Human milk cream, a lipid-rich fraction obtained during milk defatting, is typically discarded despite its potential for individualized nutritional strategies. This study evaluated the effects of Holder pasteurization (HoP) and storage conditions (refrigeration and freezing) on the microbiological profile and lipid stability of human milk-derived cream. Cream fractions from six mothers of preterm infants were analyzed for bacterial counts using Columbia Nalidixic Acid and MacConkey agar, with isolate identification by MALDI-TOF MS, and for lipid stability through free fatty acids (FFAs), triacylglycerol (TAGs) composition by GC-FID, and peroxide values (PV) determined by a rapid photometric method. Raw cream showed stable total bacterial counts across storage conditions, with a reduction in Gram-negative bacteria after freezing, while HoP samples exhibited no detectable bacterial growth. Lipolysis was significantly higher in raw cream, with increased FFAs after 72 h refrigeration, whereas HoP samples maintained lower and more stable FFA levels. TAG profiles remained largely stable under refrigeration but were significantly altered during frozen storage in raw samples, suggesting membrane disruption and selective hydrolysis; these changes were attenuated in HoP samples. PV increased over time in both groups, indicating progressive primary lipid oxidation, although values remained within moderate ranges. Overall, HoP improved microbiological safety and lipid stability, supporting the potential use of human milk cream as a controlled lipid source for individualized fortification in preterm infants.

OBJECTIVE:Endometriosis is a chronic, oestrogen-dependent inflammatory disorder affecting up to 10% of reproductive-age women, associated with pelvic pain, infertility, and reduced quality of life. Emerging evidence implicates the vaginal microbiome and oestrogen metabolism in its pathogenesis. This study aimed to evaluate the potential therapeutic role of the oral administration of Ligilactobacillus salivarius CECT 30632, a probiotic with demonstrated ability to metabolise oestrogens in vitro, to improve the quality of life in women with endometriosis. MATERIAL AND METHODS:We conducted a pilot, randomised, double-blind, placebo-controlled clinical trial assessing its effects, alongside standard dienogest treatment, in 37 women with endometriosis. Outcomes included changes in the vaginal microbiota composition (16S rRNA sequencing), in the immunological markers, in serum oestradiol levels, and in the quality-of-life of participants assessed with the EHP-30 questionnaire. RESULTS:Vaginal microbial diversity remained unchanged between groups; however, ∼20% of participants exhibited individualised microbiota shifts, including transitions to Lactobacillus-dominated profiles. In the immunological markers, the IL-10 levels decreased in 65% of probiotic supplemented participants (p = 0.042). Serum oestradiol levels were significantly reduced (∼50%) in the probiotic group (p = 0.013). The probiotic supplementation was associated with significant improvements in pain, emotional well-being, and perceived self-control (p < 0.05). CONCLUSIONS:Daily oral intake of L. salivarius CECT 30632 was associated with changes in circulating oestradiol levels and improvements in the quality of life in women with endometriosis. These preliminary findings support further powered trials to confirm efficacy and clarify underlying mechanisms.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a medical procedure to treat hematologic malignancies and restore bone marrow function. However, this approach may lead to graft-versus-host disease (GvHD), a major cause of mortality and morbidity after allogeneic HSCT. Some studies have suggested the involvement of gut microbiota in the development and prognosis of GvHD. In this context, the main objective of this study was to compare the fecal microbiome composition and short-chain profile of pediatric patients who underwent successful HSCT, developed GvHD or died. The bacterial composition was analyzed using 16S rRNA gene sequencing, while short-chain fatty acids (SCFAs) were quantified by gas chromatography. Fecal samples at engraftment were mainly characterized by a loss of bacterial diversity, a depletion of sequences belonging to the genus Blautia and significantly lower concentrations of fecal butyrate and acetate compared with those obtained before HSCT and 100 days after HSCT. Our findings confirm that children experiencing GvHD after HSCT have distinct gut microbiota and SCFA profiles, which might contribute to developing new microbiota-targeted strategies for GvHD prevention during HSCT procedures.

Artisanal cheese quality relies on a complex microbiota. The generalized use of commercial starter cultures has been associated with reduced microbial diversity, fueling interest in using indigenous lactic acid bacteria (LAB) as adjunct cultures. This study aimed to evaluate Ligilactobacillus salivarius SP36 as a starter or adjunct culture in ripened cheeses. Culture-based and culture-independent analyses were performed, together with the assessment of some physico-chemical parameters (pH, water activity, and color), including the profile of volatile compounds. All cheeses were microbiologically safe according to current EU legislation. The pH of the cheese made only with the SP36 strain was higher than those of the cheeses manufactured with a commercial starter (with or without strain SP36). L. salivarius SP36 modulated the aroma profile by increasing ethyl esters, alcohols, ketones, organic acids and sulphur compounds. LAB dominated all cheeses, with the highest microbial diversity in the cheese produced without the commercial starter. Lactiplantibacillus plantarum and Lacticaseibacillus paracasei isolates were obtained from all cheeses. Overall, L. salivarius SP36 seems a promising adjunct for mature cheeses, while autochthonous L. plantarum and L. paracasei isolates represent promising candidates for starter or adjunct cultures.

Staphylococcus aureus is the leading cause of acute mastitis during lactation. This species has a remarkable ability to form biofilms and to develop antibiotic resistance, which hampers the effectiveness of current therapeutic approaches. This study aims to evaluate the therapeutic potential of three lactic acid bacteria (LAB) strains (Limosilactobacillus fermentum I7, Limosilactobacillus reuteri 7SNG3-30 and Ligilactobacillus salivarius 22SNG3-30) to interfere with biofilms formed by two S. aureus strains isolated from milk of women with acute mastitis. Both live LAB cells and their respective cell-free supernatants were able to disrupt the S. aureus biofilm structure and significantly reduce its cellular viability. However, the effectiveness of these treatments was dependent on the S. aureus strain, the LAB strain, and the type of LAB preparation (active culture or cell-free supernatant) involved in each interaction. Overall, our results suggest that the tested LAB strains have the potential to be used either as probiotics or postbiotics complementing the current therapies against acute mastitis and other staphylococcal infections.

Acute mastitis is a common infection during lactation, primarily caused by Staphylococcus aureus, a bacterium known for its ability to form biofilms within mammary ducts and develop antibiotic resistance. This study aimed to genomically characterize S. aureus strains isolated from women with acute mastitis and healthy asymptomatic women to better understand how S. aureus strains transition from harmless components of the human milk microbiota to pathogenic agents responsible for mastitis. Whole-genome sequencing was performed on nine S. aureus strains-six from women with mastitis and three from healthy women-followed by in silico analyses of core and accessory genes, resistome, virulome, mobilome, and secondary metabolite synthesis to identify genes related to virulence, antibiotic resistance, biofilm formation, and mobile genetic elements such as plasmids, bacteriophages, and pathogenicity islands. Antimicrobial resistance profiles were evaluated using Sensititre EUST2 plates, antimicrobial activity by an agar diffusion method, biofilm formation in 96-well plates, and siderophore production with the Chrome Azurol S assay. Results showed that complete bacteriophage genomes were only present in S. aureus strains isolated from mastitis cases. Some virulence genes, including fnbB and cna, were absent in strains from healthy women. Both types of S. aureus strains exhibited biofilm formation capacity, with mastitis-associated strains SA4 and SA5 being the highest biofilm producers. Similarly, although all strains secreted siderophores, SA4 and SA55 exhibited the strongest siderophore production, indicating a link between this trait and virulence in mastitis-associated strains. The analysis of key genomic features, including virulence factors, resistance genes, and biofilm-forming capabilities, revealed some mechanisms by which S. aureus contributes to the pathogenesis of mastitis.IMPORTANCEAcute mastitis is a widespread infection in lactating women, and its main cause, Staphylococcus aureus, has developed resistance to antibiotics, making treatment challenging. The ability of this bacterium to form biofilms complicates its eradication from the mammary glands. Understanding the genomic and phenotypic characteristics of S. aureus strains associated with mastitis, compared to those isolated from asymtomatic women, is critical for developing better treatment strategies. This study provides new insights into the genetic features, such as virulence factors, antibiotic resistance profiles, and presence of bacteriophages, that make S. aureus strains pathogenic in mastitis. It also highlights the potential of biofilm formation and siderophore production as key factors in mastitis progression. These findings could guide the development of novel therapeutic approaches, such as targeted therapies or probiotics, which can more effectively treat mastitis and reduce reliance on antibiotics, ultimately improving maternal and infant health.

BackgroundThe human milk microbiota is one of the biologically active components of human milk, and factors affecting it and the effect size are not well understood. Assessments of human milk microbiota have mainly been done in small cohorts and/or in single geographical locations, and most have been restricted to the bacteriome. Here we assessed the bacterial, archaeal and fungal composition of human milk and the potential inter-kingdom interactions in milk collected from women living in a wide spectrum of countries, environments, and socio-economical settings.Materials and methodsAbout 518 human milk samples were collected in 16 countries. After DNA extraction, bacterial and fungal metataxonomic analyses were performed via amplification and sequencing of the 16S rDNA and the ITS2 genes, respectively. In parallel, the presence of methanogenic archaea was determined by qPCR.ResultsBacterial analysis revealed significant Country variations in human milk microbiota diversity and taxa distribution. Core genera such as Staphylococcus, Streptococcus, and Bifidobacterium were universally prevalent, and their abundance varied geographically. Methanogenic sequences were found in the amplicon sequences, mostly of Methanobrevibacter (11.8% of samples), while qPCR only detected 0.7% (2 out of 268) methanogens. Fungi—mostly Candida—were detected in 7% of samples, with wide country variations. Co-abundance network analysis revealed mostly positive bacterial correlations and negative inter-kingdom interactions.ConclusionThis study shows substantial global variation in the human milk microbiome with bacterial-fungal interactions, highlighting the importance of global-scale studies to understand the human microbiome and its role in maternal and infant health.

Pollinator health is increasingly threatened by multiple stressors, including climate change, nutritional imbalances, pathogens, and pesticides. Environmental Risk Assessment (ERA) aims to evaluate the potential environmental harm of pesticides and regulate their use in agriculture. However, current ERA frameworks present significant shortcomings, particularly in addressing sublethal and microbiota-mediated effects. Incorporating bee gut microbiota as a biomarker-based monitoring tool could substantially strengthen pesticide risk assessments. In this work, we advocate for the inclusion of bee gut microbiota as a novel endpoint in ERA of pesticides. Using honey bees as a model, we discuss the robustness of gut microbiota as a comparative basis across laboratory and field environments. We also highlight its potential for early detection of sublethal impacts within shorter timeframes, facilitating more sensitive and cost-effective evaluations. Finally, we propose strategies to integrate microbiota-based endpoints into existing ERA frameworks and discuss recent advances and future research directions.

Mastitis, an inflammatory condition affecting more than 25% of breastfeeding women, is usually associated with reduced milk secretion, pain, and discomfort, which often leads to early cessation of breastfeeding. Although the etiology of mastitis is multifactorial, a pro-inflammatory state of the mammary gland might be a risk factor. However, changes in milk composition, and specifically in the milk immune profile, prior to and during mastitis have not been well described. To help close this research gap, we documented the immune profiles of milk produced by both breasts of 10 women experiencing clinical (CM) and 8 women experiencing subclinical (SCM) mastitis during the week of sign/symptom development as well as the week prior and compared them with milk produced by 14 healthy controls. CM was defined as having signs/symptoms of mastitis, whereas SCM was presumed if the participant did not have signs/symptoms of CM, but her milk had a somatic cell count >400,000 cell/mL and/or sodium-to-potassium (Na/K) ratio >1.0. Concentration of 36 immune factors (including immunoglobulins, cytokines, chemokines, and growth factors) was quantified via immunoassays. Milk produced by women who developed CM had distinct immune profiles the week prior to diagnosis, particularly elevated concentrations of pro-inflammatory cytokine IL-1β and regulatory cytokines IL-2, IL-4 and IL-10. In contrast, immune profiles in milk produced by women with SCM did not differ from that produced by healthy women or those with CM the week prior to mastitis onset. Once mastitis appeared, marked changes in milk’s immune profile were observed in both CM and SCM groups. CM was characterized by elevated concentrations of 27 compounds, including pro-inflammatory cytokines (IL-1β, IL-1ra, and TNFα) and chemokines (including IL-8, eotaxin, IP-10, MCP-1, MIP1α, and MIP1β), compared to healthy controls. Milk’s immune profile during SCM was intermediate, showing higher levels of IL-6, IFNγ, and MCP-1 compared to healthy controls, suggesting a milder, more controlled immune response compared to CM. Only milk produced by the mastitis-affected breast had altered immune profiles. Further research is needed to determine if these differences in milk’s immune profiles can be used to improve mastitis risk prediction prior to onset of symptoms.

Background/Objectives: Unexplained infertility is a worldwide problem affecting a significant proportion of couples of reproductive age. Recent studies suggest that alterations in the vaginal microbiota are related to female infertility, while supplementation with some probiotic strains has been shown to improve pregnancy rates in couples experiencing this problem. This study aimed to evaluate the impact of oral administration of Ligilactobacillus salivarius CECT5713 on pregnancy success rates in couples with unexplained infertility prior to in vitro fertilization (IVF). Methods: Seventy couples were randomized to receive either a placebo or a probiotic intervention (one capsule per day containing an excipient only or 3 × 109 viable cells of L. salivarius CECT5713 plus an excipient, respectively); 57 couples completed the study. Baseline data on demographics, health status (including gynecological and reproductive history), and lifestyle habits were collected. Vaginal swabs and semen samples were obtained from each couple before the intervention and immediately prior to IVF or upon confirmed pregnancy and were analyzed for microbiological (using both culture-dependent and -independent methods) and immunological profiles. Results: Oral administration of L. salivarius CECT5713 in couples with unexplained infertility scheduled for IVF resulted in a significantly higher pregnancy success rate (48.1%) compared to the placebo group (20.0%) (one-tailed Chi-square test; p < 0.024). The probiotic intervention improved both vaginal and semen immunological profiles, with no substantial changes observed in their microbial composition. Conclusions: These preliminary findings support the potential of L. salivarius CECT5713 supplementation to enhance fertility outcomes in couples with unexplained infertility.

Adjunct cultures originating from artisanal cheese environments may play an important role in recreating and developing traditional cheese flavours, thanks to their enzymatic activities, involved in different metabolic pathways that occur during cheese ripening. In this work, Ligilactobacillus salivarius SP36, a strain isolated from an old cheese seal, was added as an adjunct culture to the cheese’s raw milk, and its effect on the microbiological, physical–chemical and sensory characteristics of the cheese was studied. The use of L. salivarius SP36 in cheese manufacturing had no significant (p > 0.05) effect on the cheese microbiota, gross composition (fat percentage, protein, total solids, moisture and NaCl concentration), colour or texture of the cheese. However, L. salivarius SP36 increased (p < 0.01) the formation of 25 volatile compounds, including 10 esters, 1 aldehyde, 8 alcohols and 6 carboxylic acids. In addition, cheeses made with L. salivarius SP36 received higher scores (p < 0.01) for aroma intensity and quality than control cheeses. L. salivarius SP36 proved to be a good candidate as an adjunct culture for cheesemaking, since it improved the cheese flavour by making it more intense and recovering typical sensorial notes of traditional cheeses.

Phelan–McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by a developmental delay and autism spectrum disorder (ASD)-like behaviors. Emerging research suggests a link between gut microbiota and neuropsychiatric conditions, including PMS. This study aimed to investigate the fecal microbiota and immune profiles of children with PMS compared to healthy controls. Fecal samples were collected from children diagnosed with PMS and age-matched healthy controls. The bacterial composition was analyzed using 16S rRNA gene sequencing, while short-chain fatty acids (SCFAs) were quantified through gas chromatography. Immunological profiling was conducted using a multiplex cytokine assay. Significant differences were observed in the gut microbiota composition between PMS patients and controls, including a lower abundance of key bacterial genera such as Faecalibacterium and Agathobacter in PMS patients. SCFA levels were also reduced in PMS patients. Immunological analysis revealed higher levels of several pro-inflammatory cytokines in the PMS group, although these differences were not statistically significant. The findings indicate that children with PMS have distinct gut microbiota and SCFA profiles, which may contribute to the gastrointestinal and neurodevelopmental symptoms observed in this syndrome. These results suggest potential avenues for microbiota-targeted therapies in PMS.

The interplay between enterohepatic circulation and the gut microbiota is the main driver determining systemic levels of estrogens and their metabolites. Nevertheless, the role of potentially probiotic microorganisms in estrogen metabolism has not been investigated so far. In this work, we have explored the ability of six Ligilactobacillus salivarius strains isolated from human milk and vaginal samples to degrade and/or conjugate parental estrogens in vitro and under aerobic conditions. The quantification of estrogens and their derivatives was carried out in cell-free supernatants by LC-QQQ-MS. All the tested L. salivarius strains achieved an average degradation rate of estrone and estriol of 98% and 55%, respectively, whereas 17β-estradiol was preferentially conjugated (up to 40%). The presence of seven out of ten genes encoding enzymes relevant for estrogen metabolism was further confirmed by PCR, highlighting their genetic potential for degrading, conjugating and/or deconjugating estrogens. The tested L. salivarius strains may be considered potential probiotics affecting the fate of endogenous estrogens. Clinical trials targeting populations with estrogen-dependent conditions will be required to elucidate the true potential of these strains for the restoration and maintenance of a healthy host estrobolome.

Breastfeeding provides many health benefits, but its impact on respiratory health remains unclear. This study addresses the complex and dynamic nature of the mother-milk-infant triad by investigating maternal genomic factors regulating human milk oligosaccharides (HMOs), and their associations with respiratory health among human milk-fed infants. Nineteen HMOs are quantified from 980 mothers of the CHILD Cohort Study. Genome-wide association studies identify HMO-associated loci on chromosome 19p13.3 and 19q13.33 (lowest P = 2.4e-118), spanning several fucosyltransferase (FUT) genes. We identify novel associations on chromosome 3q27.3 for 6'-sialyllactose (P = 2.2e-9) in the sialyltransferase (ST6GAL1) gene. These, plus additional associations on chromosomes 7q21.32, 7q31.32 and 13q33.3, are replicated in the independent INSPIRE Cohort. Moreover, gene-environment interaction analyses suggest that fucosylated HMOs may modulate overall risk of recurrent wheeze among preschoolers with variable genetic risk scores (P < 0.01). Thus, we report novel genetic factors associated with HMOs, some of which may protect the respiratory health of children.

During pregnancy, women undergo changes that affect virtually every organ, apparatus, or system, including the host microbiota. Most pregnancies progress smoothly despite the common presence of minor side-effects arising from such adaptations. However, some women may experience more serious complications, including gestational diabetes mellitus, preeclampsia, or preterm delivery. Probiotics are one of the products most used to try to prevent or treat any of the minor or severe symptoms or complications that women may experience during pregnancy or lactation; however, most of them have never been tested in such populations and, therefore, their efficacy and safety claims are frequently unsubstantiated. Overall, probiotic trials involving pregnant or lactating women have shown that these products are usually well-tolerated and safe although adverse effects may also exist. Therefore, health professionals attending pregnant or lactating women should be aware of their use and monitor their efficacy and safety. In conclusion, probiotics recommendations for pregnant or lactating women should be based on scientific evidence, opting exclusively for those products that have been designed for the specific target or condition that a pregnant or lactating woman may be experiencing or at risk of, and which efficacy and safety has already been convincingly tested in such populations.

Human milk promotes the growth of bifidobacteria in the infant gut. Adding bifidobacterial species to infant formula may contribute to increasing their presence in the gut of formula-fed infants. Therefore, the safety and anti-infectious effects of Bifidobacterium breve DSM32583, a breast milk isolate, were assessed in a pilot trial involving 3-month-old infants. The infants were randomly assigned to either the probiotic (PG) or the control (CG) groups. All the infants consumed the same formula, although it was supplemented with the strain (1 × 107 cfu/g of formula) in the PG. Overall, 160 infants (80 per group) finished the intervention. Infants in CG gained more weight compared to PG (p < 0.05), but the weights for age Z-scores at 6 months were within the normal distribution for this age group. The rates of infections affecting the gastrointestinal and respiratory tracts and antibiotic therapy were significantly lower in the PG. The bifidobacterial population and the level of short-chain fatty acids were higher (p < 0.05) in the fecal samples of PG infants. No adverse events related to formula consumption were observed. In conclusion, the administration of an infant formula with B. breve DSM32583 was safe and exerted potential beneficial effects on gut health.

Cheesemaking played a pivotal role in the life of the Pyrenean villages where cheese was a most prized commodity and the subject of much local competition. In one of them (Sasa de Sobrepuerto), Mrs. Sebastiana Palacio decided in 1877 to label all the cheeses made in her household with a seal to differentiate them from those made by other local producers. The cheese seal was last used in 1936 and, since then, it has been kept under excellent storage conditions. Since well-preserved cheese seals are rare, and bacterial cells may survive desiccation for long periods, the objective of this work was to isolate and characterize any lactic acid bacteria that survived in the seal. Analysis of the milky crust material revealed the presence of sheep caseins. Culture-based analysis led to the isolation of a strain of Bacillus licheniformis and a strain of Ligilactobacillus salivarius (L. salivarius SP36). The latter was characterized in vitro for safety and dairy-related functional properties. Its genome encodes several genes involved in protein, peptide, and amino acid catabolism, and flavor. Overall, the phenotypic and genetic features of this strain support a high potential for being used as adjunct culture in cheesemaking.

The gut microbiota of honey bees has received increasing interest in the past decades due to its crucial role in their health, and can be disrupted by pathogen infection. Nosema ceranae is an intracellular parasite that affects the epithelial cells of the midgut, altering gut homeostasis and representing a major threat to honey bees. Previous studies indicated that younger worker bees are more susceptible to experimental infection by this parasite, although the impact of infection and of age on the gut bacterial communities remains unclear. To address this, honey bees were experimentally infected with a consistent number of N. ceranae spores at various ages post-emergence (p.e.) and the gut bacteria 7 days post-infection (p.i.) were analysed using real-time quantitative PCR, with the results compared to non-infected controls. Infected bees had a significantly higher proportion and load of Gilliamella apicola. In respect to the age of infection, the bees infected just after emergence had elevated loads of G. apicola, Bifidobacterium asteroides, Bombilactobacillus spp., Lactobacillus spp., Bartonella apis, and Bombella apis. Moreover, the G. apicola load was higher in bees infected at nearly all ages, whereas older non-infected bees had higher loads of Bifidobacterium asteroides, Bombilactobacillus spp., Lactobacillus spp., Ba. apis, and Bo apis. These findings suggest that N. ceranae infection and, in particular, the age of bees at infection modulate the gut bacterial community, with G. apicola being the most severely affected species.