Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile.

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term PnimBG) in the promoter of gene nimB, resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the PnimBG mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

Randomized controlled trial demonstrates response to a probiotic intervention for metabolic syndrome that may correspond to diet.

An individual's immune and metabolic status is coupled to their microbiome. Probiotics offer a promising, safe route to influence host health, possibly via the microbiome. Here, we report an 18-week, randomized prospective study that explores the effects of a probiotic vs. placebo supplement on 39 adults with elevated parameters of metabolic syndrome. We performed longitudinal sampling of stool and blood to profile the human microbiome and immune system. While we did not see changes in metabolic syndrome markers in response to the probiotic across the entire cohort, there were significant improvements in triglycerides and diastolic blood pressure in a subset of probiotic arm participants. Conversely, the non-responders had increased blood glucose and insulin levels over time. The responders had a distinct microbiome profile at the end of the intervention relative to the non-responders and placebo arm. Importantly, diet was a key differentiating factor between responders and non-responders. Our results show participant-specific effects of a probiotic supplement on improving parameters of metabolic syndrome and suggest that dietary factors may enhance stability and efficacy of the supplement.

Gut-microbiota-targeted diets modulate human immune status.

Diet modulates the gut microbiome, which in turn can impact the immune system. Here, we determined how two microbiota-targeted dietary interventions, plant-based fiber and fermented foods, influence the human microbiome and immune system in healthy adults. Using a 17-week randomized, prospective study (n = 18/arm) combined with -omics measurements of microbiome and host, including extensive immune profiling, we found diet-specific effects. The high-fiber diet increased microbiome-encoded glycan-degrading carbohydrate active enzymes (CAZymes) despite stable microbial community diversity. Although cytokine response score (primary outcome) was unchanged, three distinct immunological trajectories in high-fiber consumers corresponded to baseline microbiota diversity. Alternatively, the high-fermented-food diet steadily increased microbiota diversity and decreased inflammatory markers. The data highlight how coupling dietary interventions to deep and longitudinal immune and microbiome profiling can provide individualized and population-wide insight. Fermented foods may be valuable in countering the decreased microbiome diversity and increased inflammation pervasive in industrialized society.

A randomized crossover trial on the effect of plant-based compared with animal-based meat on trimethylamine-N-oxide and cardiovascular disease risk factors in generally healthy adults: Study With Appetizing Plantfood-Meat Eating Alternative Trial (SWAP-MEAT).

BACKGROUND: Despite the rising popularity of plant-based alternative meats, there is limited evidence of the health effects of these products. OBJECTIVES: We aimed to compare the effect of consuming plant-based alternative meat (Plant) as opposed to animal meat (Animal) on health factors. The primary outcome was fasting serum trimethylamine-N-oxide (TMAO). Secondary outcomes included fasting insulin-like growth factor 1, lipids, glucose, insulin, blood pressure, and weight. METHODS: SWAP-MEAT (The Study With Appetizing Plantfood-Meat Eating Alternatives Trial) was a single-site, randomized crossover trial with no washout period. Participants received Plant and Animal products, dietary counseling, lab assessments, microbiome assessments (16S), and anthropometric measurements. Participants were instructed to consume ≥2 servings/d of Plant compared with Animal for 8 wk each, while keeping all other foods and beverages as similar as possible between the 2 phases. RESULTS: The 36 participants who provided complete data for both crossover phases included 67% women, were 69% Caucasian, had a mean ± SD age 50 ± 14 y, and BMI 28 ± 5 kg/m2. Mean ± SD servings per day were not different by intervention sequence: 2.5 ± 0.6 compared with 2.6 ± 0.7 for Plant and Animal, respectively (P = 0.76). Mean ± SEM TMAO concentrations were significantly lower overall for Plant (2.7 ± 0.3) than for Animal (4.7 ± 0.9) (P = 0.012), but a significant order effect was observed (P = 0.023). TMAO concentrations were significantly lower for Plant among the n = 18 who received Plant second (2.9 ± 0.4 compared with 6.4 ± 1.5, Plant compared with Animal, P = 0.007), but not for the n = 18 who received Plant first (2.5 ± 0.4 compared with 3.0 ± 0.6, Plant compared with Animal, P = 0.23). Exploratory analyses of the microbiome failed to reveal possible responder compared with nonresponder factors. Mean ± SEM LDL-cholesterol concentrations (109.9 ± 4.5 compared with 120.7 ± 4.5 mg/dL, P = 0.002) and weight (78.7 ± 3.0 compared with 79.6 ± 3.0 kg, P < 0.001) were lower during the Plant phase. CONCLUSIONS: Among generally healthy adults, contrasting Plant with Animal intake, while keeping all other dietary components similar, the Plant products improved several cardiovascular disease risk factors, including TMAO; there were no adverse effects on risk factors from the Plant products.This trial was registered at clinicaltrials.gov as NCT03718988.

High-Throughput Stool Metaproteomics: Method and Application to Human Specimens.

Stool-based proteomics is capable of significantly augmenting our understanding of host-gut microbe interactions. However, compared to competing technologies, such as metagenomics and 16S rRNA sequencing, it is underutilized due to its low throughput and the negative impact sample contaminants can have on highly sensitive mass spectrometry equipment. Here, we present a new stool proteomic processing pipeline that addresses these shortcomings in a highly reproducible and quantitative manner. Using this method, 290 samples from a dietary intervention study were processed in approximately 1.5 weeks, largely done by a single researcher. These data indicated a subtle but distinct monotonic increase in the number of significantly altered proteins between study participants on fiber- or fermented food-enriched diets. Lastly, we were able to classify study participants based on their diet-altered proteomic profiles and demonstrated that classification accuracies of up to 89% could be achieved by increasing the number of subjects considered. Taken together, this study represents the first high-throughput proteomic method for processing stool samples in a technically reproducible manner and has the potential to elevate stool-based proteomics as an essential tool for profiling host-gut microbiome interactions in a clinical setting.IMPORTANCE Widely available technologies based on DNA sequencing have been used to describe the kinds of microbes that might correlate with health and disease. However, mechanistic insights might be best achieved through careful study of the dynamic proteins at the interface between the foods we eat, our microbes, and ourselves. Mass spectrometry-based proteomics has the potential to revolutionize our understanding of this complex system, but its application to clinical studies has been hampered by low-throughput and laborious experimentation pipelines. In response, we developed SHT-Pro, the first high-throughput pipeline designed to rapidly handle large stool sample sets. With it, a single researcher can process over one hundred stool samples per week for mass spectrometry analysis, conservatively approximately 10× to 100× faster than previous methods, depending on whether isobaric labeling is used or not. Since SHT-Pro is fairly simple to implement using commercially available reagents, it should be easily adaptable to large-scale clinical studies.

Geographic variation and within-individual correlations of physiological stress markers in a widespread reptile, the common garter snake (Thamnophis sirtalis).

Characterizing the baseline and stress-induced hormonal, metabolite, and immune profiles of wild animals is important to assess the impacts of variable environments, including human-induced landscape changes, on organismal health. Additionally, the extent to which these profiles are coordinated across physiological systems within individuals remains an important question in understanding how stressors can differentially affect aspects of an individual's physiology. Here, we present data from wild populations of the common garter snake (Thamnophis sirtalis) on both baseline and stress-induced biomarkers: plasma corticosterone (CORT) concentration, plasma glucose concentration, and whole blood heterophil:lymphocyte ratio. Using a standardized restraint protocol with individuals from populations in disparate portions of this species' range - the Sierra Nevada Mountains of California and the plains of Iowa - we collected blood plasma samples at nine time points over three days. Both CORT and glucose response curves differed between georegions, with Iowa snakes attaining higher glucose concentration and maintaining elevated CORT and glucose levels for a longer duration. Additionally, both the total amount and proportional increases of CORT and glucose were lower in larger and therefore older snakes, suggesting ontogenetic shifts in stress perception or response. Within-individual correlation among the three physiological indicators was significant at the time of capture, absent after 3h in captivity, and partially restored after 3days in captivity, demonstrating the effect of stress on the relationships among these physiological systems. Together, these results provide further evidence for the great physiological flexibility of ectothermic tetrapods in maintaining homeostasis across a range of factors.