A review of human milk oligosaccharide concentrations of breast milk for infants and young children through 24 months of age

The World Health Organization and American Academy of Pediatrics both support continued breastfeeding beyond 12 months of age up to 24 months of age or beyond. Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk. HMO concentrations in early breast milk have been well-characterized, but less is known about HMO profiles later in lactation. The goals of this literature review and meta-analysis of studies that analyzed HMO concentrations at 12 months of lactation or beyond were to identify the most abundant HMOs in breast milk at various timepoints throughout lactation and assess dynamic changes in HMO concentrations over time. Literature searches were conducted to identify studies on HMO quantification following PRISMA guidelines. Only studies that measured HMOs at/beyond one year of age were analyzed. In total, thirteen studies met eligibility criteria. The identity and number of HMOs measured in each study were recorded. HMOs that appeared in at least 10 articles, termed herein as core HMOs, were selected for further analysis. Concentrations of these HMOs, as well as total HMO levels, were grouped by timepoint (colostrum, 6-, 12-, and >12-months). Core HMOs were identified as 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3′-sialyllactose (3′-SL), and 6′-sialyllactose (6′-SL). These HMOs accounted for >70% of the total HMO pool across timepoints. Total HMO concentrations decreased from colostrum to 6-months but plateaued at 12-months through 24 months. Individual core HMOs generally followed the same trend, with the exception of 3-FL, which increased in concentration over time through 12 months. Overall, HMO concentrations remained at significant levels through one year and the relative abundance of the core HMOs throughout lactation suggests biological relevance. Several studies have demonstrated associations between HMO concentrations in infants with outcomes in young children. Extending these analyses to include prolonged consumption of HMOs (> one year) would be of general interest to the field. To the best of our knowledge, this review is the first to specifically synthesize studies that analyzed HMO concentrations at 12 months lactation. Further research may enhance the understanding of the effects of HMOs beyond infancy and into young childhood

Identification of Small-Molecule Inhibitors of the Salmonella FraB Deglycase Using a Live-Cell Assay

Nontyphoidal salmonellosis is one of the most significant foodborne diseases in the United States and globally. There are no vaccines available for human use to prevent this disease, and only broad-spectrum antibiotics are available to treat complicated cases of the disease. However, antibiotic resistance is on the rise and new therapeutics are needed. We previously identified the Salmonella fraB gene, that mutation of causes attenuation of fitness in the murine gastrointestinal tract. The FraB gene product is encoded in an operon responsible for the uptake and utilization of fructose-asparagine (F-Asn), an Amadori product found in several human foods. Mutations in fraB cause an accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp), which is toxic to Salmonella. The F-Asn catabolic pathway is found only in the nontyphoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of Clostridium; it is not found in humans. Thus, targeting FraB with novel antimicrobials is expected to be Salmonella specific, leaving the normal microbiota largely intact and having no effect on the host. We performed high-throughput screening (HTS) to identify small-molecule inhibitors of FraB using growth-based assays comparing a wild-type Salmonella and a Δfra island mutant control. We screened 224,009 compounds in duplicate. After hit triage and validation, we found three compounds that inhibit Salmonella in an fra-dependent manner, with 50% inhibitory concentration (IC50) values ranging from 89 to 150 μM. Testing these compounds with recombinant FraB and synthetic 6-P-F-Asp confirmed that they are uncompetitive inhibitors of FraB with Ki′ (inhibitor constant) values ranging from 26 to 116 μM. IMPORTANCE Nontyphoidal salmonellosis is a serious threat in the United States and globally. We have recently identified an enzyme, FraB, that when mutated renders Salmonella growth defective in vitro and unfit in mouse models of gastroenteritis. FraB is quite rare in bacteria and is not found in humans or other animals. Here, we have identified small-molecule inhibitors of FraB that inhibit the growth of Salmonella. These could provide the foundation for a therapeutic to reduce the duration and severity of Salmonella infections

Assigning a function to a conserved archaeal metallo-β-lactamase from Haloferax volcanii

The metallo-β-lactamase family of enzymes comprises a large group of proteins with diverse functions in the metabolism of the cell. Among others, this superfamily contains proteins which are involved in DNA and RNA metabolism, acting as nucleases in e.g. repair and maturation. Many proteins have been annotated in prokaryotic genomes as being potential metallo-β-lactamases, but very often the function has not been proven. The protein HVO_2763 from Haloferax volcanii is such a potential metallo-β-lactamase. HVO_2763 has sequence similarity to the metallo-β-lactamase tRNase Z, a tRNA 3′ processing endonuclease. Here, we report the characterisation of this metallo-β-lactamase HVO_2763 in the halophilic archaeon Haloferax volcanii. Using different in vitro assays with the recombinant HVO_2763, we could show that the protein does not have tRNA 3′ processing or exonuclease activity. According to transcriptome analyses of the HVO_2763 deletion strain, expression of proteins involved in membrane transport is downregulated in the mutant. Therefore, HVO_2763 might be involved directly or indirectly in membrane transport

ExpI and PhzI Are Descendants of the Long Lost Cognate Signal Synthase for SdiA

SdiA of E. coli and Salmonella is a LuxR homolog that detects N-acyl homoserine lactones (AHLs). Most LuxR homologs function together with a cognate AHL synthase (a LuxI homolog), but SdiA does not. Instead, SdiA detects AHLs produced by other bacterial species. In this report, we performed a phylogenetic analysis of SdiA. The results suggest that one branch of the Enterobacteriaceae obtained a rhlR/rhlI pair by horizontal transfer. The Erwinia and Pantoea branches still contain the complete pair where it is known as expR/expI and phzR/phzI, respectively. A deletion event removed the luxI homolog from the remainder of the group, leaving just the luxR homolog known as sdiA. Thus ExpR and PhzR are SdiA orthologs and ExpI and PhzI are descendants of the long lost cognate signal synthase of SdiA

Phylogeny of LuxR and LuxI homologs.

<p>a) A phylogenetic tree of LuxR homologs. Only bootstrap values of ≥50 are displayed. A maximum likelihood tree gave similar results (not shown). Blue lines indicate species that contain a solo <i>sdiA</i>. Green lines indicate species that contain <i>sdiA</i> and an adjacent <i>luxI</i> homolog. Pink lines indicate species that contain <i>rhlR.</i> b) A phylogenetic tree of LuxI protein sequences. Only bootstrap values of ≥50 are displayed. A maximum likelihood tree gave similar results (not shown). Green lines indicate species that contain <i>sdiA</i> and an adjacent <i>luxI</i> homolog. Pink lines indicate species that contain <i>rhlR.</i></p

Genomic organization and distribution of <i>sdiA</i> in the <i>Enterobacteriaceae</i>.

<p>a) Distribution of <i>sdiA</i> on a phylogenetic tree based on 16S rDNA sequences. Only bootstrap values of ≥50 are displayed. Blue lines indicate species that contain a solo <i>sdiA</i>. Green lines indicate species that contain <i>sdiA</i> and an adjacent <i>luxI</i> homolog. A maximum likelihood tree gave similar results (not shown). b) Map of the <i>sdiA</i> region in representative organisms that encode SdiA orthologs. Any additional LuxR/LuxI pairs in those organisms are also listed. Genes depicted in white are conserved in all genera and genes in gray are not conserved. <i>sdiA</i> is represented in black and <i>sirA</i> in gray hatched lines.</p

Sugar-Phosphate Toxicities

Accumulation of phosphorylated intermediates during cellular metabolism can have wide-ranging toxic effects on many organisms, including humans and the pathogens that infect them. These toxicities can be induced by feeding an upstream metabolite (a sugar, for instance) while simultaneously blocking the appropriate metabolic pathway with either a mutation or an enzyme inhibitor.</jats:p

The Acyl Homoserine Lactone Receptor, SdiA, of Escherichia coli and Salmonella enterica Serovar Typhimurium Does Not Respond to Indole

ABSTRACT In this study, we tested the hypothesis that the SdiA proteins of Escherichia coli and Salmonella enterica serovar Typhimurium respond to indole. While indole was found to have effects on gene expression and biofilm formation, these effects were not sdiA dependent. However, high concentrations of indole did inhibit N -acyl- l -homoserine lactone (AHL) sensing by SdiA. We conclude that SdiA does not respond to indole but indole can inhibit SdiA activity in E. coli and Salmonella . </jats:p

The Role of Egg Yolk in Modulating the Virulence of Salmonella Enterica Serovar Enteritidis

Contribution of food vehicles to pathogenicity of disease-causing microorganisms is an important but overlooked research field. The current study was initiated to reveal the relationship between virulence of Salmonella enterica serovar Enteritidis and egg yolk as a hosting medium. Mice were orally challenged with Salmonella Enteritidis cultured in egg yolk or tryptic soy broth (TSB). Additionally, mice were challenged with Salmonella Enteritidis cultured in TSB, followed by administration of sterile egg yolk, to discern the difference between pre-growth of the pathogen and its mere presence in egg yolk during infection. The pathogen’s Lethal dose 50 (LD50) was the lowest when grown in yolk (2.8×102 CFU), compared to 1.1×103 CFU in TSB, and 4.6×103 CFU in TSB followed by administration of sterile yolk. Additionally, mice that orally received Salmonella Enteritidis grown in egg yolk expressed a high death rate. These findings were supported by transcriptional analysis results. Expression of promoters of virulence-related genes (sopB and sseA) in genetically modified Salmonella Enteritidis reporter strains was significantly higher (p &amp;lt; 0.05) when the bacterium was grown in the yolk, compared to that grown in TSB. Sequencing of RNA (RNA-seq) revealed 204 differentially transcribed genes in Salmonella Enteritidis grown in yolk vs. TSB. Yolk-grown Salmonella Enteritidis exhibited upregulated virulence pathways, including type III secretion systems, epithelial cell invasion, and infection processes; these observations were confirmed by RT-qPCR results. The transcriptomic analysis suggested that upregulation of virulence machinery of Salmonella Enteritidis grown in egg yolk was related to increased iron uptake, biotin utilization, flagellar biosynthesis, and export of virulence proteins encoded on Salmonella pathogenicity island 1, 2, 4, and 5. These biological responses may have acted in concert to increase the virulence of Salmonella infection in mice. In conclusion, growth in egg yolk enhanced Salmonella Enteritidis virulence, indicating the significance of this food vehicle to the risk assessment of salmonellosis.</jats:p