The authors gratefully acknowledge funding support from the Programa de Pesquisas para o SUS, PPSUS/2020, Minist&#233;rio da Sa&#250;de do Brasil; Funda&#231;&#227;o de Amparo &#224; Pesquisa e Inova&#231;&#227;o do Estado de Santa Catarina, Grant Number: 2021TR000506; Programa de Pesquisa Universal, Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq) do Minist&#233;rio de Ci&#234;ncia e Tecnologia do Brasil, Grant Number: 420996/2023-0; Programa Institucional de Bolsas de Inicia&#231;&#227;o Cient&#237;fica 2022 - 2024, Grant Number: 120815/2023-0. We also thank the volunteers for providing samples.

This protocol was performed as part of the procedures of a clinical trial conducted by our research group (Brazilian Registry of Clinical Trials, ReBEC RBR-729kr8x), and received Ethics Committee approval (process CAAE n&#186; 41063520.4.0 000.0121) and informed consent were obtained from all participants prior to randomization and sample colletction.
The procedure for reconstituting the milk microbiota is done in three steps:
1. Obtaining mother&#39;s raw milk
NOTE: The procedure for obtaining milk can be found elsewhere10,11.
<ol>
	<li>Before the procedure, obtain informed consent from the mother according to the ethical committee and be sure to explain everything that will be done to the mother, addressing any doubts, concerns, fears, and anxieties about the procedure to keep the mother relaxed.</li>
	<li>Have the mother express milk using an electric breast pump into a sterilized container under the supervision of the nurse in charge of the unit, following the established local hygiene standards of each maternity ward.</li>
	<li>After collection, label the container holding the milk with the mother&#39;s information, date, and time of collection. Keep the mother&#39;s own milk (MOM) at a temperature of -4 &#176;C or lower for up to 12 h.</li>
</ol>
2. Defrosting of pasteurized donor&#39;s milk (PDM)
NOTE: The HMBs have their own stock of PDM, frozen at a temperature below -4 &#176;C for a maximum period of up to 6 months, and the procedure is well described in the literature elsewhere12,13,14.
<ol>
	<li>Thaw raw milk or PDM stored in sterilized glass containers with plastic lids in a water bath at 40 &#176;C, ensuring temperature maintenance and a shorter processing time. 
	NOTE: Do not thaw PDM at room temperature or at refrigeration temperature (inside the refrigerator), as thawing times in these cases are extended, which promotes microbial exposure and growth.</li>
	<li>Remove human milk bottles from the water bath before the milk in the bottle reaches a temperature above 5 &#176;C.</li>
</ol>
3. Inoculation of PDM with MOM for microbiome reconstitution
NOTE: The standard volumetric ratio for the process of inoculating PDM with MOM is 90%: 10% (v/v). The steps of the procedure are as follows:
<ol>
	<li>Instruct the technician to wear a lab coat, mask, cap, and gloves.</li>
	<li>Clean the work surface with a surface cleaning solution following local institutional protocol.</li>
	<li>Arrange the materials to be used and prepare the equipment. Preheat the incubator (water bath) with water to 37 &#176;C.</li>
	<li>Plan the final volume of inoculated milk to be used and select an appropriately sized container. Place the container with fresh raw MOM and the PDM in liquid form on the work surface.</li>
	<li>Collect an aliquot of up to 1 mL of the MOM using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot in a DNase/RNase-free cryotube at -80 &#176;C.</li>
	<li>Transfer the remaining volume, equivalent to 10% of the final desired volume of milk, using a sterile volumetric instrument (e.g. syringe, pipette) to a food-safe container to be immersed in a water bath at 37 &#176;C.</li>
	<li>Collect a 1 mL aliquot of the PDM using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot in a DNase/RNase-free cryotube at -80 &#176;C.</li>
	<li>Transfer an amount of PDM equivalent to 90% of the final desired volume of milk using sterile volumetric instrument (e.g., syringe, pipette) to the container with the MOM from the previous step, now forming the inoculated mother&#39;s milk (IM).</li>
	<li>Seal the container with a lid that prevents any external liquid from contaminating the inside of the bottle and immerse it in the water bath at 37 &#176;C for 4 h to allow the microorganisms inoculated in the milk to multiply.</li>
	<li>After 4 h, remove the container with the fermented IM from the water bath, now called reconstituted milk (RM).</li>
	<li>Reserve an aliquot of up to 1 mL using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot store in a DNase/RNase-free cryotube at -80 &#176;C.</li>
	<li>Transfer the remaining volume to a sanitized container for feeding newborns.</li>
</ol>
4. Validation of the method by bacterial culturing on plates
NOTE: All procedures must be performed under sterile conditions to prevent contamination and ensure the accuracy of the dilutions. Bacterial growth can be estimated using methods described elsewhere9,15.
<ol>
	<li>Prepare serial dilutions of each milk sample (PDM, MOM, IM, RM) in sterile saline at 10-1, 10-2, 10-3, and 10-4 concentrations. Mix each dilution thoroughly to ensure uniform bacterial distribution.</li>
	<li>Pippete and spread 100 &#181;L of each diluted sample onto Man-Rogosa-Sharpe (MRS) and Mannitol Salt Agar (MSA) plates in triplicates, using a sterile Drigalski loop or glass spreader. Make sure that the volume is uniformly spread on the agar plate.</li>
	<li>Incubate MRS agar plates in an anaerobic jar containing to create a low-oxygen environment for lactic acid bacteria growth.</li>
	<li>Incubate Mannitol Salt Agar (MSA) plates aerobically to support the growth of Staphylococcus species.</li>
	<li>Incubate the plates at 37 &#177; 2 &#176;C for 48-72 h.</li>
	<li>Select the dilution with a countable number of bacterial colonies (25-250 colonies) on each type of agar</li>
	<li>Count visible colonies on each of the three replicate plates for the selected dilution.</li>
	<li>Calculate the average colony count from triplicate plates for the selected dilution in both MRS and MSA media.</li>
	<li>Determine the number of colony-forming units (CFU) per mL for each sample using the following formula and convert to a base-10 logarithmic scale (log10) to facilitate interpretation. Proceed with statistical analysis of the results. 
	Number of CFU/mL = Number of colonies &#215; total dilution factor/volume plated in mL</li>
</ol>
5. Milk microbiome analysis
NOTE: The milk microbiome analysis can be performed according to the methods and pipeline described elsewhere16. A screenshot of the R script used in this analysis is presented in Supplemental Figure S1.
<ol>
	<li>Centrifuge 500 &#181;L of the sample for 15 s at 180 &#215; g to sediment cells and particulates.</li>
	<li>Collect 350 &#181;L of the supernatant and use it for DNA extraction.</li>
	<li>Quantify the extracted DNA using a spectrophotometer.</li>
	<li>Amplify the V3 and V4 regions of the 16S ribosomal RNA using 20 ng of DNA and primers 357F/805R (see Table of Materials)17.</li>
	<li>Use magnetic beads to purify the resulting amplicons and quantify barcoded amplicons using a DNA fluorimeter.</li>
	<li>Sequence the amplicons in paired-end 250 bp mode.</li>
	<li>Process the resulting sequences by performing quality trimming and adapter removal18. Use paired-end mode (PE) with Phred33 scoring and set a minimum read length of 200 bp to ensure high-quality reads.</li>
	<li>Import paired-end sequence data into QIIME 219 using a manifest file format and the command: qiime tools import.</li>
	<li>Process the imported sequences with DADA2 for denoising, dereplication, and chimera removal by using the command: qiime dada2 denoise-paired. This step generates high-quality representative sequences and a feature table.</li>
	<li>Filter features and sequences to retain only those with sufficient representation using the commands: qiime feature-table filter-features and qiime feature-table filter-seqs.</li>
	<li>Prepare a reference database by extracting regions amplified by the primers (V3-V4) using: qiime feature-classifier extract-reads.</li>
	<li>Use the VSEARCH algorithm for taxonomic classification with the Silva reference database at a 97% identity threshold by executing the command: 
	qiime feature-classifier classify-consensus-vsearch</li>
	<li>Integrate taxonomic assignments with the feature table using BIOM tools: biom add-metadata.</li>
	<li>Perform statistical analyses as described elsewhere20,21.</li>
</ol>

Restoring Pasteurized Donor Milk Microbiota Using Mother's Own Milk

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	<li>Szyller, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+components+of+human+milk+and+their+impact+on+child's+health+and+development,+Literature+Review.">Bioactive components of human milk and their impact on child's health and development, Literature Review.</a> Nutrients. 16 (10), 1487 (2024).</li><li>Fern&#225;ndez, L., Rodr&#237;guez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+milk+microbiota:+Origin+and+potential+uses.">Human milk microbiota: Origin and potential uses.</a> Nestle Nutr Inst Workshop Ser. 94, 75-85 (2020).</li><li>Fern&#225;ndez, L., Ruiz, L., Jara, J., Orgaz, B., Rodr&#237;guez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+preservation,+restoration+and+modulation+of+the+human+milk+microbiota.+Implications+for+human+milk+banks+and+neonatal+intensive+care+units.">Strategies for the preservation, restoration and modulation of the human milk microbiota. Implications for human milk banks and neonatal intensive care units.</a> Front Microbiol. 9, 2676 (2018).</li><li>Meier, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+evidence:+Mothers'+own+milk+is+personalized+medicine+for+very+low+birthweight+infants.">More evidence: Mothers' own milk is personalized medicine for very low birthweight infants.</a> Cell Rep Med. 3 (8), 100710 (2022).</li><li>Fewtrell, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+expressed+breast+milk+volume+in+mothers+expressing+milk+for+their+preterm+infant.">Predictors of expressed breast milk volume in mothers expressing milk for their preterm infant.</a> Arch Dis Child Fetal Neonatal Ed. 101 (6), F502-F506 (2016).</li><li>Abrams, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+human+milk+for+the+high-risk+infant:+Preparation,+safety,+and+usage+options+in+the+United+States.">Donor human milk for the high-risk infant: Preparation, safety, and usage options in the United States.</a> Pediatrics. 139 (1), e20163440 (2017).</li><li>. Brasil &#233; refer&#234;ncia em doa&#231;&#227;o de leite materno Available from: <a target="_blank" href="https://www.gov.br/pt-br/noticias/saude-e-vigilancia-sanitaria/2020/02/brasil-e-referencia-em-doacao-de-leite-materno">https://www.gov.br/pt-br/noticias/saude-e-vigilancia-sanitaria/2020/02/brasil-e-referencia-em-doacao-de-leite-materno</a> (2024)</li><li>Landers, S., Updegrove, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriological+screening+of+donor+human+milk+before+and+after+holder+pasteurization.">Bacteriological screening of donor human milk before and after holder pasteurization.</a> Breastfeed Med. 5 (3), 117-121 (2010).</li><li>Cacho, N. 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Expressing and storing breast milk Available from: <a target="_blank" href="https://www.nhs.uk/conditions/baby/breastfeeding-and-bottle-feeding/breastfeeding/expressing-breast-milk/">https://www.nhs.uk/conditions/baby/breastfeeding-and-bottle-feeding/breastfeeding/expressing-breast-milk/</a> (2020)</li><li>Arslanoglu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+establishment+and+operation+of+a+donor+human+milk+bank.">Recommendations for the establishment and operation of a donor human milk bank.</a> Nutr Rev. 81 (Supplement_1), 1-28 (2023).</li><li>Moro, G. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Lactobacillus+delbrueckii+and+Streptococcus+thermophilus+strains+present+in+artisanal+raw+cow+milk+cheese+using+real-time+PCR+and+classic+plate+count+methods.">Identification of Lactobacillus delbrueckii and Streptococcus thermophilus strains present in artisanal raw cow milk cheese using real-time PCR and classic plate count methods.</a> Pol J Microbiol. 66 (4), 491-499 (2017).</li><li>Ruiz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+approaches+for+the+metataxonomic+analysis+of+the+human+milk+microbiome.">Comparison of two approaches for the metataxonomic analysis of the human milk microbiome.</a> Front Cell Infect Microbiol. 11, 622550 (2021).</li><li>Lopez Leyva, L., Brereton, N. J. B., Koski, K. 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M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+A+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: A flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>Bolyen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interactive,+scalable+and+extensible+microbiome+data+science+using+QIIME+2.">interactive, scalable and extensible microbiome data science using QIIME 2.</a> Nat Biotechnol. 37 (8), 852-857 (2019).</li><li>McMurdie, P. J., Holmes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=phyloseq:+An+R+package+for+reproducible+iInteractive+analysis+and+graphics+of+microbiome+census+data.">phyloseq: An R package for reproducible iInteractive analysis and graphics of microbiome census data.</a> PLoS ONE. 8 (4), e61217 (2013).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+language+and+environment+for+statistical+computing.">R: A language and environment for statistical computing.</a> R Foundation for Statistical Computing. , (2024).</li><li>Torrez Lamberti, M. 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The authors declare that they have no competing interests.

Here we present a protocol to be applied in maternity hospitals associated with a human milk bank, aiming to provide PDM with the reconstitution of the milk microbiota from a specific mother who, for various reasons, cannot provide sufficient milk to her newborn. The protocol is simple and essentially based on two steps. The first involves the application of procedures routinely performed in human milk banks for the proper collection and subsequent pasteurization of donor milk, as well as the collection of the mother's o...

Breast milk is widely recognized as the best source of nutrition for newborns, providing not only essential macro and micronutrients but also a complex array of elements, including various metabolites and components of the immune system, such as antibodies, cytokines, and cells1. Additionally, the beneficial properties in breast milk also arise from a community of commensal microorganisms. All these elements of breast milk play a crucial role in the development of the newborn1. The community of microorganisms, known as the microbiota, is influenced by a combination of factors such as diet, maternal lifestyle, type of delivery, and gestational age, resulting in each milk having a microbiota with particular characteristics2. The mechanisms by which the breast milk microbiota benefits neonatal development range from protecting the enteric surface through colonization by commensal bacteria to promoting immune system maturation through the production of metabolites that interact with immune cells in the intestine3. For this reason, the individualized profile of microorganisms in each mother&#39;s milk makes it a form of personalized medicine for newborns4.
The presence of a healthy microbiota can help protect the newborn against colonization by potentially pathogenic microorganisms by occupying the ecological niche and supporting nutrition, especially for premature infants, who are more vulnerable to these external impacts due to being in a hospital environment and having an immature immune system. Moreover, mothers who give birth prematurely often have difficulty producing milk in sufficient quantities for their newborns. Milk production is typically lower the younger the gestational age5. In these cases, the best option for feeding the newborn is pasteurized donor milk provided by Human Milk Banks (HMB)6.
Brazil has the largest and most extensive network of HMBs in the world7. This network collects, prepares, and distributes more than 160,000 L of pasteurized donor human milk (PDM) throughout the country at no cost to parents, through the national unified health system (Sistema &#218;nico de Sa&#250;de), managed by the Ministry of Health7. Pasteurization is essential to ensure the biosafety of the donated milk intended for newborns. This process is usually carried out using the Holder method, which involves immersing a container of raw donor breast milk in a water bath at 62.5 &#176;C for 30 min6,8. The resulting PDM is then stored under refrigeration until it is ready for consumption. However, pasteurization reduces the viability of the milk&#39;s commensal microbiota and decreases the bioavailability of various bioactive components. This is because the pasteurization process is standardized to prevent the transmission of pathogenic microorganisms to the newborn. In this context, in 2017, Cacho and colleagues described the possibility of reconstituting the commensal microbiota of a mother&#39;s own milk by inoculating it in various proportions into PDM9. Their findings demonstrated that using a 10% concentration of the mother&#39;s own milk (MOM) is the optimal concentration for maintaining the original composition of the MOM microbiome profile. This approach reduces the risk of overgrowth of specific bacteria, which, although typically commensal, could become harmful if disproportionately present in the reconstituted milk. Specifically, the study observed that a 1:10 dilution (10% MOM + 90% PDM), incubated for 4 h, resulted in a well-balanced bacterial load&#160;&#8212;&#160;approximately 60% of that found in undiluted MOM -- indicating a safer and more effective approach. In contrast, higher dilutions, such as 3:10, led to excessive bacterial growth, highlighting the critical need for controlled concentrations and conditions to ensure safety.
In this article, we demonstrate that this procedure of reconstituting the breast milk microbiota in PDM can be achieved in real-world situations within the routine care of a maternity hospital and its associated human milk bank.

<table><tbody><tr><td>Electric breast milk pump</td><td>Horigen</td><td>XN-2219M2</td><td>Soft pump dual plus</td></tr><tr><td>Pyrex&amp;nbsp;media bottles</td><td>Corning&amp;nbsp;</td><td>CLS1395100</td><td>Glass containers with plastic lids&amp;nbsp;</td></tr><tr><td>1000 &amp;micro;L pipette Labmate pro</td><td>Corning HTL SA&amp;nbsp;&amp;nbsp;</td><td>5666</td><td>Variable volume pipettor&amp;nbsp;</td></tr><tr><td>Cryogenic vial&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>CLS431417</td><td>DNase/RNase-free 2 mL vials&amp;nbsp;</td></tr><tr><td>15 mL centrifuge tubes</td><td>Corning&amp;nbsp;</td><td>CLS431470</td><td>DNase/RNase-free 15 mL tubes&amp;nbsp;</td></tr><tr><td>Water bath</td><td>EcoSonics</td><td>Q3.0/40A</td><td></td></tr><tr><td>Man&amp;ndash;Rogosa&amp;ndash;Sharpe (MRS)&amp;nbsp;</td><td>Difco</td><td>288210</td><td>Lactic acid bacteria growth media</td></tr><tr><td>Mannitol Salt Agar (MSA)</td><td>Himedia</td><td>MH118</td><td>Staphylococcus&amp;nbsp; growth media</td></tr><tr><td>Anaerobic Jar</td><td>Permution</td><td></td><td>Create a low-oxygen environment for lactic acid bacteria growth</td></tr><tr><td>Extracta&amp;nbsp; Kit &amp;ndash; DNA e RNA</td><td>Loccus</td><td>MPTA-PV16-B Y</td><td>DNA extraction Kit</td></tr><tr><td>Nanodrop</td><td>Promega</td><td>E6150</td><td>Quantus DNA</td></tr><tr><td>GoTaqG2</td><td>Promega</td><td>M7841</td><td>PCR amplication System</td></tr><tr><td>Quantus Fluorometer</td><td>Promega</td><td>E5150</td><td>DNA / RNA quantitation kit</td></tr><tr><td>MiSeq System</td><td>Illumina Inc.</td><td>M-GL-00006 v4.0</td><td>Sequencing equipment</td></tr><tr><td>MiSeq Reagent Kit v2 (300-cycles)</td><td>Illumina Inc.</td><td>MS-102-2002</td><td>Sequencing kits and reagents</td></tr><tr><td>&lt;strong&gt;Software name&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trimmomatic</td><td>http://www.usadellab.org/cms/index.php?page=trimmomatic</td><td></td><td>Read trimming tool for Illumina NGS data</td></tr><tr><td>QIIME 2&amp;nbsp;</td><td>https://docs.qiime2.org/2024.10/</td><td></td><td>Microbiome analysis package&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Primer name&lt;/strong&gt;</td><td>&lt;strong&gt;Primer sequence&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>16S_357F&amp;nbsp;</td><td>TCGTCGGCAGCGTCAGA&lt;br/&gt; TGTGTATAAGAGACAGC&lt;br/&gt; CTACGGGNGGCWGCAG</td><td></td><td></td></tr><tr><td>16S_805R</td><td>GTCTCGTGGGCTCGGAG&lt;br/&gt; ATGTGTATAAGAGACAGG&lt;br/&gt; ACTACHVGGGTATCTAATC</td><td></td><td></td></tr></tbody></table>

individualized reconstitution of human milk microbiota: a feasible approach in real-world settings

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot breastfeed, the preferred alternative is pasteurized donor human milk (PDM), which is routinely provided by human milk banks. PDM offers a superior range of nutritional and immunological elements compared to any commercially available formula. However, to ensure biosafety, PDM undergoes pasteurization, a process that inactivates commensal microbiota and reduces certain bioactive compounds. This study presents a protocol designed to restore the microbiota of PDM using MOM as a microbial source, adapting the approach to a real-world clinical setting.
The protocol was implemented in a clinical trial conducted at a maternity hospital and its associated human milk bank, with the aim of providing personalized donor milk to preterm infants whose mothers cannot produce sufficient milk. The methodology involves inoculating PDM with 10% of MOM, followed by incubation at 37 &#176;C for 4 h. Microbiological analysis demonstrated successful bacterial growth in the inoculated milk (IM) post incubation, with the microbiota profile of the reconstituted milk (RM) closely resembling that of MOM, indicating effective microbiota restoration. These results suggest that the reconstitution protocol is feasible for implementation in neonatal care, with the potential to enhance the nutritional and immunological quality of PDM, thereby supporting the health and development of non-breastfed newborns.

Bacterial growth analysis by plate culturing 
The microbiological tests conducted to validate the method of inoculating PDM with the MOM, as proposed by Cacho et al.9, demonstrated that, based on the CFU/mL count, there were no significant differences between the inoculated milk samples before and after the 4 h incubation. However, differences in the number of colonies were observed at these two time points, indicating bacterial growth during incubation on MSA for Staphyl...

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

The article describes a protocol for reconstituting pasteurized donor milk microbiota using the mother's own milk microbiota in practical, real-world settings. It demonstrates effective bacterial growth and microbiome modulation, supporting the feasible application of this procedure within the routine care of a maternity hospital and its associated human milk bank.

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot ...

individualized-reconstitution-human-milk-microbiota-feasible-approach

Research

JoVE Journal

Medicine

395 Views.  Federal University of Santa Catarina. We onderzoeken of het mogelijk is om de microbiota van een specifieke moedermelkconserverende voedingsstructuur in gepasteuriseerde melk te herstellen door middel van een methodologie die in ziekenhuizen kan worden geïmplementeerd. Ons doel is om een nieuwe nutritionele superstrategie voor pasgeborenen te ontwikkelen en de zichtbaarheid ervan te beoordelen. Wij zijn van mening dat de grootste uitdaging bij het experimenteel reconstrueren van de microbiota va...

Video: Geïndividualiseerde reconstitutie van de microbiota van moedermelk: een haalbare aanpak in de praktijk

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Bioengineering

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

Immunology and Infection

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of the specific role of individuals, defined or complex groups of microorganisms in host response. Traditionally bred and reared in flexible-film or semi-rigid isolators, germ-free mouse husbandry and experimental manipulation are costly and require numerous trained staff and a large space footprint in animal housing facilities. The IsoPositive caging system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages (isocages), reducing cost and enabling greater flexibility in experimental manipulations. 
Here, a protocol is described for transferring germ-free mice from breeding isolators to isocages and subsequent fecal transfer from human donor stool into mice to create stable long-term gut "humanized" mice for future studies. The materials and preparation needed for the utilization of the isocage system are described, including the use of chlorine-dioxide sterilant chemical sterilant to clean cages, supplies, equipment, and personal protective equipment. The methods for confirming the germ-free status of transferred mice and how to determine contamination in the caging system are discussed. The procedure for husbandry, including bedding, food, and water supply, is further discussed. The protocol for human fecal slurry preparation and gavage into germ-free mice to create gut "humanized" mice, along with stool collection to monitor the microbial community composition of these mice, are described. An experiment illustrates that two weeks post-human fecal transplant allows for stable colonization of donor microbiota in the murine hosts, enabling subsequent experimental usage. Furthermore, the collection of humanized mouse feces in viability preservation media, enabling use in further functional experiments, is described. Overall, these methods allow for the safe and effective establishment of humanized mouse communities in experimental gnotobiotic cages for further manipulation.

This protocol describes the best practices for germ-free mouse transfer to and housing in experimental single-cage isolators (isocages) while maintaining sterile conditions. Methods for fecal transplant into germ-free mice and the collection of viable bacteria from these gut "humanized" mice for further applications are discussed.

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of ...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

Biology

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

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