The Effect of Oral Iron Supplementation on Gut Microbial Composition: a Secondary Analysis of a Double-Blind, Randomized Controlled Trial among Cambodian Women of Reproductive Age.

The World Health Organization recommends untargeted iron supplementation for women of reproductive age (WRA) in countries where anemia prevalence is greater than 40%, such as Cambodia. Iron supplements, however, often have poor bioavailability, so the majority remains unabsorbed in the colon. The gut houses many iron-dependent bacterial enteropathogens; thus, providing iron to individuals may be more harmful than helpful. We examined the effects of two oral iron supplements with differing bioavailability on the gut microbiomes in Cambodian WRA. This study is a secondary analysis of a double-blind, randomized controlled trial of oral iron supplementation in Cambodian WRA. For 12 weeks, participants received ferrous sulfate, ferrous bisglycinate, or placebo. Participants provided stool samples at baseline and 12 weeks. A subset of stool samples (n = 172), representing the three groups, were randomly selected for gut microbial analysis by 16S rRNA gene sequencing and targeted real-time PCR (qPCR). At baseline, 1% of women had iron-deficiency anemia. The most abundant gut phyla were Bacteroidota (45.7%) and Firmicutes (42.1%). Iron supplementation did not alter gut microbial diversity. Ferrous bisglycinate increased the relative abundance of Enterobacteriaceae, and there was a trend towards an increase in the relative abundance of Escherichia-Shigella. qPCR detected an increase in the enteropathogenic Escherichia coli (EPEC) virulence gene, bfpA, in the group that received ferrous sulfate. Thus, iron supplementation did not affect overall gut bacterial diversity in predominantly iron-replete Cambodian WRA, however, evidence does suggest an increase in relative abundance within the broad family Enterobacteriaceae associated with ferrous bisglycinate use. IMPORTANCE To the best of our knowledge, this is the first published study to characterize the effects of oral iron supplementation on the gut microbiomes of Cambodian WRA. Our study found that iron supplementation with ferrous bisglycinate increases the relative abundance of Enterobacteriaceae, which is a family of bacteria that includes many Gram-negative enteric pathogens like Salmonella, Shigella, and Escherichia coli. Using qPCR for additional analysis, we were able to detect genes associated with enteropathogenic E. coli, a type of diarrheagenic E. coli known to be present around the world, including water systems in Cambodia. The current WHO guidelines recommend blanket (untargeted) iron supplementation for Cambodian WRA despite a lack of studies in this population examining iron's effect on the gut microbiome. This study can facilitate future research that may inform evidence-based global practice and policy.

Quantitative proteomics reveals the crucial role of YbgC for Salmonella enterica serovar Enteritidis survival in egg white.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a food-borne bacterial pathogen that can cause human salmonellosis predominately by contamination of eggs and egg products. However, its survival mechanisms in egg white are not fully understood, especially from a proteomic point of view. In this study, the proteomic profiles of S. Enteritidis in Luria-Bertani (LB) broth containing 50% and 80% egg white, and in whole egg white were compared with the profile in LB broth using iTRAQ technology to identify key proteins that were involved in S. Enteritidis survival in egg white. It was found that there were 303, 284 and 273 differentially expressed proteins in S. Enteritidis after 6 h exposure to whole, 80% and 50% egg white, respectively. Most of up-regulated proteins were primarily associated with iron acquisition, cofactor and amino acid biosynthesis, transporter, regulation and stress responses, whereas down-regulated proteins were mainly involved in energy metabolism, virulence as well as motility and chemotaxis. Three stress response-related proteins (YbgC, TolQ, TolA) of the tol-pal system responsible for maintaining cell membrane stability of Gram-negative bacteria were up-regulated in S. Enteritidis in response to whole egg white. Interestingly, deletion of ybgC resulted in a decreased resistance of S. Enteritidis to egg white. Compared with the wild type and complementary strains, a 3-log population reduction was observed in △ybgC mutant strain after incubation in whole egg white for 24 h. Cellular morphology of △ybgC mutant strain was altered from rods to spheres along with cell lysis in whole egg white. Furthermore, deletion of ybgC decreased the expression of tol-pal system-related genes (tolR, tolA). Collectively, these proteomic and mutagenic analysis reveal that YbgC is essential for S. Enteritidis survival in egg white.

Heat resistance of Salmonella enterica is increased by pre-adaptation to peanut oil or sub-lethal heat exposure.

Cross-protection represents a considerable challenge in the food industry where hurdled interventions are often employed to reduce Salmonella contamination. The heat resistance of Salmonella strains from five serotypes (i.e., Typhimurium, Enteritidis, Tennessee, Thompson and Hartford) at 70 °C was determined by measurement of viable cell populations before and after adaptation to two common stresses employed in low-water activity food processing, desiccation and sub-lethal heat treatment. Survival of Salmonella at 70 °C significantly increased (p < 0.05) following the six-day incubation in peanut oil (aw 0.52 ± 0.00) and/or the exposure to a sub-lethal heat treatment at 45 °C for 3 min. Quantitative PCR revealed upregulation of two desiccation stress-related genes, fadA and otsB, following the peanut oil incubation, whereas heat treatment induced upregulation of a heat-resistance gene, dnaK. Invasion gene invA and alternative sigma factor rpoE were downregulated following either of the treatments. Interestingly, different Salmonella strains yielded different transcriptional profiles. The strain-specific resistance phenotypes and transcriptional profiles provided further insights into the mechanisms employed to tolerate desiccation and heat stresses in the food industry.

Strain-Specific Survival of Salmonella enterica in Peanut Oil, Peanut Shell, and Chia Seeds.

In North America, outbreaks of Salmonella have been linked to low-water activity (aw) foods, such as nuts and seeds. These outbreaks have implicated an assortment of Salmonella serotypes. Some Salmonella serotypes (e.g., Enteritidis and Typhimurium) cause high proportions of salmonellosis. Nevertheless, there has recently been an emergence of uncommon Salmonella serotypes and strains (e.g., Tennessee, Hartford, and Thompson) in low-aw foods. The aim of this study was to evaluate the survival characteristics of Salmonella serotypes Enteritidis, Typhimurium, Tennessee, Hartford, and Thompson in three low-aw food ingredients with varying aw: peanut oil (aw = 0.521 ± 0.003), peanut shell (aw = 0.321 ± 0.20), and chia seeds (aw = 0.585 ± 0.003). The survival of individual Salmonella strains on each food matrix was monitored for a maximum of 150 days by spreading the bacterial cells onto Luria-Bertani and/or xylose lysine deoxycholate agar. Overall, Salmonella survived for the longest periods of time in peanut oil (96 ± 8 days), followed by chia seeds (94 ± 46 days). The survival period was substantially reduced on the surface of peanut shell (42 ± 49 h), although PCR after 70 days of incubation revealed the presence of Salmonella cells. In addition, Salmonella exhibited a strain-specific response in the three low-aw foods tested. Salmonella Hartford was identified as highly persistent in all low-aw food matrices, whereas Salmonella Typhimurium was the least persistent. The current research emphasizes the adaptable nature of Salmonella to low-aw food ingredients. This may pose additional problems owing to the downstream production of various end products. Additionally, unique survival characteristics among Salmonella strains highlight the need for tailored mitigation strategies regarding high-risk Salmonella strains in the food industry.