Studying the urine microbiome in superficial bladder cancer: samples obtained by midstream voiding versus cystoscopy.

BACKGROUND: Preliminary data suggest that the urinary microbiome may play a role in bladder cancer. Information regarding the most suitable method of collecting urine specimens is needed for the large population studies needed to address this. To compare microbiome metrics resulting from 16S ribosomal RNA gene sequencing between midstream, voided specimens and those obtained at cystoscopy. METHODS: Adults, with a history of superficial urothelial cell carcinoma (non-muscle invasive bladder cancer) being followed with periodic surveillance cystoscopy had a urine sample collected by a mid-stream, voided technique and then from the bladder at cystoscopy. Urine samples underwent 16S ribosomal RNA gene sequencing on the Illumina MiSeq platform. RESULTS: 22 subjects (8 female, 14 male) were included. There was no significant difference in beta diversity (diversity between samples) in all samples between collection methods. However, analysis by sex revealed a difference between voided and cystoscopy samples from the same individual in males (p = 0.006, Adonis test) but not in females (p = 0.317, Adonis test). No differences were seen by collection method in any alpha diversity (diversity within a sample) measurement or differential abundance of taxa. CONCLUSIONS: Beta diversity of the urine microbiome did differ by collection method for males only. This suggests that the urinary microbiomes of the two collection methods are not equivalent to each other, at least in males, which is the sex that bladder cancer occurs most frequently in. Therefore, the same collection method within a given study should be used.

Assessment of the Urinary Microbiome in Children Younger Than 48 Months.

BACKGROUND: The urinary tract was once thought to be sterile, and little is known about the urinary microbiome in children. This study aimed to examine the urinary microbiome of young children across demographic and clinical factors. METHODS: Children <48 months, undergoing a urinary catheterization for clinical purposes in the Pediatric Emergency Department were recruited and urine samples collected. Detailed demographic and clinical information were recorded. Urine samples underwent DNA extraction and 16S ribosomal RNA gene sequencing, urinalysis and urine culture. RESULTS: Eighty-five children were included; a urinary microbiome was identified in every child. Nine children had Escherichia coli urinary tract infections (UTIs) identified. Those with UTIs had a significantly decreased alpha diversity (t test, P < 0.001) and the composition of the microbiome clustered separately (P = 0.001) compared with those without UTIs. CONCLUSIONS: A urinary microbiome was identified in every child, even neonates. Differences in microbiome diversity and composition were observed in patients with a standard culture positive UTI. The urinary microbiome has just begun to be explored, and the implications on long-term disease processes deserve further investigation.

Prenatal and Peripartum Exposure to Antibiotics and Cesarean Section Delivery Are Associated with Differences in Diversity and Composition of the Infant Meconium Microbiome.

The meconium microbiome may provide insight into intrauterine and peripartum exposures and the very earliest intestinal pioneering microbes. Prenatal antibiotics have been associated with later obesity in children, which is thought to be driven by microbiome dependent mechanisms. However, there is little data regarding associations of prenatal or peripartum antibiotic exposure, with or without cesarean section (CS), with the features of the meconium microbiome. In this study, 16S ribosomal RNA gene sequencing was performed on bacterial DNA of meconium samples from 105 infants in a birth cohort study. After multivariable adjustment, delivery mode (p = 0.044), prenatal antibiotic use (p = 0.005) and peripartum antibiotic use (p < 0.001) were associated with beta diversity of the infant meconium microbiome. CS (vs. vaginal delivery) and peripartum antibiotics were also associated with greater alpha diversity of the meconium microbiome (Shannon and Simpson, p < 0.05). Meconium from infants born by CS (vs. vaginal delivery) had lower relative abundance of the genus Escherichia (p < 0.001). Prenatal antibiotic use and peripartum antibiotic use (both in the overall analytic sample and when restricting to vaginally delivered infants) were associated with differential abundance of several bacterial taxa in the meconium. Bacterial taxa in the meconium microbiome were also differentially associated with infant excess weight at 12 months of age, however, sample size was limited for this comparison. In conclusion, prenatal and peripartum antibiotic use along with CS delivery were associated with differences in the diversity and composition of the meconium microbiome. Whether or not these differences in the meconium microbiome portend risk for long-term health outcomes warrants further exploration.

Comparison of Infant Gut and Skin Microbiota, Resistome and Virulome Between Neonatal Intensive Care Unit (NICU) Environments.

Background: There is a growing move to provide care for premature infants in a single family, private room neonatal intensive care unit (NICU) in place of the traditional shared space, open bay NICU. The resultant effect on the developing neonatal microbiota is unknown. Study Design: Stool and groin skin swabs were collected from infants in a shared-space NICU (old NICU) and a single-family room NICU (new NICU) on the same hospital campus. Metagenomic sequencing was performed and data analyzed by CosmosID bioinformatics software package. Results: There were no significant differences between the cohorts in gestational age, length of stay, and delivery mode; infants in the old NICU received significantly more antibiotics (p = 0.03). Differentially abundant antimicrobial resistance genes and virulence associated genes were found between the cohorts in stool and skin, with more differentially abundant antimicrobial resistance genes in the new NICU. The entire bacterial microbiota analyzed to the genus level significantly differed between cohorts in skin (p = 0.0001) but not in stool samples. There was no difference in alpha diversity between the two cohorts. DNA viruses and fungi were detected but did not differ between cohorts. Conclusion: Differences were seen in the resistome and virulome between the two cohorts with more differentially abundant antimicrobial resistance genes in the new NICU. This highlights the influence that different NICU environments can have on the neonatal microbiota. Whether the differences were due to the new NICU being a single-family NICU or located in a newly constructed building warrants exploration. Long term health outcomes from the differences observed must be followed longitudinally.

Collection of non-meconium stool on fecal occult blood cards is an effective method for fecal microbiota studies in infants.

BACKGROUND: Effective methods are needed to collect fecal samples from children for large-scale microbiota studies. Stool collected on fecal occult blood test (FOBT) cards that can be mailed provides an effective solution; however, the quality of sequencing resulting from this method is unknown. The aim of this study is to compare microbiota metrics of 16S ribosomal RNA (rRNA) gene sequencing from stool and meconium collected on FOBT cards with stool collected in an Eppendorf tube (ET) under different conditions. METHODS: Eight stool samples from children in diapers aged 0 month-2 years and three meconium samples were collected and stored as follows: (1) ≤ 2 days at room temperature (RT) in an ET, (2) 7 days at - 80 °C in an ET, (3) 3-5 days at RT on a FOBT card, (4) 7 days at RT on a FOBT card, and (5) 7 days at - 80 °C on a FOBT card. Samples stored at - 80 °C were frozen immediately. Each specimen/condition underwent 16S rRNA gene sequencing with replicates on the Illumina MiSeq. Alpha and beta diversity measures and relative abundance of major phyla were compared between storage conditions and container (ET vs. FOBT card), with pairwise comparison between different storage conditions and the "standard" of 7 days at - 80 °C in an ET and fresh stool in an ET. RESULTS: Stool samples clustered mainly by individual diaper (P < 10-5, Adonis), rather than by storage condition (P = 0.42) or container (P = 0.16). However, meconium samples clustered more by container (P = 0.002) than by individual diaper (P = 0.009) and storage condition (P = 0.02). Additionally, there were no differences in alpha diversity measures and relative abundance of major phyla after Bonferroni correction between stool stored on a FOBT card at RT for 7 days with stool stored in an ET tube at - 80 °C; differences in alpha diversity were seen however when compared to fresh stool in an ET. Overall, based on the intraclass correlation coefficient (ICC), the different storage containers/conditions are reliable in preserving the microbial memberships and slightly less reliable in preserving the alpha diversity and relative microbial composition of infant stool. CONCLUSIONS: Acknowledging certain limitations, FOBT cards may be a useful tool in large-scale stool microbiota studies in children requiring outpatient follow-up where only small amounts of stool can be obtained, but should not be used when studying meconium.

The Microbiome in Necrotizing Enterocolitis: A Case Report in Twins and Minireview.

Our case describes the serial microbiome changes in twins discordant for necrotizing enterocolitis (NEC), who shared similar intrauterine and early environmental exposures. The key findings were that the 2 neonates had distinctly different microbiome compositions from the first stool samples collected. Also, in the twin who developed NEC there was a decrease in bacterial diversity and an increase in Proteobacteria a week before developing any clinical symptoms, suggesting an early role of the intestinal microbiome in the development of NEC. Here we briefly review the literature on the role of the intestinal microbiome in NEC and how a greater understanding of the neonatal microbiome and host interactions may help mitigate this devastating disease.