removed based on a combined in vitro and in silico workflow. In order to reach the required specificity to unambiguously address the question of vertical transmission of microbial community structure and function from mother to neonate, the use of curated, high-resolution metagenomic sequencing data (rather than solely performing 16S rRNA gene amplicon sequencing) is imperative. More specifically, the applied methodological approach allows the highly specific tracking of individual microbial functions and strains from mother to neonates on a case-by-case basis. Our results based on both 16S rRNA gene amplicon and metagenomic sequencing, and supported by multivariate analyses, demonstrate that early differences exist in the gut microbiomes of neonates and that these differences are predominantly driven by the mode of delivery. Our data agrees with previously reported differences in microbial composition related to birth mode, notably the increased relative abundance of 42. Notably, a selection of enteric strains from the mother was found to be exclusively transferred to the VD neonate . When measuring population differentiation (FST), we found evidence that strains acquired from the mother are capable of quickly adapting to the new environment, as observed previously5. In contrast, vaginal strains harboured a low potential to stably colonize the neonatal gut thereby further adding to recently published data demonstrating that vaginal taxa do not play a prominent role in the initial colonization of the neonatal gut5. This may in part be explained by the distinct niches of the anaerobic gastrointestinal environment and the microaerophilic vaginal environment. With respect to ongoing clinical interventions aimed at restoring the earliest neonatal gut microbiome in the case of caesarean section43, our findings raise questions over the expected efficiency of microbiota engraftment from a purely vaginal source and suggest that gut-derived strains may be more efficacious.In line with the broad taxonomic differences between VD and CSD, our findings demonstrate that CSD significantly affects the functional gene complement of the earliest neonatal gut microbiome by impeding the vertical transfer of specific bacterial strains from the maternal gut microbiome to the neonate. Consequently, the gut of CSD neonates is most likely colonized by strains derived from other sources, such as breast milk, skin or saliva, as suggested in previous studies44. On the basis of the observed immunogenicity of the purified LPS fractions, it has not escaped our attention that other factors, including the actual LPS composition, may additionally contribute to the difference in the immunostimulatory potential of the colonizing gut microbiome  could confer protective effects towards allergy development46. In this context, LPS is likely closely involved in the priming of the neonatal immune system and the subsequent tolerance towards the colonizing gut microbiome during a most critical window in early neonatal life14. Using a mouse model, it has been shown that strongly immunostimulatory LPS can contribute to the protection from immune-mediated diseases such as diabetes47 and that disruption of host-commensal interactions in early-life can lead to persistent defects in the development of specific immune subsets12. On the basis of additional cytokine measurements in neonatal plasma, VD neonates displayed higher levels of IL-18 and TNF-α, thereby indicating a link between the immunostimulatory potential of microbial LPS in the gut and the overall immune status of the neonatal host early on. Investigations of the longer-term consequences of these differences between CSD and VD neonates will be necessary to assess their possible impact on the development of chronic diseases in later life.Our study highlights differences in immunostimulatory potential of the earliest gut microbiome according to delivery mode. This occurs during a critical window of immune system priming. Notably, alterations to early immune system stimulation may be linked to the higher propensity of CSD infants to develop chronic diseases in later life48, which may suggest that they play a role in the activation of the neonatal immune system. Additionally, enrichments in pathways relating to bacterial motility were observed. These included the two-component system pathway, which is an important mediator of signal transduction, flagellar assembly and bacterial chemotaxis. These pathways are essential for bacterial motility in response to external stimuli and consequently also for competition with other members of the gut microbiome49. Additionally, flagellin, the main structural component of the flagellum, is an effective stimulator of innate immunity50 and promotes mucosal immunity through the activation of TLR551. Another functional pathway that is potentially interacting with the human immune system early on is the resistance to cationic antimicrobial peptides (CAMP). While this resistance has been observed in all major commensal phyla and across all members of the phylum Bacteroidetes, this pathway is essential to evade detection by the human immune system through the modification of the microbial LPS structure52. In the context of our study, an enrichment in CAMP resistance may therefore prevent the dominant colonizers  from being recognized by the immune system and subsequently removed from the VD neonatal gut. Future studies are needed to assess whether the gut microbiome of VD neonates harbours more modified LPS moieties linked to CAMP resistance and which potential effects the altered LPS structures may have on the neonatal immune system. In accordance with the observation of an apparent enrichment in flagellar biosynthesis, bacterial chemotaxis and CAMP resistance, other microbiota-derived molecular factors, apart from LPS, may be involved in early immune system priming.Apart from LPS biosynthesis, other pathways that were significantly enriched in the gut microbiome of VD neonates included genes involved in membrane transport, i.e., ATP-binding cassette (ABC) transporters. On the one hand this may reflect the adaptation of the colonizing microbiome of VD neonates to the gut environment through enhanced nutrient intake. On the other hand, associated ABC transporter proteins for both Gram-positive and Gram-negative bacteria have previously been shown to be immunogenic42. An additional focus should be placed on uncovering the source and mode of transfer of gut strains from mothers to neonates. Such mechanistic understanding will be important for devising future clinical interventions principally aimed at restoring a VD-like pioneering microbiota in the case of CSD. An alternative approach may consist of ensuring appropriate early priming of the neonatal immune system by the controlled provision of microbial antigens. Both avenues may provide the basis for the development of preventative strategies for adverse health effects in CSD neonates in the future.Our results imply that a more comprehensive understanding of the effect of the earliest microbial exposure on innate and adaptive immune responses and the different molecular factors involved in neonatal immune system priming is necessary. Future long-term follow-up studies based on larger cohorts, high-resolution multi-omic analyses, detailed immunological screening and tracking of health status will be essential to unravel the interdependencies between mode of delivery, other potential confounding factors, mother-to-neonate transmission, microbiome colonization, exposure to microbial factors, immune system priming and long-term health status. Furthermore, additional sources of maternal strains of importance in relation to microbiome-conferred molecular factors, besides the maternal vagina and gut, have to be considered to assess their relative importance in relation to their impact on neonate physiology. For this, additional samples may be obtained from maternal milk, skin, the oral cavity, and the hospital environmentWritten informed consent was obtained before specimen collection from all enrolled mothers after a detailed consultation. All aspects of recruitment as well as collection, handling, processing and storing of samples and data were approved by the Luxembourg ethics board, the Comité national d’éthique de recherche, under reference number 201110/06 and by the Luxembourg National Commission for Data Protection under reference number A005335/R000058.53. Enrolled pairs of mothers and neonates (n = 16 pairs) included one twin birth (C115 (CSD) and C116 (CSD + SGA)).All study participants were enrolled and gave birth at the Centre Hospitalier de Luxembourg (CHL). Exclusion criteria for mother–neonate pairs included the administration of antibiotics to neonates immediately postpartum, birth prior to 34 weeks of gestation, and maternal gestational diabetes. Clinical metadata for all the analysed time points  are listed in Supplementary Data 7. For a subset of neonates, the mother was sampled additionally. Maternal samples  were collected less than 24 h before delivery. Samples were collected into sterile plastic tubes, immediately flash-frozen in liquid nitrogen and stored at −80 °C until further processing. Neonatal blood was collected by capillary or venous sampling, and plasma was isolated and stored at −80 °C from 31 healthy neonates  at day 3 (28 samples) or day 5 (three samples) after birth, including 15 of the 16 neonates for whom metagenomic data were analysed , two neonates for whom no metagenomic but 16S rRNA gene amplicon sequencing data were available (two CSD) and 14 neonates  that were sampled under the same conditions7  with an additional step to increase extraction yield involving the incubation of the samples in PowerSoil tubes with solution C1 at 65 °C for 10 min prior to homogenization for 5 min at 20 Hz in an Oscillating Mill MM 400 . DNA was subsequently extracted following the manufacturer’s instructions.−1 in a FastPrep-24 5 G . Samples were centrifuged for 1 min at 12,000 g. Solutions C2 (250 µl) and C3 (100 µl) were added to the supernatant and incubated at 4 °C for 5 min, centrifuged for 1 min at 12,000 g, then 700 µl of solution C4 and 600 µl of 100% ethanol were added to the supernatant and mixed. 650 µl were loaded onto a Spin Filter and centrifuged at 10,000 × g for 1 min. This step was repeated until all lysate had passed through the filter. For the higher input-mass maternal faecal samples, the same isolation procedure was followed except that the filters were washed with a mix of 300 µl solution C4 and 370 µl 100% ethanol, with centrifugation at 10,000 × g for 1 min. This latter step was omitted for the low input neonatal samples. All filters were washed with 650 µl 100% ethanol, then 500 µl solution C5. After drying, 60 µl solution C6 was added to the centre of the filter and incubated at room temperature for 5 min. DNA was eluted by centrifugation at 10,000 × g for 30 s. RNase A  was added and incubated at 37 °C for ≥ 30 min. Then, one-tenth volume 3 M sodium acetate (pH 6.8) and two volumes isopropanol were added to precipitate the DNA on ice prior to centrifugation. The pellet was washed with 150 µl 70% ethanol, before the dried DNA was dissolved in 50 µl  or 100 µl  RNase-free water. To obtain an artefact control sample, DNA was extracted from 800,000 trypsinized Caco-2 cells/ml. Caco-2 cells were grown in Dulbecco’s Modified Eagle’s Medium  containing 20% v/v foetal bovine serum and 1% penicillin–streptomycin (Invitrogen) to prevent microbial growth. DNA was extracted with the low-biomass protocol described above, subsequently titrated and samples with 480, 240, 120, 60 and 30 ng total mass were sequenced. DNA integrity and quantity were determined for extracted samples of all origins on 1% agarose gels and in a Qubit 2.0 fluorometer (Thermo Fisher Scientific). Extracted DNA was stored at −80 °C until further use.Faecal samples and cell-culture pellets were processed with the Powerlyzer PowerSoil DNA isolation kit (MO BIO Laboratories), optimized for low-yield samples. Bead solution (500 µl), C1 (60 µl), UltraPure™ Phenol:Chloroform:Isoamyl Alcohol  and 50 mg neonatal stool or 150 mg maternal stool were added to a dry glass bead tube, incubated at 65 °C for 10 min, and homogenized by milling for 45 s at 4 m sAll DNA samples  underwent standard amplicon sequencing of the V4 region of 16S rRNA genes using primers 515F- 5′-GTGBCAGCMGCCGCGGTAA-3′ and 805R- 5′-GACTACHVGGGTATCTAATCC-3′ at the Center for Analytical Research and Technology–Groupe Interdisciplinaire de Génoprotéomique Appliquée . Selected DNA samples of maternal , neonatal  and cell-culture origins were subjected to random shotgun sequencing , and the de novo assembly step used the MEGAHIT54 metagenome assembler. The IMP parameters were customized for different sample types: default parameters were retained for maternal faecal samples; for low-biomass samples , the integrated VizBin29 sequence cut-off length was set to 1.Metagenomic data sets were processed with the Integrated Meta-omic Pipeline , which performs pre-processing, assembly, functional annotation of predicted genes and downstream analyses of Illumina next-generation sequencing metagenomic data in a single, reproducible workflowMycoplasma were negative, and that the metagenomic sequencing data did not include any Mycoplasma sequences, any bacterial contamination of the mammalian cell culture could be confidently excluded. Then, metagenomic reads from each study sample were mapped against these contigs using Bowtie 255 (version 2.0.2). Matching sequences were excluded prior to taxonomic profiling of metagenomic reads by phylogenetic markers35. As the artefactual sequences identified in the control samples did not represent full genomes, we further used a binning-based approach to identify additional potential artefactual sequences of the same organism among the de novo assembled contigs of the study samples. After removing the rRNA sequences from the contigs56, we performed joint binning of control cell-culture contigs with each of the samples’ contigs individually using VizBin29 without any length cut-off. Bins were identified based on VizBin embeddings56 using density-based spatial clustering of applications with noise (DBSCAN), without correction for the depth of coverage and completeness. All distinct bins  that contained > 0.01% of the total contig length of the cell-culture control sample were considered putative reconstructed genomes of artefactual DNA, and the corresponding contigs were removed from the study samples in silico.To identify and exclude artefactual sequences in the low biomass samples, contigs were assembled from the sequencing reads obtained from the DNA extracts of the Caco-2 cells after the removal of human reads. Given that the Caco-2 cells were cultured in the presence of 1% penicillin–streptomycin, that the routine surveys for 56 trained for all KO57 groups. The functional KO HMMs were aligned using HMMER 3.159. The best hit KO  for every gene was assigned if the bit score was higher than the binary logarithm of the number of target genes. The FeatureCounts60 tool with arguments –p and –O was used to extract the number of reads per KO 56. The reconstructed genomes of all samples belonging to a mother–neonate pair were merged into a union set. For each sample set, predicted amino acid sequences were searched against and annotated using a defined set of essential marker genes61 using HMMER 3.158. Protein sequences assigned to 35 specific marker genes that form the cross-section of previously suggested sets of phylogenetic marker genes62 were selected. These marker amino acid sequences were clustered with CD-HIT63 at 97.5% identity. The frequencies of genes from different genome reconstructions co-occurring in the same clusters were determined. A simple graph network representation was constructed with the reconstructed genomes as nodes and counts of co-occurrences between two reconstructed genomes as weighted, undirected edges. Highly interlinked sub-networks, representing related reconstructed genomes, were detected with the cluster_fast_greedy algorithm64 implemented in the R package igraph (v.1.0.1). The resulting reconstructed genomes from a given sub-network were manually inspected, and the taxonomy of reconstructed genomes was assigned using PhyloPhlAn30 . To visualize the positions of markers in genome reconstructions, the reference markers of the species assigned to the reconstructed genomes in StrainPhlAn were aligned to the genome reconstructions post hoc, using blastn and an E value cut-off of 1 × 10−10, as in StrainPhlAn.Strains that occurred in multiple samples were determined with StrainPhlAnFST) and the intra-population diversity (π) were assessed by the presence of SNVs. Metagenomic sequencing reads were mapped against the reconstructed genomes using MOSAIK66 (version 2.2), with default parameters. A minimum alignment identity of 95% was applied to restrict the mapping to reads of the same species67. Genome–sample combinations generating alignments with a median coverage < 20X and/or a breadth < 40% were not included in downstream analyses. To reduce bias stemming from variation in coverage, alignments were down-sampled to a median coverage of 20X using Picard tools . SNV calling was performed with FreeBayes68 (version 1.1.0) using the -pooled-continuous option on the merged alignment files containing all samples for the same genome. Potential SNVs were required to be supported by four or more reads and to have an allele frequency ≥ 1%.For all neonatal reconstructed genomes that were estimated to be > 65% complete and linked to at least one other sample of the same neonate or their mother, the fixation index (https://github.com/EnvGen/POGENOM), a Perl-based tool that enables population-genomic analysis of metagenome samples. POGENOM was used to calculate the intra-population nucleotide diversity (π), which is defined as the average number of nucleotide differences per site between any two sequence reads chosen randomly from the sample population (0 ≤ π < 1). When reads of two or more samples mapped with sufficient coverage to the same genome, the fixation index (FST) was calculated, reflecting the population differentiation between a pair of samples. FST is defined as one minus the average intra-population diversity of the samples divided by the nucleotide diversity between the samples (inter-population diversity). POGENOM was tuned to include only the loci recovered in all samples mapped to the same reference genome, assuring a valid comparison of the intra-species variation.The output from FreeBayes (VCF-file) was used as input for POGENOM  were assigned to the taxonomy in an open reference approach, using USEARCH69 against the SILVA70 16S rRNA gene amplicon reference database  as covariates, was performed with the R package DESeq2 version 1.10.133. KOs were considered significantly differentially abundant in VD and CSD (±SGA) if the FDR-adjusted P value of the Wald test was < 0.05 for at least one comparison (CSD vs. VD or CSD + SGA versus VD) and the directionality of change in both comparisons was the same. Principal coordinate analysis (PCoA) graphs were generated using Jensen-Shannon distances as implemented in the R package phyloseq72. Differentially abundant pathways were detected through pathway enrichment analysis using a custom R script56. Tests for the enrichment of reconstructed genomes with differentially abundant KOs were performed using Fisher’s exact test and FDR-adjustment for multiple testing in R.The R statistical software package (version 3.3.3) was used for statistical analyses and visualization of the taxonomic profiles derived from metagenomic and amplicon sequencing. Sum normalization and calculations of taxon richness (number of metagenomic OTUs (mOTUs) for metagenomic data or OTUs for amplicon sequencing data), diversity (Shannon), evenness (Pielou) indices and Spearman correlation coefficients were performed using the vegan R package. To discover differences in the data sets between the birth modes at the different collection time points postpartum, Wilcoxon rank-sum tests were applied, with FDR multiple-testing adjustment if applicable. Differential taxonomic abundances (according to delivery mode) were also calculated using ANCOMEscherichia coli strain K-12 (sub-strain MG1655). To maximise yields, LPS was purified from three aliquots of 50 mg of each neonatal faecal sample using the hot phenol–water method73 and further purification was performed using a modified phenol re-extraction protocol74. For the E. coli control samples, three 5 ml overnight cultures were diluted to an optical density (600 nm) of 0.5 and centrifuged. LPS was isolated from cell pellets by the same protocol as above. LPS for each individual was pooled and quantified using an ELISA-based endotoxin detection assay . From the 16 neonatal faecal samples, 11 produced measurable amounts of LPS, whereas 5 were under the detection limit  was used to prepare standard curves for universal prokaryotic and specific E. coli primers, respectively. Reaction mixture, measurements and calculations of bacterial load  were performed as previously described7  to a concentration of 1 ng l77. Most importantly, the NF-κB-dependent pathway in TLR-4 signalling is intact in neonatal MoDCs as they produce pro-inflammatory cytokines upon LPS stimulation, while adult and neonatal DCs are both able to produce comparable levels of TNF-α, IL-6 and IL-8 in response to LPS78. Isolated monocytes were differentiated into dendritic cells (MoDCs) in 12-well plates for 5 days in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% foetal bovine serum (Thermo Fisher Scientific), 20 ng ml−1 each of granulocyte-macrophage colony-stimulating factor , 20 ng ml−1 IL-4 (Peprotech) and 1% penicillin–streptomycin (Invitrogen). To assess the immune stimulatory potential of isolated LPS, we treated MoDCs for 24 h with LPS extracted from VD or CSD (±SGA) neonatal faecal samples using two different methods; one based on LPS volume and one based on the normalization of LPS concentration with the bacterial load (see below for more information).Primary human monocytes were isolated from blood samples obtained from the Luxembourg Red Cross originating from twelve healthy adult donors. Human neonatal dendritic cells (DCs) were previously shown to be competent in MHC class I antigen processing and presentation to the same extent than adult DCs5 MoDCs was added per well. For the negative control, MoDCs were incubated with 7.5 µl of LPS extraction blank, and for the positive control, MoDCs were treated with 15 endotoxin units (EU) LPS isolated from E. coli cultures. MoDCs were treated for 24 h to assess the immunostimulatory potential of the isolated LPS. Treatments were performed in duplicates and tested on at least three different donors. Culture supernatants from stimulated MoDCs were diluted 1/10  and analysed for the presence of TNF-α using a commercial ELISA reagent set  and a microplate reader .As we started from the same amount of material for all the neonatal stool samples and used the exact same extraction protocol to isolate all LPS fractions for all samples, we assumed that if we treated MoDCs from the same donor with the exact same volume of yielded LPS (independent of the concentration of LPS present), we would realistically emulate the microbial LPS load which immune cells would be exposed to in vivo and thus be representative of the immunostimulatory potential of a given sample at 3 days postpartum. To stimulate MoDCs, 7.5 µl of LPS extract per 105 MoDCs were incubated with 15 µl of LPS extraction blank, and for the positive control 2 × 105 MoDCs were treated with 100 EU LPS isolated from E. coli cultures. Treatments were performed on cells from four distinct MoDCs donors (2 × 105 MoDCs/donor), except for LPS isolated from C120, which was only sufficient to stimulate donor 4’s MoDCs in duplicate. MoDCs and isolated LPS samples were incubated for 24 h. Culture supernatants from stimulated MoDCs were diluted twofold and analysed for the presence of seven cytokines  using a Human Premixed Multi-Analyte Kit  and a MagPix multiplex reader , according to the manufacturers’ instructions  were due to the fact that some samples have a much lower bacterial load or if also the bacterial composition (and proportion of Gram-negative bacteria) plays a role in the immunostimulation, we normalized the amount of LPS used to stimulate MoDCs with the bacterial load. For example, the bacterial load was highest for VD neonate C105  and 10 µg of E. coli protein extract were used. A precast gel was loaded with the LPS samples and stained with Coomassie  to check for protein contaminations. Silver staining of the gel was performed using a corresponding kit  according to the manufacturer’s instructions.On the basis of availability of sufficient extracted LPS material, 0.5 µg of extracted LPS from the stool samples of two VD neonates (C007 and C111) collected on day 3 after birth, were prepared with Laemmli sample buffer , heated for 5 min at 95 °C and separated on 12 % Bis-Tris precast gel  at 200 V for 45 min. As positive controls, 0.5 µg, 1 µg and 10 µg of commercially available LPS  were used to compare the purity of the LPS samples. As a positive control, 100 ng of E. coli DNA extract was used. The gel was stained with ethidium bromide, separated at 100 V for 50 min and analysed using a BioDocAnalyse system . To check if nucleic acid contaminations could be identified in isolated LPS samples and would result in a TNF-α response, agarose bands were cut out  were cut out and purified. In addition, a purification blank was generated. The purified DNA fractions were used to stimulate MoDCs following the same protocol as for the stimulation with extracted LPS.To check if LPS extracts were contaminated with immunostimulatory nucleic acids, 0.5 µg of extracted LPS from the stool samples of two VD neonates (C007 and C111), which presented highly concentrated LPS fractions that could be visualised on agarose gel, were prepared with DNA loading dye  and loaded onto a 1% agarose gel. In addition, 0.5, 1 and 10 µg of commercially available LPS , were stimulated with LPS extracted from five selected neonatal faecal samples (three VD and two CSD), which presented sufficient amounts of extractable LPS. HEK-Blue™ TLR and NOD cells are designed to detect stimulants of the human receptors by induction of secreted embryonic alkaline phosphatase (SEAP). For all the cell lines, the levels of SEAP were determined with HEK-Blue™ Detection , a cell culture medium that allows for real-time detection of SEAP.−1 glucose, l-glutamine, Sigma-Aldrich, Belgium), supplemented with 10% foetal bovine serum (Thermo Fisher Scientific), 1% penicillin–streptomycin , 100 µg ml−1 Normocin  and respective selective antibiotics according to the user’s manual.While the hTLR4 receptor only recognizes LPS, hTLR2 recognizes peptidoglycan, lipoteichoic acid and lipoprotein from gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from the yeast cell wall, the receptor NOD1 binds to bacterial molecules containing the D-glutamyl-meso-diaminopimelic acid (iE-DAP) moiety and NOD2 recognizes bacterial molecules (peptidoglycans) and stimulates an immune reaction. HEK-Blue™ cells were grown and maintained in DMEM (4.5 g l−1) was added per well, and second, using the same volume of LPS, where 7.5 µl extracted LPS was added to 105 HEK-Blue™ cells. To convert endotoxin activity (EU) into mass (ng), we considered that around 10 EU are equivalent to 1 ng endotoxin79. For positive controls, HEK-Blue™ NOD1 cells were stimulated with 1 µl TriDAP , HEK-Blue™ NOD2 cells with 1 µl Murabutide , HEK-Blue™ hTLR2 cells with 1 µl of Pam3CSK4  and HEK-Blue™ hTLR4 cells with 1 µl ultrapure LPS . In addition, all cell lines were treated with 1 µl ultrapure LPS  and 1 µl ultrapure LPS  as well as with commercially available LPS : 1 µl of 5 µg µl−1 and 1 µl of 0.01 ng µl−1. For the negative control, HEK-Blue™ cells were incubated with 1 µl of endotoxin-free H2O . All conditions were performed in duplicates and SEAP expression was monitored using a microplate reader at 655 nm  except for LPS isolated from C117 where only 7.5 µl extracted LPS/105 HEK-Blue™ cells was added to the cells and tested in duplicates.To monitor the activation of NF-κB, HEK-Blue™ cells were seeded according to the user’s manual in HEK-Blue™ Detection medium , in flat-bottom 96-well plates and stimulated for 22 h with LPS samples. We used two conditions: first, using the same concentration of LPS, where 1 µl of extracted LPS (0.01 ng µln = 31) collected 3 or 5 days postpartum (13 VD, 13 CSD and five CSD + SGA; 28 samples collected at day 3, 3 samples collected at day 5 postpartum; Supplementary Data Plasma samples n = 3 collectehttps://git-r3lab.uni.lu/Cosmic/Earliest.All custom scripts written for this study are available online at Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14Supplementary Data 15Supplementary Data 16Supplementary Data 17Reporting Summary


We suggest that this method has particular utility for the recovery of bacteriophages for use as natural antimicrobial agents in aquaculture.This work details a protocol for recovering bacteriophages from intertidal sessile mussels and testing their lytic activity against pathogenic bacteria. Although bacteriophages were highly abundant in coastal filter-feeding organisms, they were not detectable in the surrounding water column. This difference reflects the high filtering rate of the mussels, which capture and concentrate high amounts of bacteria, generating an ideal environment for bacteriophages. We validated the protocol providing a mean concentration of 4E + 04 PFU mL Specification TableValue of the ProtocolVibrio ordalii from the Chilean coastal zone. We selected an abundant filter-feeding intertidal mussel (Perumytilus purpuratus) and recovered a high number of bacteriophages. Vibrio ordalii was grown at 20 °C in tryptone-soy agar (TSA) or tryptone-soy broth (TSB)  in which 50 % of the water was replaced with filter-sterilized seawater.Although large numbers of bacteriophages are reported from seawater, the successful recovery of bacteriophages capable of killing specific pathogenic bacteria is dependent on host density and surrounding environmental conditions. Most existing protocols to recover lytic bacteriophages from coastal zones require large volumes of water inoculated with host bacteria to enrich the target bacteriophages followed by the concentration of viral particles through tangential flow filtration or by ultracentrifugation ,2. TheseA is the filtration area; X is the number of counted cells; f is the dilution factor, and a is the area of the field observed under the microscope.To count the bacterial cells in the suspension, the sample was treated with DAPI fluorescent dye  . After Dg. Finally, 500 μL of the aqueous phase corresponding to the virion suspension was recovered, carefully avoiding the uptake of chloroform.Benthic filter-feeder mollusks (mytilid mussels) were collected from the coastal zone of Antofagasta, Chile. These were placed in sterile containers and processed on the same day of collection. First, 100 g of soft tissue were extracted from the shells and homogenized, adding 100 mL of seawater into a sterile blender for 1 min. The product was centrifuged for 10 min at 4000 g and ∼ 50 mL of supernatant was carefully collected, avoiding the movement or inclusion of solid debris. At this stage, a viscous supernatant was obtained due to the presence of elevated amounts of suspended solids, in addition to microorganisms and viral particles. Bacterial counts were conducted during this stage. Two mL of supernatant was sequentially filtered through nitrocellulose membranes of 5 μm, 0.45 μm, and 0.22 μm pore size (Millipore) to eliminate major particles. 100 μL of chloroform was added to 1 mL of the filtrate to remove the remaining bacterial remnants. After 30 s of vortex shaking, the tubes were centrifuged for 5 min at 10,000 2 was added to the culture medium (TSB), to provide the divalent cations required for phage multiplication [Specific lytic phages were isolated by the double layer agar culture technique ,4. For tlication . Two-lay2. They were incubated overnight at 20 °C and the isolation process was repeated at least three times to ensure the presence of a single lytic phage per tube. The phages obtained from the process were named according to the nomenclature norms proposed by Kropinski et al. [http://rebase.neb.com/rebase/rebase.html). Presence of genes associated with a temperate life cycle (i.e. integration/excision genes) must be evaluated by molecular techniques (PCR-Sequencing) to finally ensure that our isolated is an obligately lytic bacteriophage [At least 30 clearly defined lysis plates were selected by gently picking them with sterile wooden sticks (toothpicks), and used to inoculate 1 mL of early exponential bacterial culture containing 10 mM MgCli et al. , followiriophage .As an optional step to increase the number of specific phages for a bacterium, 200 μL of the suspension of (putative) virions could be added to 20 mL of early exponential bacterial culture, supplemented with 10 mM of MgCl2. Then, keep it at 20 °C until observing a medium clarification, for a maximum period of 48 h. To recover the phages, follow the procedure described previously.Vibrio ordalii ATCC-33509 in the intertidal zone of San Jorge bay . We sampled at five points along a 50 m transect, with each point separated by 10 m. At each point, 5 L of sea water and 20 bivalve mussel (Perumytilus purpuratus) were collected and subsequently processed to assess bacterial counts and lytic phage recovery for Vibrio ordalii, following the protocol detailed above. Water samples were subject to a prior enrichment stage to increase the number of lytic specific phages for Vibrio ordalii. Mean (± SD) cell counts were 1E + 09 (± 2E + 08) cell/mL in Perumytilus and 7E + 5 (±3E+05) cell/mL in the surrounding sea water.We undertook a search for lytic bacteriophages for the bacterial strain Vibrio ordalii was observed for all the extracts from Perumytilus, with a mean (±SD) total count of lysis plaques, of 4E + 4 (±8E+03) PFU/mL (Lytic activity against ) PFU/mL . No lysi) PFU/mL b. A tota) PFU/mL a. Seven


For increasing concentrations of direct chlorination, the median total cell count (TCC) values were 9 × 104, 2.9 × 104 and 1.8 × 103 cells/mL, respectively. Electrolysis treatment produced very similar TCC concentrations, 8.8 × 104, 1.1 × 104 and 2.3 × 103 cells/mL. The TCC concentrations were lower than the concentration of the water entering each tank (∼3 × 105 cells/mL). Intact cell count (ICC) measurements indicated that the viable cell concentrations, were less than 10% of the TCC values. Though electrolysis treatment can produce powerful oxidants, such as hydroxyl radical, there was no evidence that electrolysis in this system provided additional benefits beyond chlorine production for control of total or intact cell counts. Oxidation of bacteria by chlorine was the dominant anti-bacterial mechanism in our system. Monitoring of dissolved organic carbon (DOC) and assimilable organic carbon (AOC) did not suggest that carbon-limitation significantly impacted cell counts when chlorination or electrolysis treatment was applied. This work demonstrates that either direct chlorination or electrolysis treatment are able to reduce bacteria concentrations over long-term operation of a hand washing water treatment system. We recommend selecting chlorine residual targets such that a chlorine residual is maintained during periods of challenging operating conditions. We observed that a target residual of 1 mg Cl2/L, in our system, maintained the TCC below the concentration found in Zürich drinking water.Innovative solutions are necessary to enable the decentralized recycling of greywater for applications requiring high-quality water, such as hand washing. While physical barriers such as ultrafiltration membranes effectively prevent the passage of bacteria, and chemical and biological treatments can effectively reduce the carbon content of the treated water, there exists a knowledge gap regarding the application of anti-bacterial strategies to prevent the growth of harmful bacteria following treatment. In this study, the effluent water from a household-scale greywater treatment system was fed to seven parallel experimental post-treatment tanks: three receiving direct chlorination with free chlorine residuals of 0.2, 1 or 5 mg Cl  •Electrolysis and chlorination cell counts were below drinking water concentrations.•Electrolysis treatment did not yield lower cell counts than chlorination.•Inactivation attributed to oxidation damage of cell membranes and nucleic material.•Chlorine residual targets should provide residual during disruptive operation.•We recommend a free chlorine residual target of 1 mg/L in our system. Yet, suEscherichia coli and Pseudomonas aeruginosa . A significant concern however, is the growth of bacteria that may be able to enter into the water after the membrane, while the water is stored until use. Pathogen growth testing has demonstrated that the permeate of a BAMBi system, treating a wastewater similar in composition to greywater, can still contain significant concentrations of assimilable organic carbon (AOC), dissolved organic carbon (DOC) and other nutrients, sufficient to support the growth of ruginosa . Thereforuginosa .Escherichia coli within 5 min of exposure (Free chlorine (HOCl) is a strong oxidant, which affects a cell in many ways, including damaging membranes, disrupting membrane functions and damaging DNA. Batch testing with permeate water from a BAMBi system treating hand washing water mixed with flushing water from a source-separating toilet, demonstrated that a free chlorine concentration of 0.5 mg/L could inactivate 5-log of spiked exposure . March eexposure . Chlorinexposure , which mexposure , or may exposure , which cThe specific goals of this study were to (i) evaluate bacterial content in systems employing different dosages of direct chlorination and electrolysis treatment, (ii) compare the anti-bacterial mechanisms employed in each condition , and (iii) recommend optimal post-treatment solutions for systems treating hand washing water or similar water types such as rainwater or greywater from other sources. Understanding the performance and mechanisms for each post-treatment, as well as how this performance could be affected by challenging operating conditions were ultimately necessary to determine an optimal process configuration.22.12 total surface area polyethersulfone ultrafiltration membrane . Aeration (20 L/min) was delivered with coarse bubbles (∼3 mm diameter) introduced directly below the membrane module. An interruption in the aeration was discovered on the 43rd day of operation, in which the air supply had been unintentionally disconnected. The aeration was resumed at this time, but the duration of the outage is not known. The only pressure driving water though the membrane was generated by the water head on the feeding side (approximately 35 mbar on average).A BAMBi system  has beenThe BAMBi was fed 75 L/day of a synthetic greywater solution Section . The dai2.2The recipe for the synthetic feed was taken directly from, and is described in great detail by, 2.32 (aq) for low (0.2 mg/L), medium (1 mg/L) and high (5 mg/L) final free available chlorine concentrations. The chlorine working solutions were prepared in chilled (4 °C) de-ionized (DI) water with final pH adjusted to 11 using NaOH. Every 15 min, about 0.6 mL of working solution was dosed to each tank. The average flow rate of chlorine solution was approximately 0.6% of the flow rate of water entering each post-treatment tank. Water in control and chlorination tanks were mixed by constant recirculation pumping at a rate of 0.5 L/min.Chlorine was dosed using peristaltic pumps from refrigerated working solutions (4 °C) of 125, 400 and 1200 mg/L Cl2 surface area parallel-plate electrodes and a 3-mm separation distance . Water was pumped from the post-treatment tank, passed through an electrolysis cell and then returned to the post-treatment tank at a rate of 0.5 L/min. Electrolysis was operated at 0.3 A and 6 V with continuous on/off cycles of approximately 1/89, 1/14, and 1/4 (minutes on)/(minutes off) for low, medium and high doses, respectively. The amperage was selected though trial and error such that the all target doses could be achieved with the same amperage without imposing too lengthy off times for the lowest dose. Unintended variation occurred in electrolysis dosing between days 47 and 57 of operation. The degree of electrolysis dosage that occurred during this period is unknown. Proper control was resumed on day 57 and dosage rates were intentionally modified to better achieve the target chlorine concentrations in each tank. Data collected between (not including) days 47 and 57 for electrolysis treatment and all data from days 43 and 44 (when an aeration interruption occurred in BAMBI) were not included in statistical analysis. Monitoring of chlorine concentrations, resulting from the direct chlorination and the electrolysis treatment, suggested that stable dosages of electrolysis resulted in increasing free chlorine production over time. The on/off times were modified to return the free chlorine residual concentrations to the desired targets. The dosing was reduced 6.7% for the medium dose and 33% for the high dose on day 57. The medium dose was reduced an additional 10% and the low dosage was increased 19% on day 61. The polarity of the current was reversed between each on/off cycle. Recirculation pumps were operated continuously. Electrolysis cells were examined for fouling every 1–2 weeks during testing, but no significant fouling was observed.Boron-doped diamond (BDD) electrolysis flow cells were employed for electrolysis treatment, with 85 cm2.4N,N-diethyl-p-phenylenediamine) method .DOC was monitored using a TOC/DOC analyzer . Ammonium was measured with gas-diffusion flow injection  or Hach Lange kits . Nitrite, nitrate and chloride were measured by ion chromatography . Free and combined chlorine were measured using Hach Lange kits based on the DPD  and a flow cytometer  with 30 s reads on 30 μL samples. Intact cell counts (ICC) were conducted as with TCC, except propidium iodide (PI) was also added during incubation. Flow cytometry analysis was selected for bacterial quantification because of the simplicity and speed of processing high quantities of samples (relative to sequencing or qPCR techniques), the ability to quantify bacteria that are non-culturable (and not captured by heterotrophic plate count), and the ability to differentially identify cells with intact cell membranes (TCC) from cells with damaged cell membranes (ICC)  Evian water , 2) water treated by a BAMBi system fed the synthetic greywater described in this study, 3) water treated by a BAMBi system fed a mixture of soap, urine and feces, and 4) water collected from the Chriesbach stream in Dübendorf, Switzerland. Bacteria from the BAMBi waters and the stream water were twice washed and re-suspended in mineral buffer (TCC in the post-treatment tanks and TCC for AOC measurements were conducted using SYBRes (ICC) . Flow cyes (ICC) . The AOCl buffer  by centr33.12/L for chlorine dosed directly, or produced through electrolysis. In each case, the free chlorine concentrations observed were generally close to the target concentrations. For direct chlorination, the median free chlorine concentrations were 0.21, 0.95 and 4.8 mg Cl2/L, respectively for increasing dosages  chlorine concentrations for each dosage.The target residual free chlorine concentrations in the low, medium and high dose conditions were 0.2, 1 and 5 mg Cl dosages a. For el dosages b. With t3.25 cells/mL. For increasing concentrations of direct chlorination, the median TCC values were 9.0 × 104, 2.8 × 104 and 1.6 × 103 cells/mL, respectively. For increasing dosages of electrolysis treatment, the median TCC values were 8.6 × 104, 1.0 × 104 and 1.7 × 103 cells/mL, respectively. Direct chlorination and electrolysis treatments produced similar TCC values for low, medium and high residual free chlorine concentrations, respectively.The TCC includes all cells that can be identified by the flow cytometer. TCC and ICC results are presented in 5 cells/mL with a standard deviation of 5 × 104 cells/mL  while the total chlorine meets the expected 0.2 mg Cl2/L target value. 5 cells/mL, which is significantly higher than the median value over the course of the experiment (9.0 × 104 cells/mL), and higher than the concentration of Zürich drinking water (8.97 × 104 cells/mL)  .Fig. 4BA4The discussion is divided into three sections. The first section focuses on understanding the anti-bactreial mechanisms that produce the reductions in bacterial concentrations observed with both direct chlorination and electrolysis treatments. The second section discusses why we do not see significant differences between direct chlorination and electrolysis treatments operating at similar concentrations of chlorine residual. The third section looks specifically at how the different dosages of direct chlorination and electrolysis treatment perform with respect to bacterial concentrations and how this work advances our ability to design water recycling systems that ensure microbial safety.4.1The three potential anti-bacterial mechanisms that we might expect to observe with direct chlorination or electrolysis treatments are: (1) direct inactivation of bacteria, (2) direct growth inhibition of viable bacteria, or (3) indirect growth inhibition by removal of carbon substrate. Our results demonstrate that inactivation is the dominant anti-bacterial mechanism. In every experimental condition tested, we observed a net reduction in TCC and ICC of at least one order of magnitude compared to the bacterial concentrations of the water entering each tank. We cannot quantify from our data to what degree some growth might still occur in the presence of the different chlorine residual concentrations, however any growth that does occur is less significant than the amount of inactivation. The potential for growth in the presence of chlorine is likely significantly less than the potential for growth in the control, which is itself quite small (population doubles over 24-hour residence time). A specific comparison between direct chlorination and electrolysis treatments is provided in Section ® Green I are generally able to permeate through a cellular membrane, access and then stain the nucleic materials within a cell. PI is used for ICC staining because it is less permeable through the cellular membrane. Positive staining with PI, indicates cells that have suffered membrane damage. Cells which stain positive with a TCC stain but do not stain positive with a membrane integrity stain (like PI) are generally considered intact. Reduction in ICC is often credited as being a conservative indicator of losing viability because a cell must usually undergo significant membrane damage before it becomes permeable to the PI stain  in bacteria is a complex scientific question, as is how best to measure the concentration and viability of cells . The FCMPI stain . More exPI stain . In our 2/L, Pseudomonas aeruginosa, in simulated drinking water, displayed reductions of 4 or more log in ICC and approximately 1 log in TCC. These results are comparable with the moderate reductions in TCC and the much more significant reductions in ICC observed in our study. Pseudomonas aeruginosa inactivation was conducted in pure culture, while our study utilized a community of bacteria. The presence of a community of bacteria introduces the possibility that some bacteria in our system may be inactivated in chlorine at faster, comparable, or slower rates than the Pseudomonas aeruginosa tested by The work of 2/L). Previous research has indicated that chlorine oxidation is capable of transforming a portion of the DOC to AOC in drinking water  examined by 2). Increasing the current density in our application might better promote mineralization, but this might not overcome the influence of the chloride/DOC ratio of our water. Another study seeking to optimize BDD electrode disinfection, this time in treated wastewater, found that increasing current density incurred greater energy costs but did not improve inactivation of bacteria, , of both direct chlorination and electrolysis treatment, generally matched the TCC concentration (8.97 × 104 cells/mL) in drinking water in Zürich Switzerland  failing to maintain TCC values below the drinking water concentration, while the medium and high chlorine dosing still maintained enough chlorine residual to keep the TCC below drinking water concentration.There is not a scientific consensus for what must be done to water before it is safe for hand washing. While it might not be necessary for hand washing water to reach the same level of microbial safety as drinking water, drinking water quality could be considered as a conservative goal for recycled hand washing water. Drinking water is frequently used for hand washing in many well-developed countries and is generally regarded as safe for human exposure. In terms of organic carbon, our BAMBi + GAC treatment system achieved >99% removal of DOC and ultimately achieved a DOC concentration consistent with drinking water levels (0.05 mg DOC/L) . Chlorintzerland . A detaitzerland . We expe2/L would be an effective treatment in our system. This residual chlorine target of 1 mg Cl2/L is a common design target or minimum threshold for many laboratory and case studies, and has generally been observed to be sufficient anti-bacterial control by different means of testing  may mitigate demands on chlorine, and these factors must also be considered. Though this study examined a residual chlorine concentration of 5 mg Clng water . Beyond ng water .2/L. This study has demonstrated that both chlorine and electrolysis treatments complement well our primary BAMBi + GAC treatment to deliver hand washing water that achieves drinking water quality in terms of DOC and TCC concentrations. Choosing between direct chlorination or electrolysis treatments, and possible inclusion of any feedback dosing, should likely be done in accordance with cost and service constrains within specific applications.Our results demonstrate that sufficient quantities of chlorine can be delivered by either direct chlorination or produced through electrolysis. We have discussed that it may be possible to optimize electrolysis to promote mineralization of carbon and possible removal of AOC, however it is not clear if this potential anti-bacterial mechanism provides a benefit in a system that is currently dominated by destructive oxidation of the bacteria. A more productive strategy could be to better understand the relationship between electrolysis and chlorine production in our system. We have manually adjusted the dosages of electrolysis treatment to achieve our target residual concentrations to compensate for an apparent increase over time in chlorine production efficiency. A thorough understanding of what caused this increase would make the application of electrolysis more accurate and require less monitoring. Automatic feedback on electrolysis or direct chlorination could allow the dosing of either post-treatment to better adapt to variations or disruptions and perhaps allow the chorine residual target to be < 1 mg Cl5•The combined treatment of a BAMBi, GAC and either direct chlorination or electrolysis disinfection is suitable to reduce DOC and TCC concentrations from hand washing water to values below drinking water concentrations in Zürich, Switzerland.•The electrolysis treatment resulted in the production of chlorine and concentrations of TCC, ICC, DOC and AOC similar to systems in which the same concentration of chlorine was added directly. This indicates that bacterial inactivation through oxidation by chlorine was the dominant anti-bacterial mechanism and that more powerful oxidants such as hydroxyl radicals were either not produced or did not have any impact on the concentration or bioavailability of carbon or bacterial inactivation.•The dosing of chlorine through direct chlorination or electrolysis must include a factor of safety such that a free chlorine residual is maintained during challenging and disruptive operation.All authors confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process . He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


MdMYB1, which is a major gene regulating fruit skin anthocyanin production. ‘Honeycrisp’ is heterozygous for red fruit skin overcolor at MdMYB1. The ‘Honeycrisp’ haplotype at the LG9 QTL associated with increased ZLC is in linkage phase with the allele at MdMYB1 associated with red color. Selection for the red allele from ‘Honeycrisp’ at MdMYB1 will result in most offspring also inheriting the haplotype at the LG9 QTL associated with high ZLC. The occurrence of two copies of this haplotype was sub-lethal in seedlings of a family where both parents inherited both the red overcolor allele at MdMYB1 and the haplotype at the LG9 QTL associated with high ZLC. This is the first study to have identified a genetic component of ZLC with clear breeding implications.‘Honeycrisp’ is a widely grown and acclaimed apple cultivar that is commonly used in breeding programs. It also has a well-documented tendency to develop the physiological disorder, zonal leaf chlorosis (ZLC). This disorder causes reduced photosynthetic capacity and is thought to be due to a problem with phloem loading, although the underlying genetics of the disorder have not previously been discerned. In order to understand the breeding implications of the disorder, six families with ‘Honeycrisp’ as a parent and one family with ‘Honeycrisp’ as both a maternal and paternal grandparent were evaluated for ZLC incidence over two years. One major quantitative trait locus (QTL) for ZLC incidence was identified on linkage group (LG) 9. A haplotype in ‘Honeycrisp’ that originated from grandparent ‘Duchess of Oldenburg’ was associated with increased ZLC incidence in offspring in both years and all families evaluated. The LG9 QTL was 5 to 10 cM from  Malus × domestica) cultivar in U.S. markets [‘Honeycrisp’ has become a popular apple  apple breeding program that have ‘Honeycrisp’ as a parent have been observed to segregate for ZLC. Considering the importance of ‘Honeycrisp’ as a breeding parent and grower concerns over ZLC, an understanding of the inheritance of ZLC could be useful in apple breeding. With that in mind, the focus of this study was to identify major QTL for ZLC and breeding implications of identified QTL.Two populations were evaluated in this study  Tables. The second population consisted of 152 genotypes from a single family from a cross between ‘Minneiska’ (‘Honeycrisp’ x ‘Minnewashta’)  and ‘MN5Venturia inaequalis) or cedar apple rust (Gymnosporangium juniperi-virginianae) could not be distinguished from ZLC. Zonal leaf chlorosis ratings for genotypes were averaged over replicates for use in quantitative trait loci (QTL) analyses.Trees were rated in 2014, 2015, and 2016 for ZLC on a 1–5 scale similar to that described in Schupp , where 0Seedlings evaluated in 2018 were characterized into 4 classes: extreme dwarf, dwarf, normal, or intermediate at 4 weeks post germination. Extreme dwarf seedlings were less than less than 20 mm tall with extremely small and chlorotic leaves the size of cotyledons and no or undetectable internodes  using Kompetitive Allele Specific PCR (KASP) for SNPs ss475882938, ss475879516, ss475879521, and ss475879580 following the protocol in Simko et al.  modified5 were used for each analysis. Every 250th sample was stored for a total of 1000 samples for use in posterior QTL inferences. QTL positions were identified based on QTL intensity estimates via posterior distributions of QTL locations. QTL regions were recorded as consecutive 2 cM bins where each bin had 2*ln Bayes factors [The QTL analysis methodology used and the rationale for the use of this methodology in this study closely follows that of Howard et al., 2018 . As desc factors  that wer factors . QTL peaSeparate QTL analyses were performed for each population per year and for ZLC ratings averaged over all three years for the ‘Honeycrisp’ families. Two QTL analyses were performed for each population-year combination using different starting seed numbers for the Markov chain Monte Carlo simulation to ensure the results for the identified QTL and their positions were reproducible (data from only one simulation is reported).https://iris.angers.inra.fr/gddh13/) with BLASTN 2.6.0+ [The physical location of SNPs near identified QTL were determined by blasting the 50 bp flanking sequence of each SNP against version 1.1 of the GDDH13 apple reference genome   and traced through known pedigrees to their furthest ancestor. Additional markers not originally included in the genetic map used in this study  were incThe methodology for comparing haplotype effects among and within families closely follows that of Howard et al., 2018 . The effZLC ratings tended to be highest in the ‘Honeycrisp’ x ‘Pitmaston Pineapple’ family . ZLC ratOne major QTL for ZLC was consistently identified in all QTL analyses near the bottom of linkage group (LG) 9 . The peaMdMYB1 gene [MdMYB1 at 5 to 10 cM from the LG9 QTL for ZLC, depending on the year of evaluation , as well as for the first 24.6 cM and last 22.0 cM of LG12 and because MN1764 contains multiple long haplotypes that are identical by state with haplotypes in ‘Duchess of Oldenburg’. MN1764 itself does not exhibit ZLC and the ‘Honeycrisp’ x MN1764 family is not associated with higher average ZLC rating compared to other families evaluated in this study.‘Monark’ also likely inherited its LG9-H5 haplotype from ‘Duchess of Oldenburg’, as one homolog of LG9 in ‘Monark’ consists of haplotypes from ‘Duchess of Oldenburg’ from 16.8 cM to the end of the LG, with a recombination between 41.5 cM and 42 cM. Similarly long haplotypes that are identical between both cultivars exist on LGs 2, 5, 6, 10, and 17, indicating ‘Duchess of Oldenburg’ is a likely ancestor of the chronologically much more recent ‘Monark’. Progeny of ‘Monark’ that inherited the LG9-H5 haplotype were found to have a significantly higher ZLC rating compared to progeny that inherited the LG9-H8 haplotype in 2016, but only when coupled with the LG9-H1 haplotype from ‘Honeycrisp’ .Several cultivars released from the UMN apple breeding program contain one copy of the LG9-H5 haplotype from ‘Duchess of Oldenburg’, including ‘Haralson’, ‘Regent’, and ‘Red Baron’, as well as multiple UMN breeding selections, but no cultivars or selections have been identified with the LG9-H11 from ‘Duchess of Oldenburg’, which would correspond with the first 15 markers of the LG9-H1 haplotype associated with higher ZLC incidence.Segregation distortion at the LG9 QTL was observed in two families. The ‘Honeycrisp’ x ‘Pitmaston Pineapple’ family was deficient of individuals with the LG9-H1 haplotype. This genomic region exhibited the most severe segregation distortion of anywhere across the genome for ‘Honeycrisp’ haplotypes in this family. Additionally, although the diplotype of ‘Pitmaston Pineapple’ is LG9-H3 + LG9-H6 , the ‘PiThe ‘Minneiska’ x ‘MN55’ family evaluated in the field in 2016 contained no individuals with two copies of the LG9-H1 haplotype, though an approximately equal proportion of individuals with the other three diplotype combinations was observed. Seed germination and seedling propagation records indicated that a higher percentage of individuals from this family and other families where both parents were heterozygous for LG9-H1 were stunted and either died or were culled due to lack of vigor several weeks after germination when compared to other families in the breeding program.Of the 460 seedlings from the ‘Minneiska’ x ‘MN55’ cross evaluated in the greenhouse in 2018, 380 lived to the 4-week post germination period. Data for 44 seedlings was discarded for having failed SNP call test results and 10 were discarded for being in the intermediate size category. Of the remaining 326 seedlings, 60 of them were categorized as dwarf or extreme dwarf. Three of the four SNPs used for genotyping the seedlings had consistent genotype ratings. SNP ss475879580 had far more heterozygous SNP calls than expected. These SNP calls were inconsistent with the other three SNPs used for genotyping the seedlings and thus were excluded from analysis. The genotype ratings for seedlings characterized as dwarf and extreme dwarf were similar, so their genotype ratings were combined for comparison to the genotype ratings for seedlings characterized as normal. Few of the seedlings characterized as normal were homozygous LG9-H1, whereas all individuals characterized as dwarf or extreme dwarf were homozygous LG9-H1 .Five different haplotypes were observed at the location of the LG9 QTL for fruit skin overcolor . ‘HoneycThe LG5-H4 haplotype in ‘Pitmaston Pineapple’  at the LA major QTL for ZLC was identified on LG9 in all years of evaluation in this study , making The genetics and expression of ZLC in ‘Honeycrisp’ and its progeny were not explained solely by the LG9-H1 haplotype at the LG9 QTL for ZLC incidence. The majority of the ‘Honeycrisp’ progeny with the LG9-H1 haplotype did not express ZLC at the same severity as ‘Honeycrisp’, which was consistently rated a 4 in each year for the two trees grown alongside its progeny evaluated in this study. It is possible that the specific diplotype of ‘Honeycrisp’ at the LG9 QTL for ZLC is responsible for its high level of ZLC. Some evidence for other alleles at the LG9 QTL for ZLC influencing severity of the disorder can be found in the ‘Honeycrisp’ x MN1702, ‘Honeycrisp’ x ‘Monark’, and the ‘Honeycrisp’ x ‘Pitmaston Pineapple’ families. In the ‘Honeycrisp’ x MN1702 and ‘Honeycrisp’ x ‘Monark’ families, the LG9-H1 + LG9-H5 diplotype was found to be associated with higher average ZLC ratings, particularly in 2016 where ZLC ratings for this diplotype were significantly higher . Severe f apple scab resistance [The observation of severe segregation distortion disfavoring the LG9-H1 haplotype from ‘Honeycrisp’ associated with higher ZLC ratings in offspring indicates a detrimental effect of ZLC and should be considered in breeding decisions, particularly when both parents hold one copy of the haplotype. We deduce this because of the observed segregation distortion in the ‘Honeycrisp’ x ‘Pitmaston Pineapple’ family, because there were no individuals from the ‘Minneiska’ x ‘MN55’ family under field observation that possessed two copies of the LG9-H1 haplotype, and because most seedlings evaluated in 2018 with two copies of the LG9-H1 haplotype were severely dwarfed , will lisistance . These rMdMYB1, which has been demonstrated to regulate anthocyanin biosynthesis in apple skin color [MdMYB1 (MDP0000259614) is located on linkage group 9 between 35,545,015 and 35,549,069 bp [MdMYB1 as close as 5.92 cM or as far as 10.6 cM away from the QTL peaks identified for ZLC Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 TableThe 2*ln Bayes factors are interpreted as having hardly any (0–2), positive (2–5), strong (5–10), and decisive (>10) evidence for a 1 QTL model vs. a 0 QTL model per linkage group. The darker colors on this table represent the larger Bayes factors.(XLSX)Click here for additional data file.S6 TablePhysical and genetic coordinates for each SNP can be found in (XLSX)Click here for additional data file.S7 TablePhysical and genetic coordinates for each SNP can be found in (XLSX)Click here for additional data file.S8 TablePhysical and genetic coordinates for each SNP can be found in (XLSX)Click here for additional data file.S9 TablePhysical and genetic coordinates for each SNP can be found in (XLSX)Click here for additional data file.S1 Fig(JPG)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 FigChromosome numbers are indicated at the top of each graph. Green lines at the bottom of each graph represent SNP marker coverage. Numbers below the series of graphs indicate the cM position at the end of each linkage group.(PNG)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.