Influencia del procesamiento de la leche humana donada sobre la microbiota intestinal, la expresión genómica y el equilibrio oxidativo en recién nacidos pretérmino menores de 32 semanas de edad gestacional

A nivel mundial, 15 millones de bebés nacen prematuros cada año. El período neonatal es un período de vida excepcionalmente vulnerable durante el cual los recién nacidos a término (RNT) pero especialmente los prematuros tienen, un mayor riesgo de mortalidad y morbilidad que puede conducir a secuelas neurocognitivas, motoras y sensoriales permanentes, constituyendo así un grave problema de salud económico y social. Una colonización microbiana adecuada durante este periodo es fundamental para la maduración apropiada del sistema inmune, el metabolismo así como el desarrollo del sistema nervioso central, favoreciendo la maduración de funciones cognitivas y sensoriales, como la visión. Las alteraciones de la microbiota pueden tener consecuencias importantes para la salud. Los RNT cuentan con un sistema de defensa antioxidante que les protegerá frente al aumento en la producción de radicales libres de oxígeno (ROS) y de nitrógeno (RNS). Sin embargo, los recién nacidos prematuros tienen un sistema de defensa antioxidante inmaduro. La leche materna (LM) ha sido reconocida como el estándar de oro para la nutrición humana. Tiene compuestos bioactivos se consideran no solo protectores sino que también estimulan el crecimiento y neurodesarrollo junto con la maduración del sistema inmune inmaduro. La leche humana donada (LHD) pasteurizada es la mejor alternativa en los prematuros menores de 32 semanas de edad gestacional, menores de 1500 gramos, cuando la leche de la propia madre (LM) no está disponible. Los beneficios demostrados de alimentar a los recién nacidos con LHD frente a las fórmulas artificiales son a corto plazo: su protección frente a la enterocolitis necrotizante la infección nosocomial y mejor tolerancia enteral. Y a largo plazo, presentan un mejor neurodesarrollo que aquellos alimentados con fórmula y un menor riesgo cardiovascular durante la adolescencia. La pasteurización causa la pérdida de algunas de las propiedades biológicas, estructurales y funcionales de la LM. OBJETIVOS: Nuestro objetivo fue determinar el impacto de la LHD sobre la microbiota intestinal, el estrés oxidativo en orina y la expresión genómica en células intestinales epiteliales exfoliadas (CIEE), en recién nacidos prematuros ingresados en una unidad de cuidados intensivos neonatales de referencia. POBLACIÓN Y MÉTODOS: Se realizó un estudio de cohortes, prospectivo, observacional y unicéntrico dónde se incluyen, durante un periodo de 12 meses, todos los recién nacidos ≤ 32 semanas de edad gestacional, ≤ 1500 gramos, ingresados en la Unidad de Neonatología del Hospital Universitario y Politécnico La Fe (Valencia, España), Se recogieron muestras fecales y de orina de 69 prematuros, cuando se consiguió la alimentación enteral completa (definida como ≥150 cc / kg / día) con LM, LHD o fórmula de prematuro (FP). La composición de la microbiota intestinal se analizó mediante secuenciación del gen de ARNr 16S. La determinación de biomarcadores de daño oxidativo al ADN y a las proteínas en muestras de orina de RNPT se realizó siguiendo un método de cromatografía líquida Ultra Performance previamente validado - espectrometría de masas en tándem (UPLC-MS / MS). El ARN total de CIEE se hibridó en el micromatriz Clariom S Human. RESULTADOS: A pesar de una mayor variabilidad, no se encontraron diferencias en la diversidad y riqueza microbiana, aunque el tipo de alimentación influyó significativamente en la composición de la microbiota intestinal en los prematuros y en los perfiles funcionales predictivos. Por otra parte, la evaluación in vivo no invasiva del estrés oxidativo no mostró diferencias estadísticamente significativas en ninguno de los 22 biomarcadores en orina, entre ambos grupos (LM vs LHD) en el momento en que se consiguió la nutrición enteral completa (150 ml / kg / día). Los genes expresados diferencialmente (DEG) derivados del análisis ANOVA (valor p <0.05) revelaron cambios estadísticamente significativos en 1629 transcripciones de CIE en neonatos alimentados con LM versus LHD. El grupo de LM sobreexpresó el gen de la lactoalbúmina alfa (LALBA), el gen de la subunidad I de la citocromo C oxidasa (COX1) y el gen kappa de las caseínas (CSN3), el gen beta (CSN2) y el gen alfa (CSN1S1) y el gen del factor citosólico de neutrófilos 1 (NCF1) infraexpresado en comparación al grupo LHD. Esto explica la falta de activación de las vías inflamatorias, la formación de citocinas no inflamatorias y el bloqueo de la generación de radicales libres de oxígeno (ROS). CONCLUSIONES: 1. La leche humana de donante favorece el desarrollo de un microbioma intestinal que se asemeja más al microbioma adquirido por los lactantes alimentados con LM propia que el microbioma de los lactantes alimentados con fórmula. 2. A pesar de estas diferencias, la leche humana de donante confiere efectos beneficiosos potenciales sobre la funcionalidad intestinal, el sistema inmunitario y las actividades metabólicas. 3. Los neonatos prematuros alimentados con leche humana de donante tienen una capacidad antioxidante similar a la de los prematuros alimentados con leche materna propia, como se refleja en el nivel de biomarcadores urinarios de daño oxidativo en proteínas, ADN o lípidos. 4. La variabilidad en términos de expresión transcriptómica en las células intestinales exfoliadas fue del 12,1% entre la LM propia y la LHD. Sin embargo, 1629 genes se expresaron de forma diferencial entre ambos grupos. 5. Los genes candidatos con diferente expresión entre los prematuros alimentados con LM propia y con LHD fueron Lactoalbúmina alfa (LALBA), Caseína kappa (CSN3), Caseína beta (CSN2), Caseína alfa-1 (CSN1S1), Citocromo C oxidasa subunidad 1 (COX1), Factor citosólico de neutrófilos 1 (NCF1). 6. El análisis biológico de estos genes indica que la LHD tiene un menor potencial antiinflamatorio que la leche materna propia. Esto es importante en cuanto a su capacidad de protección contra los patógenos y el desarrollo de la enterocolitis necrotizante. 7. A pesar de las diferencias, nuestros estudios muestran muchos datos a favor de las importantes ventajas de la LHD sobre la leche de fórmula. Sin embargo, deberían desarrollarse nuevos métodos de pasteurización más respetuosos con la integridad de la leche humana de donante.Worldwide, 15 million babies are born prematurely each year, which is to be estimated at approximately 11% of all births, with an increasing tendency in most of the countries. The neonatal period is an exceptionally vulnerable period of life during which not only full-term newborns but also, and especially premature ones have, compared to other stages of life, an increased risk of mortality and morbidity that can lead to permanent neurocognitive, motor and sensory sequelae, thus constituting a major economic and social problem. An adequate microbial colonization during this period is essential for proper maturation of the immune system, metabolism as well as the development of the central nervous system, encouraging the maturation of cognitive and sensory functions, such as vision. Microbiota alterations can have important health consequences. Full-term newborns have an antioxidant defense system that will protect them against increased production of oxygen-free radicals (ROS) and nitrogen (RNS). Nevertheless, premature newborns have an immature antioxidant defense system. Breast milk (BM) has been recognized as the gold standard for human nutrition. Those bioactive compounds are considered not only protectors but also stimulators of growth and neurodevelopment along with the maturation of the immature immune system. Pasteurized donated human milk (DHM) is the best alternative in premature children under 32 weeks of gestational age, lighter than 1.500 grams, when mother's own milk (OMM) is not available. The proven benefits of feeding newborns with DHM versus artificial formulas are for a short-term: protection against necrotizing enterocolitis, nosocomial infection and better enteral tolerance. For the long term, they have a better neurodevelopment than those fed with formula and a lower cardiovascular risk during adolescence. Pasteurization causes the loss of some of the biological, structural and functional properties. OBJECTIVES: Our objective was to determine the impact of DHM on the gut microbiota, oxidative stress in urine and genomic expression in exfoliated epithelial intestinal cells (EEIC), in premature newborns admitted to a reference neonatal intensive care unit. POPULATION AND METHODS: A cohort, prospective, observational and unicentric study was carried out where all newborns are included for a period of 12 months, ≤ 32 weeks gestational age, ≤ 1.500 grams, admitted in the Neonatology Unit of the University and Polytechnic Hospital La Fe (Valencia, Spain). Fecal and urine samples of 69 preterm were collected, when the neonates reached complete enteral feeding (defined as ≥150 cc/ kg / day). There were 3 groups: neonates fed with OMM, DHM or premature formula (FP). The composition of the gut microbiota was analyzed by sequencing the 16S RNAr gene. The determination of biomarkers of oxidative damage to DNA and proteins in urine samples of premature newborns, as well as lipid peroxidation biomarkers mediated by individual free radicals, was performed following a pre-validated Ultra Performance liquid chromatography method - tandem mass spectrometry (UPLC-MS/MS). The total RNA of CIEE was processed with the Clariom S Human micro-matrix. RESULTS: Despite greater variability, no differences in diversity and microbial wealth were found, even though the type of diet significantly influenced the composition of the gut microbiota in preterm ones and in the predictive functional profiles. Moreover, the non-invasive in vivo evaluation of oxidative stress revealed no statistically significant differences in any of the 22 biomarkers in urine, between the two groups (OMM vs. DHM) when they reached full enteral nutrition (150 ml / kg / day). The OMM group overexpressed the lactalbumin alpha gene (LALBA), the cytochrome C oxidase subunit I gene (COX1) and the casein kappa gene (CSN3), the beta gene (CSN2) and the alpha gene (CSN1S1) and the neutrophil cytosolic factor gene 1 (NCF1) was down-expressed in comparison with the DHM group. This explains the lack of activation of inflammatory pathways, the formation of non-inflammatory cytokines and the blocking of oxygen-free radical generation (ROS). CONCLUSIONS: 1. Donor human milk favors the development of a gut microbiome that resembles the microbiome acquired by infants nourished by their own mother’s milk more than microbiome in formula fed infants. Nonetheless, preterm infants nourished donor human milk, have a distinct microbial profile but shortage in the microbial diversity and richness present in the gut microbiome of infants fed with own mother’s milk. 2. Despite these differences, donor human milk confers beneficial potential effects upon intestinal functionality, immune system and metabolic activities. 3. Preterm infants nourished with donor human milk have similar antioxidant capacity as preterm fed own mother's milk as reflected in the level of urinary biomarkers of oxidative damage to proteins, DNA or lipids. 4. The variability in terms of transcriptomic expression in exfoliated intestinal cells was 12.1% between own mother´s milk and donor human milk. Nevertheless, 1629 genes were differentially expressed between both groups. 5. The candidate genes with different expression between preterm nourished with owns mother milk and donor human milk were Lactalbumin alpha (LALBA),)Casein kappa (CSN3), Casein beta (CSN2), Casein alpha-1 (CSN1S1), Cytochrome C oxidase subunit 1 (COX1), Neutrophil cytosolic factor 1 (NCF1). 6. The biological analysis of these genes indicates that donor human milk possesses a lower anti-inflammatory potential than its own mother´s milk. This is important in terms of its ability to protect against pathogens and the development of necrotizing enterocolitis. 7. Notwithstanding the differences, our studies show many data in favor of important advantages of donor human milk over formula milk. Conversely, new pasteurization methods that preserve the integrity of donor human milk should be developed

Reference ranges for SpO2, respiratory rate, and tidal volume in term newborn infants after birth: a narrative review

Background and Objective: Post-natal adaptation implies respiratory and hemodynamic changes that contribute to the successful switching from transplacental to lung-based respiration and from serial to parallel circulation after cord clamping. Initial respiratory efforts extrude lung fluid to the interstitial space, facilitate alveolar aeration, and alveolar-capillary gas exchange contributing to an increase in the arterial partial pressure of oxygen (paO2). Consequently, there is a decrease in pulmonary vascular resistance and the closure of the intra-and-extracardiac shunts. The establishment of a functional residual capacity contributes to the stabilization of the respiratory rate (RR) and the arterial paO2. This study sought to update the reference ranges for respiratory function, oxygen saturation, and heart rate (HR) to guide neonatologists in the stabilization and/or resuscitation of newborn infants in the delivery room (DR). Methods: Data on the respiratory function and oxygenation pattern of normal term infants in the stabilization period during the first minutes after birth were retrieved and stored by employing continuous pre-ductal pulse oximetry and respiratory function monitoring. The electronic databases searched included MEDLINE, Embase, CINAHL, and Scopus. Statistical methods were employed to establish the percentile curves for each parameter. Key Content and Findings: We provide data of HR and oxygen saturation measured by pulse oximetry and RR and tidal volume (VT) measured by a respiratory function monitor and recorded during the first 10 minutes after birth in healthy term infants. Post-natal respiratory and cardiorespiratory adaptation in healthy term babies is enhanced by delayed cord clamping. HR and oxygen saturation achieved values of 140–160 bpm and 90–95% in the first 2–3 minutes after birth, respectively, and a VT and RR between 5–10 minutes. Conclusions: Updated reference ranges for clinical parameters in the DR constitute a useful tool for guiding neonatologists in the stabilization of newborn infants.M Vento received PI20/00964 and RD16/0022/0001 grants, and the ILC CM20/00187 grant from the Health Research Institute Carlos III (Ministry of Science and Innovation; Kingdom of Spain).Medicin

The effect of Holder pasteurization on the lipid and metabolite composition of human milk

Human milk (HM) is the gold standard for newborn nutrition. When own mother's milk is not sufficiently available, pasteurized donor human milk becomes a valuable alternative. In this study we analyzed the impact of Holder pasteurization (HoP) on the metabolic and lipidomic composition of HM. Metabolomic and lipidomic profiles of twelve paired HM samples were analysed before and after HoP by liquid chromatography-mass spectrometry (MS) and gas chromatography-MS. Lipidomic analysis enabled the annotation of 786 features in HM out of which 289 were significantly altered upon pasteurization. Fatty acid analysis showed a significant decrease of 22 out of 29 detectable fatty acids. The observed changes were associated to five metabolic pathways. Lipid ontology enrichment analysis provided insight into the effect of pasteurization on physical and chemical properties, cellular components, and functions. Future research should focus on nutritional and/or developmental consequences of these changes

Effects of Sepsis on Immune Response, Microbiome and Oxidative Metabolism in Preterm Infants

This is a narrative review about the mechanisms involved in bacterial sepsis in preterm infants, which is an illness with a high incidence, morbidity, and mortality. The role of the innate immune response and its relationship with oxidative stress in the pathogenesis are described as well as their potential implementation as early biomarkers. Moreover, we address the impact that all the mechanisms triggered by sepsis have on the dysbiosis and the changes on neonatal microbiota

Comparing Targeted vs. Untargeted MS2 Data-Dependent Acquisition for Peak Annotation in LC-MS Metabolomics

One of the most widely used strategies for metabolite annotation in untargeted LCMS is based on the analysis of MSn spectra acquired using data-dependent acquisition (DDA), where precursor ions are sequentially selected from MS scans based on user-selected criteria. However, the number of MSn spectra that can be acquired during a chromatogram is limited and a trade-off between analytical speed, sensitivity and coverage must be ensured. In this research, we compare four different strategies for automated MS2 DDA, which can be easily implemented in the frame of standard QA/QC workflows for untargeted LC-MS. These strategies consist of (i) DDA in the MS working range; (ii) iterated DDA split into several m/z intervals; (iii) dynamic iterated DDA of (pre)selected potentially informative features; and (iv) dynamic iterated DDA of (pre)annotated metabolic features using a reference database. Their performance was assessed using the analysis of human milk samples as model example by comparing the percentage of LC-MS features selected as the precursor ion for MS2, the number, and class of annotated features, the speed and confidence of feature annotation, and the number of LC runs required

Galectins-1, -3 and -9 Are Present in Breast Milk and Have a Role in Early Life Development

Galectins (Gal) are a family of conserved soluble proteins with high affinity for β-galactoside structures. They have been recognized as important proteins for successful pregnancy. However, little is known about their presence in breast milk and their role in early infancy. Gal-1, -3 and -9 concentrations were evaluated by Multiplex immunoassays in mother-infant pairs from the MAMI cohort in maternal plasma (MP) (n = 15) and umbilical cord plasma (UCP) (n = 15) at birth and in breast milk samples (n = 23) at days 7 and 15 postpartum. Data regarding mother and infant characteristics were collected. Gal-9 was present in a lower concentration range than Gal-1 and Gal-3 in plasma, specifically in UCP. A major finding in the current study is that Gal-1, -3 and -9 were detected for the first time in all the transitional breast milk samples and no differences were found when comparing the two breastfeeding time points. Finally, Gal levels were associated with some maternal and infant characteristics, such as gestational age, pregnancy weight gain, maternal diet, the gender, infant growth and infant infections. In conclusion, Gal levels seem to be involved in certain developmental aspects of early life. Keywords: galectin; breast milk; umbilical cord plasma; maternal plasm

Association of Maternal Microbiota and Diet in Cord Blood Cytokine and Immunoglobulin Profiles

Mothers confer natural passive immunization to their infants through the transplacental pathway during the gestation period. The objective of the present study was to establish at birth the maternal and cord plasma concentration and relationship of immunoglobulins (Igs), cytokines (CKs), and adipokines. In addition, the impact of the maternal microbiota and diet was explored. The plasma profile of these components was different between mothers and babies, with the levels of many CKs, IgM, IgG2a, IgE, IgA, and leptin significantly higher in mothers than in the cord sample. Moreover, the total Igs, all IgG subtypes, IgE, and the Th1/Th2 ratio positively correlated in the mother-infant pair. Maternal dietary components such as monounsaturated fatty acids-polyunsaturated fatty acids and fiber were positively associated with some immune factors such as IgA in cord samples. The microbiota composition clustering also influenced the plasma profile of some factors (i.e., many CKs, some Ig, and adiponectin). In conclusion, we have established the concentration of these immunomodulatory factors in the maternal-neonatal pair at birth, some positive associations, and the influence of maternal diet and the microbiota composition, suggesting that the immune status during pregnancy, in terms of CKs and Igs levels, can influence the immune status of the infant at birth. Keywords: breast milk; cord blood; cytokine; diet; enterotype; immunoglobulin; microbiota

SARS-CoV-2 RNA and antibody detection in breast milk from a prospective multicentre study in Spain

This study has been supported by a research grant from Fundacion La Marato-TV3 (MilkCORONA, ref 202106).Objectives To develop and validate a specific protocol for SARS-CoV- 2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV- 2 infection on the presence, concentration and persistence of specific SARS-CoV- 2 antibodies. Design and patients This is a prospective, multicentre longitudinal study (April–December 2020) in 60 mothers with SARS-CoV- 2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included. Setting Seven health centres from different provinces in Spain. Main outcome measures Presence of SARS-CoV- 2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV- 2-specific immunoglobulins (Igs)—IgA, IgG and IgM—in breast milk samples from patients with COVID-19. Results All breast milk samples showed negative results for presence of SARS-CoV- 2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV- 2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%–87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation). Conclusions Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV- 2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence.Fundacion La Marato-TV3 20210

SARS-CoV-2 RNA and antibody detection in human milk from a prospective multicenter study in Spain

Objectives To develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies. Design and patients This is a prospective, multicentre longitudinal study (April-December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included. Setting Seven health centres from different provinces in Spain. Main outcome measures Presence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)¿IgA, IgG and IgM¿in breast milk samples from patients with COVID-19. Results All breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%-87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation). Conclusions Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence

Breastfeeding during the COVID-19 pandemic: analysis of the breastmilk antibodies, neutralization capacity and microbiota profile from infected and vaccinated wome

Resumen del póster presentado a las III Jornadas Científicas PTI+ Salud Global, celebradas en el Centro de Ciencias Humanas y Sociales (CCHS), CSIC (Madrid) del 20 al 22 de noviembre de 2023.[Background] Breastmilk is considered the gold standard in infant nutrition and provides bioactive compounds to the neonate, among them antibodies and microbiota. In the context of the COVID- 19 pandemics, there were great concerns about a possible mother-to-infant transfer of SARS-CoV-2, since limited knowledge about the safety of breastfeeding after natural infection or vaccination, as well as the transfer of protective antibodies and their neutralization capacity, was available. Additionally, there are concerns about potential short- and long-term adverse effects of SARS-CoV-2 infection and vaccine-induced changes to the breastmilk microbiome composition, which contributes in shaping the early-life microbiome.[Methods] This study included 60 mothers which had a confirmed SARS-CoV-2 infection and also, 86 mothers vaccinated with mRNA-based (Comirnaty, mRNA-1273) and adenoviral-vectored vaccines (ChAdOx1 nCoV-19) were recruited and breastmilk samples were collected longitudinally from baseline up to 30 days after the second dose at seven or eight time points (depending on vaccine type). In COVID-19 lactating mothers, the presence of SARS-CoV-2 was assessed by RT-qPCR targeting the N1 region of the nucleocapsid gene and the envelope (E) gene. In both studies, the levels of SARS-CoV-2 RBD-specific IgA, IgM and IgG were determined by ELISA. The neutralization capacity was tested using pseudotyped vesicular stomatitis virus carrying either the Wuhan-Hu-1, Delta, or BA.1 Omicron spike proteins. To assess the microbiome composition, DNA from breastmilk samples was extracted and the V3-V4 region of the 16S rRNA gene was sequenced using the MiSeq system of Illumina.[Results] After SARS-CoV-2 infection, no virus-specific RNA was detected in breastmilk samples. Determination of antibody levels in mothers with confirmed SARS-CoV-2 infection showed that 82.9% (58 of 70) of milk samples were positive for at least one of the three tested antibody isotypes. Vaccination elicited also a strong induction of SARS-CoV-2-specific antibodies, which was higher in IgG when compared to COVID-19 convalescent women and was strongly increased after the 2nd dose. mRNA-based vaccines induced higher IgG and IgA levels when compared to the adenovirus- vectored vaccine, and women with previous virus exposure increased their IgG antibodies levels after the first dose to a similar level observed in vaccinated women after the second dose. When assessing the neutralization capacity, natural infection resulted in higher neutralizing titers that correlated positively with levels of SARS-CoV-2-specific immunoglobulin A in breastmilk. Breastmilk samples from COVID-19 convalescent mothers infected during the first wave (Wuhan-Hu-1 strain) neutralized less effectively Omicron BA.1 than the Wuhan-Hu-1 variant. In addition, significant differences in the capacity to produce neutralizing antibodies were observed between both mRNA- based vaccines and the adenovirus-vectored ChAdOx1 COVID-19 vaccine. First results of the analysis of the breastmilk microbiome found no significant differences in the mean diversity of species (alpha-diversity) after natural SARS-CoV-2 infection, whereas some specific bacterial groups were increased (e.g. Enterobacteriaceae).[Conclusions] Overall, our results indicate that breastmilk from naturally infected women or those vaccinated with mRNA-based vaccines contain SARS-CoV-2 neutralizing antibodies that could potentially provide protection to breastfed infants from infection.Peer reviewe