The degree of protection conferred after receiving an oral cholera vaccine (OCV) varies based on age, prior exposure to Vibrio cholerae , and unknown factors. Recent evidence suggests that the microbiota may mediate some of the unexplained differences in oral vaccine responses. We used metagenomic sequencing of the microbiota at the time of vaccination, and then related microbial features to immune responses after OCV using a reference-independent gene-level analysis. We found that the presence of sphingolipid-producing bacteria was associated with the development of protective immune responses after OCV. We experimentally tested these results by stimulating human macrophages with Bacteroides xylanisolvens metabolites and found that sphingolipid-containing extracts increased innate immune responses to OCV antigens. Our findings demonstrate a new analytic method for translating metagenomic sequencing data into strain-specific results associated with a biological outcome, and in validating this tool, we identified that microbe-derived sphingolipids impact in vitro immune responses to OCV antigens.

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that the V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from ten cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than co-infection with divergent V. cholerae O1 lineages. The number of single nucleotide variants decreased between vomit and stool in four patients, increased in two, and remained unchanged in four. The number of genes encoded in the V. cholerae genome decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract. IMPORTANCE: Vibrio cholerae O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed V. cholerae genomes are usually studied, but V. cholerae isolated from vomit may be more representative of where V. cholerae colonizes in the upper intestinal epithelium. V. cholerae may experience bottlenecks, or large reductions in bacterial population sizes or genetic diversity, as it passes through the gut. Passage through the gut may select for distinct V. cholerae mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in V. cholerae genetic diversity, and only in some patients. These results fill a gap in our understanding of the V. cholerae life cycle, transmission, and evolution.

Structured illumination (SI) involves projecting controlled spatial light patterns onto a scene or sample and processing the captured response to recover information such as the phase, surface shape, or optical contrast. Three-Step Phase Shifting (TPS), also known as three-phase demodulation (TPD) in the spatial frequency domain imaging (SFDI) field, is a common SI workflow in which three phase-shifted sinusoidal patterns are projected and captured sequentially for one demodulation. To achieve acceptable demodulation quality, all three phase-shifted patterns must be captured correctly. However, achieving this quality introduces a trade-off since each phase must be captured at the correct time, increasing acquisition time and requiring precise projector–camera synchronization. In real-time TPD-based SI, low pattern-generation throughput, synchronization uncertainty, and often bulky desktop implementations remain major limitations. Therefore, this work investigates, designs, and validates a deterministic, low-latency, and portable projection–capture synchronization system for TPD-based SI. First, a Hyperspectral (HS) Python-based SI (HSPy-SI) system, representative of common state-of-the-art (SOTA) implementations, is evaluated. It uses an HS snapshot camera and a fixed-delay desktop synchronizer. Then, the proposed HyperSI system is introduced as a real-time projection–capture synchronizer implemented as a bench-top prototype on heterogeneous embedded platforms: a Single-Board Computer (SBC) and a System-on-Chip (SoC) board. Its core contribution is a tunable parameter, W (Frame Count to Wait), which counts frame-generation interrupts before capture and reduces the delay-search space. Compared with the SOTA, HyperSI achieves over 8× higher pattern-generation throughput, increases polarized acquisition by 7× to nearly 4 FPS, reaches about 12 FPS without polarizers, and reduces waiting time by 88×.