Noninvasive prediction models of intra-amniotic infection in women with preterm labor.

BACKGROUND: Among women with preterm labor, those with intra-amniotic infection present the highest risk of early delivery and the most adverse outcomes. The identification of intra-amniotic infection requires amniocentesis, perceived as too invasive by women and physicians. Noninvasive methods for identifying intra-amniotic infection and/or early delivery are crucial to focus early efforts on high-risk preterm labor women while avoiding unnecessary interventions in low-risk preterm labor women. OBJECTIVE: This study modeled the best performing models, integrating biochemical data with clinical and ultrasound information to predict a composite outcome of intra-amniotic infection and/or spontaneous delivery within 7 days. STUDY DESIGN: From 2015 to 2020, data from a cohort of women, who underwent amniocentesis to rule in or rule out intra-amniotic infection or inflammation, admitted with a diagnosis of preterm labor at <34 weeks of gestation at the Hospital Clinic and Hospital Sant Joan de Déu, Barcelona, Spain, were used. At admission, transvaginal ultrasound was performed, and maternal blood and vaginal samples were collected. Using high-dimensional biology, vaginal proteins (using multiplex immunoassay), amino acids (using high-performance liquid chromatography), and bacteria (using 16S ribosomal RNA gene amplicon sequencing) were explored to predict the composite outcome. We selected ultrasound, maternal blood, and vaginal predictors that could be tested with rapid diagnostic techniques and developed prediction models employing machine learning that was applied in a validation cohort. RESULTS: A cohort of 288 women with preterm labor at <34 weeks of gestation, of which 103 (35%) had a composite outcome of intra-amniotic infection and/or spontaneous delivery within 7 days, were included in this study. The sample was divided into derivation (n=116) and validation (n=172) cohorts. Of note, 4 prediction models were proposed, including ultrasound transvaginal cervical length, maternal C-reactive protein, vaginal interleukin 6 (using an automated immunoanalyzer), vaginal pH (using a pH meter), vaginal lactic acid (using a reflectometer), and vaginal Lactobacillus genus (using quantitative polymerase chain reaction), with areas under the receiving operating characteristic curve ranging from 82.2% (95% confidence interval, ±3.1%) to 85.2% (95% confidence interval, ±3.1%), sensitivities ranging from 76.1% to 85.9%, and specificities ranging from 75.2% to 85.1%. CONCLUSION: The study results have provided proof of principle of how noninvasive methods suitable for point-of-care systems can select high-risk cases among women with preterm labor and might substantially aid in clinical management and outcomes while improving the use of resources and patient experience.

The Microbiota and Malnutrition: Impact of Nutritional Status During Early Life.

According to the developmental origins of health and disease hypothesis, our health is determined by events experienced in utero and during early infancy. Indeed, both our prenatal and postnatal nutrition conditions have an impact on the initial architecture and activity of our microbiota. Recent evidence has underlined the importance of the composition of the early gut microbiota in relation to malnutrition, whether it be undernutrition or overnutrition, that is, in terms of both stunted and overweight development. It remains unclear how early microbial contact is linked to the risk of disease, as well as whether alterations in the microbiome underlie the pathogenesis of malnutrition or are merely the end result of it, which indicates that thequestion of causality must urgently be answered. This review provides information on the complex interaction between the microbiota and nutrition during the first 1,000 days of life, taking into account the impact of both undernutrition and overnutrition on the microbiota and on infants' health outcomes in the short- and long-term.

Association of Maternal Secretor Status and Human Milk Oligosaccharides With Milk Microbiota: An Observational Pilot Study.

BACKGROUND AND OBJECTIVES: Breast milk contains several bioactive factors including human milk oligosaccharides (HMOs) and microbes that shape the infant gut microbiota. HMO profile is determined by secretor status; however, their influence on milk microbiota is still uncovered. This study is aimed to determine the impact of the FUT2 genotype on the milk microbiota during the first month of lactation and the association with HMO. METHODS: Milk microbiota from 25 healthy lactating women was determined by quantitative polymerase chain reaction and 16S gene pyrosequencing. Secretor genotype was obtained by polymerase chain reaction-random fragment length polymorphisms and by HMO identification and quantification. RESULTS: The most abundant bacteria were Staphylococcus and Streptococcus, followed by Enterobacteriaceae-related bacteria. The predominant HMO in secretor milk samples were 2'FL and lacto-N-fucopentaose I, whereas non-secretor milk was characterized by lacto-N-fucopentaose II and lacto-N-difucohexaose II. Differences in microbiota composition and quantity were found depending on secretor/non-secretor status. Lactobacillus spp, Enterococcus spp, and Streptococcus spp were lower in non-secretor than in secretor samples. Bifidobacterium genus and species were less prevalent in non-secretor samples. Despite no differences on diversity and richness, non-secretor samples had lower Actinobacteria and higher relative abundance of Enterobacteriaceae, Lactobacillaceae, and Staphylococcaceae. CONCLUSIONS: Maternal secretor status is associated with the human milk microbiota composition and is maintained during the first 4 weeks. Specific associations between milk microbiota, HMO, and secretor status were observed, although the potential biological impact on the neonate remains elusive. Future studies are needed to reveal the early nutrition influence on the reduction of risk of disease.

Breast Milk Polyamines and Microbiota Interactions: Impact of Mode of Delivery and Geographical Location.

BACKGROUND/AIMS: The aim of the present study was to identify and quantify the polyamine levels in human milk obtained from different countries and through different modes of delivery, and to investigate their association with breast milk microbes. METHODS: Mature breast milk samples were obtained from 78 healthy mothers after 1 month of lactation from 4 different geographical locations: Finland, Spain (Europe); South Africa (Africa); and China (Asia). Polyamines were determined using HPLC after dansyl derivatization and milk microbiota was obtained by 16S rRNA gene sequencing. RESULTS: The mean values of polyamines in breast milk were 70.0, 424.2, and 610.0 nmol/dL for putrescine, spermidine and spermine, respectively, and 1,170.9 nmol/dL of total polyamines. The levels of putrescine were significantly higher in Spain (p < 0.05) and spermidine levels were significantly higher in Finland (p < 0.05) compared with other countries. Cesarean delivery had an impact on polyamine levels and it was related to an increase in the putrescine concentration being significant in Spanish samples (p < 0.01). Furthermore, putrescine levels were correlated positively with Gammaproteobacteria (r = 0.46, p < 0.001), especially with Pseudomonas fragi (r = 0.40, p < 0.001). CONCLUSIONS: The results demonstrate significant effect of geographical variations in human milk polyamine concentrations, being correlated with human milk microbiota composition. These differences may have an impact on infant development during lactation.

Revealing microbial recognition by specific antibodies.

BACKGROUND: Recognition of microorganisms by antibodies is a vital component of the human immune response. However, there is currently very limited understanding of immune recognition of 50 % of the human microbiome which is made up of as yet un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples, and its interaction with the immune system. RESULTS: We show the power of the technique in human faecal, saliva, oral biofilm and breast milk samples, labeled with fluorescent anti-IgG or anti-IgA antibodies. Using Fluorescence-Activated Cell Sorting (FACS), bacterial cells were separated depending on whether they are coated with IgA or IgG antibodies. Each bacterial population was PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which were recognized by each immunoglobulin. CONCLUSIONS: The application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases.

Resembling breast milk: influence of polyamine-supplemented formula on neonatal BALB/cOlaHsd mouse microbiota.

Infant microbiota is influenced by numerous factors, such as delivery mode, environment, prematurity and diet (breast milk or formula). In addition to its nutritional value, breast milk contains bioactive substances that drive microbial colonisation and support immune system development, which are usually not present in infant formulas. Among these substances, polyamines have been described to be essential for intestinal and immune functions in newborns. However, their effect on the establishment of microbiota remains unclear. Therefore, the aim of the present study was to ascertain whether an infant formula supplemented with polyamines has an impact on microbial colonisation by modifying it to resemble that in breast-fed neonatal BALB/c mice. In a 4 d intervention, a total of sixty pups (14 d old) were randomly assigned to the following groups: (1) breast-fed group; (2) non-enriched infant formula-fed group; (3) three different groups fed an infant formula enriched with increasing concentrations of polyamines (mixture of putrescine, spermidine and spermine), following the proportions found in human milk. Microbial composition in the contents of the oral cavity, stomach and small and large intestines was analysed by quantitative PCR targeted at fourteen bacterial genera and species. Significantly different (P< 0·05) microbial colonisation patterns were observed in the entire gastrointestinal tract of the breast-fed and formula-fed mice. In addition, our findings demonstrate that supplementation of polyamines regulates the amounts of total bacteria, Akkermansia muciniphila, Lactobacillus, Bifidobacterium, Bacteroides-Prevotella and Clostridium groups to levels found in the breast-fed group. Such an effect requires further investigation in human infants, as supplementation of an infant formula with polyamines might contribute to healthy gastrointestinal tract development.

Role of vertical and horizontal microbial transmission of antimicrobial resistance genes in early life: insights from maternal-infant dyads.

Early life represents a critical window for metabolic, cognitive and immune system development, which is influenced by the maternal microbiome as well as the infant gut microbiome. Antibiotic exposure, mode of delivery and breastfeeding practices modulate the gut microbiome and the reservoir of antibiotic resistance genes (ARGs). Vertical and horizontal microbial gene transfer during early life and the mechanisms behind these transfers are being uncovered. In this review, we aim to provide an overview of the current knowledge on the transfer of antibiotic resistance in the mother-infant dyad through vertical and horizontal transmission and to highlight the main gaps and challenges in this area.

Intake of Natural, Unprocessed Tiger Nuts (Cyperus esculentus L.) Drink Significantly Favors Intestinal Beneficial Bacteria in a Short Period of Time.

Horchata is a natural drink obtained from tiger nut tubers (Cyperus esculentus L.). It has a pleasant milky aspect and nutty flavor; some health benefits have been traditionally attributed to it. This study evaluated the effects of an unprocessed horchata drink on the gut microbiota of healthy adult volunteers (n = 31) who consumed 300 mL of natural, unprocessed horchata with no added sugar daily for 3 days. Although there were no apparent microbial profile changes induced by horchata consumption in the studied population, differences could be determined when volunteers were segmented by microbial clusters. Three distinctive enterogroups were identified previous to consuming horchata, respectively characterized by the relative abundances of Blautia and Lachnospira (B1), Bacteroides (B2) and Ruminococcus and Bifidobacterium (B3). After consuming horchata, samples of all volunteers were grouped into two clusters, one enriched in Akkermansia, Christenellaceae and Clostridiales (A1) and the other with a remarkable presence of Faecalibacterium, Bifidobacterium and Lachnospira (A2). Interestingly, the impact of horchata was dependent on the previous microbiome of each individual, and its effect yielded microbial profiles associated with butyrate production, which are typical of a Mediterranean or vegetable/fiber-rich diet and could be related to the presence of high amylose starch and polyphenols.

Metallomic and Untargeted Metabolomic Signatures of Human Milk from SARS-CoV-2 Positive Mothers.

SCOPE: Lack of information about the impact of maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the elemental and metabolomic profile of human milk (HM). METHODS AND RESULTS: An observational study on HM from mothers with COVID-19 is conducted including a prepandemic control group. Maternal-infant clinical records and symptomatology are recorded. The absolute quantification of elements and untargeted relative metabolomic profiles are determined by inductively coupled plasma mass spectrometry and gas chromatography coupled to mass spectrometry, respectively. Associations of HM SARS-CoV-2 antibodies with elemental and metabolomic profiles are studied. COVID-19 has a significant impact on HM composition. COVID-19 reduces the concentrations of Fe, Cu, Se, Ni, V, and Aluminium (Al) and increases Zn compared to prepandemic control samples. A total of 18 individual metabolites including amino acids, peptides, fatty acids and conjugates, purines and derivatives, alcohols, and polyols are significantly different in HM from SARS-CoV-2 positive mothers. Aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine, and linoleic acid pathways are significantly altered. Differences are obtained depending on COVID-19 symptomatic and asymptomatic status. CONCLUSIONS: This study provides unique insights about the impact of maternal SARS-CoV-2 infection on the elemental and metabolomic profiles of HM that warrants further research due the potential implications for infant health.

Glyphosate-based herbicide affects the composition of microbes associated with Colorado potato beetle (Leptinotarsa decemlineata).

Here, we examined whether glyphosate affects the microbiota of herbivores feeding on non-target plants. Colorado potato beetles (Leptinotarsa decemlineata) were reared on potato plants grown in pots containing untreated soil or soil treated with glyphosate-based herbicide (GBH). As per the manufacturer's safety recommendations, the GBH soil treatments were done 2 weeks prior to planting the potatoes. Later, 2-day-old larvae were introduced to the potato plants and then collected in two phases: fourth instar larvae and adults. The larvae's internal microbiota and the adults' intestinal microbiota were examined by 16S rRNA gene sequencing. The beetles' microbial composition was affected by the GBH treatment and the differences in microbial composition between the control and insects exposed to GBH were more pronounced in the adults. The GBH treatment increased the relative abundance of Agrobacterium in the larvae and the adults. This effect may be related to the tolerance of some Agrobacterium species to glyphosate or to glyphosate-mediated changes in potato plants. On the other hand, the relative abundances of Enterobacteriaceae, Rhodobacter, Rhizobium and Acidovorax in the adult beetles and Ochrobactrum in the larvae were reduced in GBH treatment. These results demonstrate that glyphosate can impact microbial communities associated with herbivores feeding on non-target crop plants.

Exercise Training Modulates Gut Microbiota Profile and Improves Endotoxemia.

INTRODUCTION: Intestinal metabolism and microbiota profiles are impaired in obesity and insulin resistance. Moreover, dysbiotic gut microbiota has been suggested to promote systemic low-grade inflammation and insulin resistance through the release of endotoxins particularly lipopolysaccharides. We have previously shown that exercise training improves intestinal metabolism in healthy men. To understand whether changes in intestinal metabolism interact with gut microbiota and its release of inflammatory markers, we studied the effects of sprint interval (SIT) and moderate-intensity continuous training (MICT) on intestinal metabolism and microbiota in subjects with insulin resistance. METHODS: Twenty-six, sedentary subjects (prediabetic, n = 9; type 2 diabetes, n = 17; age, 49 [SD, 4] yr; body mass index, 30.5 [SD, 3]) were randomized into SIT or MICT. Intestinal insulin-stimulated glucose uptake (GU) and fatty acid uptake (FAU) from circulation were measured using positron emission tomography. Gut microbiota composition was analyzed by 16S rRNA gene sequencing and serum inflammatory markers with multiplex assays and enzyme-linked immunoassay kit. RESULTS: V˙O2peak improved only after SIT (P = 0.01). Both training modes reduced systematic and intestinal inflammatory markers (tumor necrosis factor-α, lipopolysaccharide binding protein) (time P < 0.05). Training modified microbiota profile by increasing Bacteroidetes phylum (time P = 0.03) and decreasing Firmicutes/Bacteroidetes ratio (time P = 0.04). Moreover, there was a decrease in Clostridium genus (time P = 0.04) and Blautia (time P = 0.051). Only MICT decreased jejunal FAU (P = 0.02). Training had no significant effect on intestinal GU. Colonic GU associated positively with Bacteroidetes and inversely with Firmicutes phylum, ratio Firmicutes/Bacteroidetes and Blautia genus. CONCLUSIONS: Intestinal substrate uptake associates with gut microbiota composition and whole-body insulin sensitivity. Exercise training improves gut microbiota profiles and reduces endotoxemia.

Bacterial analysis of breast milk: a tool to differentiate Raynaud's phenomenon from infectious mastitis during lactation.

Lactational Raynaud's syndrome may be misdiagnosed as infectious mastitis on the basis of the breast pain. The objective of this work was to elucidate if microbiological analysis of milk may contribute to the differentiation of both conditions. Ten lactating women clinically diagnosed by Spanish lactation consultants were included in the study. Of these, five suffered from mastitis and the remaining five suffered from Raynaud's syndrome. Breast milk samples were inoculated on diverse culture media. Seventy isolates were selected and identified by 16S rDNA PCR sequencing. Parallel, PCR-DGGE and quantitative real-time PCR were used to assess the presence of bacterial DNA in the samples. Neither bacteria nor yeasts could be detected in the milk samples provided by the women suffering from Raynaud's syndrome. In contrast, large numbers of bacteria were isolated from those with infectious lactational mastitis. Globally, the levels of bacterial DNA were significantly higher in the milk of mastitis-suffering women. Bacteriological analysis of milk can be an useful tool to facilitate the differential diagnosis between the infectious mastitis and Raynaud's syndrome during lactation.

The Mucin degrader Akkermansia muciniphila is an abundant resident of the human intestinal tract.

A 16S rRNA-targeted probe, MUC-1437, was designed and validated in order to determine the presence and numbers of cells of Akkermansia muciniphila, a mucin degrader, in the human intestinal tract. As determined by fluorescent in situ hybridization, A. muciniphila accounted more than 1% of the total fecal cells and was shown to be a common bacterial component of the human intestinal tract.

Effect of glucose in removal of microcystin-LR by viable commercial probiotic strains and strains isolated from dadih fermented milk.

The removal of the cyanobacterial peptide toxin microcystin-LR at 4 and 37 degrees C by six commercial probiotic strains and Lactobacillus plantarum strains IS-10506 and IS-20506 isolated from dadih, Indonesian traditional fermented milk, was assessed in this study. The aim was to evaluate the main factors influencing the viability and metabolic activity of the probiotic strains, as well as their capacity to remove microcystin-LR. Both L. plantarum strains isolated from dadih, as well as Bifidobacterium lactis strains Bb12 and 420, were shown to be more resistant, and >85% remained viable in phosphate-buffered saline (PBS) solution for 48 h of incubation at either temperature, while the viability of the other four commercial bacteria decreased markedly over time. The effect of glucose on viability and removal of toxin was shown to be a strain-specific and strain-dependent property, but in general, the efficiency of microcystin-LR removal increased when glucose was added to the solution. A maximum removal of 95% was observed for L. plantarum strain IS-20506 (37 degrees C, 10 (11) colony-forming units mL(-1)) with 1-2% glucose supplementation and 75% in PBS alone.

Intestinal integrity and Akkermansia muciniphila, a mucin-degrading member of the intestinal microbiota present in infants, adults, and the elderly.

Fluorescence in situ hybridization and real-time PCR analysis targeting the 16S rRNA gene of Akkermansia muciniphila were performed to determine its presence in the human intestinal tract. These techniques revealed that an A. muciniphila-like bacterium is a common member of the human intestinal tract and that its colonization starts in early life and develops within a year to a level close to that observed in adults (10(8) cells/g) but decreases (P < 0.05) in the elderly.

Measurement of aggregation properties between probiotics and pathogens: in vitro evaluation of different methods.

Aggregation properties of probiotics with pathogens are of importance for both food preservation and therapeutic impact of food on intestinal microbiota. We assessed spectrophotometry, fluorescence and radioactivity techniques to characterize and quantify co-aggregation. Probiotic strains tested showed co-aggregation abilities, which were strain-specific and dependent on time and incubation conditions. Co-aggregation may be useful for preliminary screening in order to identify potentially probiotic strains suitable for food, human or animal use.

Characterization of the gastrointestinal mucosa-associated microbiota of pigs and chickens using culture-based and molecular methodologies.

The microbiota of the gastrointestinal tract (GIT) can play an important role in the healthy status of farm animals and in the safety of the whole food chain. In this study, the mucosa-associated microbiota of the GIT of pigs and chickens was analyzed by culture methods and fluorescence in situ hybridization combined with flow cytometry (FCM-FISH). In all pig GIT sections, lactic acid bacteria, Enterobacteriaceae, Atopobium, Bacteroides, and Clostridium histolyticum were the predominant bacterial groups. Atopobium, Bifidobacterium, Bacteroides, and Lactobacillus were detected at higher levels (P < 0.05) in the intestine than in the stomach. In all broilers' GIT sections, lactic acid bacteria, Atopobium, Bacteroides, and Escherichia coli were the predominant bacterial groups. Atopobium, Bifidobacterium, E. coli, and Eubacterium rectale--Clostridium coccoides counts were significantly higher (P < 0.05) in the crop, while those of Bacteroides and Lactobacillus were higher (P < 0.05) in the large intestine. Lactic acid bacteria were one of the predominant GIT mucosa-associated bacteria of pigs and broilers, which could be an index of their healthy status. FCM-FISH analysis also allowed the detection of bacterial groups hard to cultivate yet quantitatively important. The distribution of Lactobacillus and Bacteroides followed the same trend in both animal species, whereas that of Atopobium and Bifidobacterium was the opposite. These results contribute to the knowledge on the diversity and distribution of the animal GIT mucosa-associated microbiota.

Potential probiotic characteristics of Lactobacillus and Enterococcus strains isolated from traditional dadih fermented milk against pathogen intestinal colonization.

Traditional fermented buffalo milk in Indonesia (dadih) has been believed to have a beneficial impact on human health, which could be related to the properties of the lactic acid bacteria (LAB) involved in its fermentation process. In previous studies, it was discovered that strains of dadih lactic isolates possessed some beneficial properties in vitro. In the present study, the adhesion capacity of specific LAB isolates from dadih to intestinal mucus was analyzed. Further, the ability to inhibit model human pathogens and displace them from mucus was assessed. The adhesion of tested LAB strains was strain-dependent and varied from 1.4 to 9.8%. The most adhesive Lactobacillus plantarum strain was IS-10506, with 9.8% adhesion. The competition assay between dadih LAB isolates and pathogens showed that a 2-h preincubation with L. plantarum at 37 degrees C significantly reduced pathogen adhesion to mucus. All tested LAB strains displaced and inhibited pathogen adhesion, but the results were strain-specific and dependent on time and pathogen strains. In general, L. plantarum IS-10506 showed the best ability against pathogen adhesion.

Method for direct selection of potentially probiotic Bifidobacterium strains from human feces based on their acid-adaptation ability.

A method for direct selection of acid-resistant Bifidobacterium strains was developed by prolonged exposure of human feces to homologous lethal stress conditions. The recovered strains were intrinsically resistant to acidic gastric conditions (pH 2.0) and also showed good tolerance to high concentrations of bile salts and NaCl. Bifidobacterium breve and Bifidobacterium adolescentis were, respectively, the infant- and adult-type bifidobacterial species showing the highest ability to develop an acid-tolerant phenotype. Therefore, this procedure could be applicable to the direct selection of Bifidobacterium strains with improved stability in adverse environments and, probably, contribute to expand the spectra of probiotic species of human origin currently marketed.

Adhesion properties and competitive pathogen exclusion ability of bifidobacteria with acquired acid resistance.

The adhesion properties of Bifidobacterium longum and Bifidobacterium catenulatum strains with an acquired resistance to acid and their ability to competitively exclude Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, Enterobacter sakazakii, and Clostridium difficile from adhering to human intestinal mucus were evaluated and compared with the results when the same experiments were run with the original acid-sensitive strains. In half of the four studied cases, the acid-resistant derivative showed a greater ability to adhere to human intestinal mucus than the original strain. The ability of bifidobacteria to inhibit pathogen adhesion to mucus was not generally improved by the acquisition of acid resistance. In contrast, three of the four acid-resistant strains showed a greater ability to displace preadhered pathogens than the original strains, especially preadhered Salmonella Typhimurium and C. difficile. Overall, the induction of acid resistance in bifidobacteria could be a strategy when selecting strains with enhanced stability and improved surface properties that favor their potential functionality as probiotics against specific pathogens.