Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model.

Bacteriophage therapy is a promising approach to address antimicrobial infections though questions remain regarding the impact of the immune response on clinical effectiveness. Here, we develop a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target Vancomycin-Resistant Enterococcus gut colonization. Phage treatment significantly reduces fecal bacterial loads of Vancomycin-Resistant Enterococcus. We also characterize immune responses elicited following administration of the phage cocktail. While minimal innate responses are observed after phage administration, two rounds of treatment induces phage-specific neutralizing antibodies and accelerate phage clearance from tissues. Interestingly, the myophages in our cocktail induce a more robust neutralizing antibody response than the siphophages. This anti-phage immunity reduces the effectiveness of the phage cocktail in our murine model. Collectively, this study shows phage-specific immune responses may be an important consideration in the development of phage cocktails for therapeutic use.

Nanobodies as potential tools for microbiological testing of live biotherapeutic products.

Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.

Application of MALDI-TOF MS for enumerating bacterial constituents of defined consortia.

Characterization of live biotherapeutic product (LBP) batches typically includes a measurement of viability, such as colony forming units (CFU). However, strain-specific CFU enumeration assays can be complicated by the presence of multiple organisms in a single product with similar growth requirements. To overcome specific challenges associated with obtaining strain-specific CFU values from multi-strain mixtures, we developed a method combining mass spectrometry-based colony identification with a traditional CFU assay. This method was assessed using defined consortia made from up to eight bacterial strains. Among four replicate batches of an eight-strain mixture, observed values differed from expected values by less than 0.4 log10 CFU among all strains measured (range of differences, -0.318 to + 0.267). The average difference between observed and expected values was + 0.0308 log10 CFU, with 95% limits of agreement from -0.347 to 0.408 (Bland-Altman analysis). To estimate precision, a single batch of eight-strain mixture was assayed in triplicate by three different users, for a total of nine measurements. Pooled standard deviation values ranged from 0.067 to 0.195 log10 CFU for the eight strains measured, and user averages did not differ significantly. Leveraging emerging mass-spectrometry-based colony identification tools, a novel method for simultaneous enumeration and identification of viable bacteria from mixed-strain consortia was developed and tested. This study demonstrates the potential for this approach to generate accurate and consistent measurements of up to eight bacterial strains simultaneously and may provide a flexible platform for future refinements and modifications. KEY POINTS: • Enumeration of live biotherapeutics is essential for product quality and safety. • Conventional CFU counting may not differentiate between strains in microbial products. • This approach was developed for direct enumeration of mixed bacterial strains simultaneously.

Longitudinal sampling sheds light on SARS-CoV-2 fecal shedding dynamics.

Persistent fecal shedding of SARS-CoV-2 viral RNA has remained a clinical feature of interest throughout the COVID-19 pandemic. In this issue of Med, Natarajan et al. report fecal shedding dynamics of individuals diagnosed with mild-to-moderate COVID-19 disease and sampled longitudinally for up to 10 months1.

Evaluating Cefoperazone-Induced Gut Metabolic Functional Changes in MR1-Deficient Mice.

Mucosal-associated invariant T cells are activated following the recognition of bacterial antigens presented by the major histocompatibility complex class I-related molecule (MR1). Previous metagenomics data showed that MR1-/- knock-out (KO) mice had distinct microbiota and displayed a resistance to Clostridioides difficile (CDI) colonization vs. wild-type (WT) mice. In the present study, LC/MS-based untargeted metabolomics are applied to evaluate the changes in metabolic activities, in accordance with the changes in gut microbiota caused by cefoperazone (Cef) treatment. Adult C57Bl/6J WT and MR1-/- KO mice were given sterile drinking water or spiked with 0.5 mg/mL Cef ad libitum for five days. Fecal pellets were collected daily, and both small intestinal and cecal contents were harvested at sacrifice. The PLS-DA score plots of the metabolomic data indicate that the microbiota is relatively less disturbed by Cef treatment in KO mice, which is consistent with the metagenomics data. The most noticeable differences in the metabolome of KO and WT mice were the increases in carbohydrates in the WT mice, but not in the KO mice. Metabolic functional biomarkers were identified through the correlation analysis of gamma-aminobutyric acid (GABA) and riboflavin. These detected metabolic functional biomarkers could provide information complementary to metagenomics data.

A method for detection of SARS-CoV-2 RNA in healthy human stool: a validation study.

BACKGROUND: Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool. METHODS: In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at -80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing. FINDINGS: The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at -80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at -80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2. INTERPRETATION: This is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic. FUNDING: National Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.

Bile Acid Profile and its Changes in Response to Cefoperazone Treatment in MR1 Deficient Mice.

Mucosal associated invariant T-cells (MAIT cells) are activated following recognition of bacterial antigens (riboflavin intermediates) presented on major histocompatibility complex class I-related molecule (MR1). Our previous study showed that MR1-/- knock-out (KO) mice (lacking MAIT cells) harbor a unique microbiota that is resistant to antibiotic disruption and Clostridioides difficile colonization. While we have characterized the microbiota of this mouse strain, changes in global metabolic activity in these KO mice have not been assessed. Here, LC/MS-based untargeted metabolomics was applied to investigate the differences in the metabolome, specifically in the bile acid (BA) profile of wild-type (WT) and MR1-/- KO mice, as well as how antibiotics change these profiles. BA changes were evaluated in the intestinal content, cecum content, and stool samples from MR1-/- mice and WT mice treated with cefoperazone (Cef). Fecal pellets were collected daily and both intestinal and cecal contents were harvested at predetermined endpoints on day 0 (D0), day 1 (D1), day 3 (D3), and day 5 (D5). KO mice exhibited no changes in 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH2OH; an MR1-restricted riboflavin derivative) in the stool samples at either time point vs. D0, while WT mice showed significant decreases in rRL-6-CH2OH in the stool samples on all treatment days vs. D0. Metabolomics analysis from cecal and stool samples showed that KO mice had more total BA intensity (KO/WT = ~1.7 and ~3.3 fold higher) than that from WT mice prior to Cef treatment, while the fold change difference (KO/WT = ~4.5 and ~4.4 fold) increased after five days of Cef treatment. Both KO and WT mice showed decreases in total BA intensity in response to Cef treatment, however, less dramatic decreases were present in KO vs. WT mice. Increases in taurocholic acid (TCA) intensity and decreases in deoxycholic acid (DCA) intensity in the stool samples from WT mice were associated with the depletion of certain gut bacteria, which was consistent with the previously reported microbiome data. Furthermore, the non-detected TCA and relatively higher DCA intensity in the KO mice might be related to Clostridioides difficile infection resistance, although this needs further investigation.

The Human Gut Microbiome's Influence on Arsenic Toxicity.

PURPOSE OF REVIEW: Arsenic exposure is a public health concern of global proportions with a high degree of interindividual variability in pathologic outcomes. Arsenic metabolism is a key factor underlying toxicity, and the primary purpose of this review is to summarize recent discoveries concerning the influence of the human gut microbiome on the metabolism, bioavailability, and toxicity of ingested arsenic. We review and discuss the current state of knowledge along with relevant methodologies for studying these phenomena. RECENT FINDINGS: Bacteria in the human gut can biochemically transform arsenic-containing compounds (arsenicals). Recent publications utilizing culture-based approaches combined with analytical biochemistry and molecular genetics have helped identify several arsenical transformations by bacteria that are at least possible in the human gut and are likely to mediate arsenic toxicity to the host. Other studies that directly incubate stool samples in vitro also demonstrate the gut microbiome's potential to alter arsenic speciation and bioavailability. In vivo disruption or elimination of the microbiome has been shown to influence toxicity and body burden of arsenic through altered excretion and biotransformation of arsenicals. Currently, few clinical or epidemiological studies have investigated relationships between the gut microbiome and arsenic-related health outcomes in humans, although current evidence provides strong rationale for this research in the future. SUMMARY: The human gut microbiome can metabolize arsenic and influence arsenical oxidation state, methylation status, thiolation status, bioavailability, and excretion. We discuss the strength of current evidence and propose that the microbiome be considered in future epidemiologic and toxicologic studies of human arsenic exposure.

The gut microbiome is required for full protection against acute arsenic toxicity in mouse models.

Arsenic poisons an estimated 200 million people worldwide through contaminated food and drinking water. Confusingly, the gut microbiome has been suggested to both mitigate and exacerbate arsenic toxicity. Here, we show that the microbiome protects mice from arsenic-induced mortality. Both antibiotic-treated and germ-free mice excrete less arsenic in stool and accumulate more arsenic in organs compared to control mice. Mice lacking the primary arsenic detoxification enzyme (As3mt) are hypersensitive to arsenic after antibiotic treatment or when derived germ-free, compared to wild-type and/or conventional counterparts. Human microbiome (stool) transplants protect germ-free As3mt-KO mice from arsenic-induced mortality, but protection depends on microbiome stability and the presence of specific bacteria, including Faecalibacterium. Our results demonstrate that both a functional As3mt and specific microbiome members are required for protection against acute arsenic toxicity in mouse models. We anticipate that the gut microbiome will become an important explanatory factor of disease (arsenicosis) penetrance in humans, and a novel target for prevention and treatment strategies.