CYP8B1 downregulation mediates the metabolic effects of vertical sleeve gastrectomy in mice.

BACKGROUND AND AIMS: Although the benefits of vertical sleeve gastrectomy (VSG) surgery are well known, the molecular mechanisms by which VSG alleviates obesity and its complications remain unclear. We aim to determine the role of CYP8B1 (cytochrome P450, family 8, subfamily B, polypeptide 1) in mediating the metabolic benefits of VSG. APPROACH AND RESULTS: We found that expression of CYP8B1, a key enzyme in controlling the 12α-hydroxylated (12α-OH) bile acid (BA) to non-12α-OH BA ratio, was strongly downregulated after VSG. Using genetic mouse models of CYP8B1 overexpression, knockdown, and knockout, we demonstrated that overexpression of CYP8B1 dampened the metabolic improvements associated with VSG. In contrast, short hairpin RNA-mediated CYP8B1 knockdown improved metabolism similar to those observed after VSG. Cyp8b1 deficiency diminished the metabolic effects of VSG. Further, VSG-induced alterations to the 12α-OH/non-12α-OH BA ratio in the BA pool depended on CYP8B1 expression level. Consequently, intestinal lipid absorption was restricted, and the gut microbiota (GM) profile was altered. Fecal microbiota transplantation from wild type-VSG mice (vs. fecal microbiota transplantation from wild-type-sham mice) improved metabolism in recipient mice, while there were no differences between mice that received fecal microbiota transplantation from knockout-sham and knockout-VSG mice. CONCLUSIONS: CYP8B1 is a critical downstream target of VSG. Modulation of BA composition and gut microbiota profile by targeting CYP8B1 may provide novel insight into the development of therapies that noninvasively mimic bariatric surgery to treat obesity and its complications.

A Phase II Clinical Trial of Pembrolizumab and Enobosarm in Patients with Androgen Receptor-Positive Metastatic Triple-Negative Breast Cancer.

LESSONS LEARNED: The combination of enobosarm and pembrolizumab was well tolerated and showed a modest clinical benefit rate of 25% at 16 weeks. Future trials investigating androgen receptor-targeted therapy in combination with immune checkpoint inhibitors are warranted. BACKGROUND: Luminal androgen receptor is a distinct molecular subtype of triple-negative breast cancer (TNBC) defined by overexpression of androgen receptor (AR). AR-targeted therapy has shown modest activity in AR-positive (AR+) TNBC. Enobosarm (GTx-024) is a nonsteroidal selective androgen receptor modulator (SARM) that demonstrates preclinical and clinical activity in AR+ breast cancer. The current study was designed to explore the safety and efficacy of the combination of enobosarm and pembrolizumab in patients with AR+ metastatic TNBC (mTNBC). METHODS: This study was an open-label phase II study for AR+ (≥10%, 1+ by immunohistochemistry [IHC]) mTNBC. Eligible patients received pembrolizumab 200 mg intravenous (IV) every 3 weeks and enobosarm 18 mg oral daily. The primary objective was to evaluate the safety of enobosarm plus pembrolizumab and determine the response rate. Peripheral blood, tumor biopsies, and stool samples were collected for correlative analysis. RESULTS: The trial was stopped early because of the withdrawal of GTx-024 drug supply. Eighteen patients were enrolled, and 16 were evaluable for responses. Median age was 64 (range 36-81) years. The combination was well tolerated, with only a few grade 3 adverse events: one dry skin, one diarrhea, and one musculoskeletal ache. The responses were 1 of 16 (6%) complete response (CR), 1 of 16 (6%) partial response (PR), 2 of 16 (13%) stable disease (SD), and 12 of 16 (75%) progressive disease (PD). Response rate (RR) was 2 of 16 (13%). Clinical benefit rate (CBR) at 16 weeks was 4 of 16 (25%). Median follow-up was 24.9 months (95% confidence interval [CI], 17.5-30.9). Progression-free survival (PFS) was 2.6 months (95% CI, 1.9-3.1) and overall survival (OS) was 25.5 months (95% CI, 10.4-not reached [NR]). CONCLUSION: The combination of enobosarm and pembrolizumab was well tolerated, with a modest clinical benefit rate of 25% at 16 weeks in heavily pretreated AR+ TNBC without preselected programmed death ligand-1 (PD-L1). Future clinical trials combining AR-targeted therapy with immune checkpoint inhibitor (ICI) for AR+ TNBC warrant investigation.

Whole-Genome Sequencing to Determine Origin of Multinational Outbreak of Sarocladium kiliense Bloodstream Infections.

We used whole-genome sequence typing (WGST) to investigate an outbreak of Sarocladium kiliense bloodstream infections (BSI) associated with receipt of contaminated antinausea medication among oncology patients in Colombia and Chile during 2013-2014. Twenty-five outbreak isolates (18 from patients and 7 from medication vials) and 11 control isolates unrelated to this outbreak were subjected to WGST to elucidate a source of infection. All outbreak isolates were nearly indistinguishable (<5 single-nucleotide polymorphisms), and >21,000 single-nucleotide polymorphisms were identified from unrelated control isolates, suggesting a point source for this outbreak. S. kiliense has been previously implicated in healthcare-related infections; however, the lack of available typing methods has precluded the ability to substantiate point sources. WGST for outbreak investigation caused by eukaryotic pathogens without reference genomes or existing genotyping methods enables accurate source identification to guide implementation of appropriate control and prevention measures.

Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study.

Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.

Francisella tularensis subsp. tularensis group A.I, United States.

We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage.

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum.

Real-time PCR assays for genotyping of Cryptococcus gattii in North America.

BACKGROUND: Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen. METHODS: We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human. RESULTS: Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA. CONCLUSIONS: The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.

Cabozantinib and nivolumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial.

Supplementation with CBM588, a bifidogenic live bacterial product, has been associated with improved clinical outcomes in persons with metastatic renal cell carcinoma (mRCC) receiving nivolumab and ipilimumab. However, its effect on those receiving tyrosine kinase inhibitor-based combinations is unknown. In this open-label, randomized, investigator-initiated, phase 1 study, 30 participants with locally advanced or mRCC with histological confirmation of clear cell, papillary or sarcomatoid component were randomized in a 2:1 fashion to receive cabozantinib (an inhibitor of vascular endothelial growth factor receptor, MET and AXL) and nivolumab (anti-programmed cell death protein 1) with or without CBM588 as first-line treatment. Metagenomic sequencing was performed on stool samples to characterize their gut microbiome at baseline and 13 weeks into treatment. The primary endpoint was a change in the relative abundance of Bifidobacterium spp.; secondary endpoints included objective response rate (ORR), progression-free survival (PFS) and toxicity profile. The primary endpoint of the study was not met and the addition of CBM588 to cabozantinib and nivolumab did not result in a difference in the relative abundance of Bifidobacterium spp. or alpha diversity (as measured by the Shannon index). However, ORR was significantly higher in participants treated with CBM588 compared to those in the control arm (14 of 19, 74% versus 2 of 10, 20%; P = 0.01). PFS at 6 months was 84% (16 of 19) and 60% (6 of 10) in the experimental and control arms, respectively. No significant difference in toxicity profile was seen between the study arms. Our results provide a preliminary signal of improved clinical activity with CBM588 in treatment-naive participants with mRCC receiving cabozantinib and nivolumab. Further investigation is needed to confirm these findings and better characterize the underlying mechanism driving this effect.ClinicalTrials.gov identifier: NCT05122546.

Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition.

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles.

White-nose syndrome restructures bat skin microbiomes.

IMPORTANCE: Inherent complexities in the composition of microbiomes can often preclude investigations of microbe-associated diseases. Instead of single organisms being associated with disease, community characteristics may be more relevant. Longitudinal microbiome studies of the same individual bats as pathogens arrive and infect a population are the ideal experiment but remain logistically challenging; therefore, investigations like our approach that are able to correlate invasive pathogens to alterations within a microbiome may be the next best alternative. The results of this study potentially suggest that microbiome-host interactions may determine the likelihood of infection. However, the contrasting relationship between Pd and the bacterial microbiomes of Myotis lucifugus and Perimyotis subflavus indicate that we are just beginning to understand how the bat microbiome interacts with a fungal invader such as Pd.

Comparison of Brucella abortus population structure based on genotyping methods with different levels of resolution.

Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission dynamics of brucellosis worldwide. We aimed to compare the population structure of the genomes of 53 Brazilian Brucella abortus isolates using eight different genotyping methods: multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphism (SNP) detection (parSNP and NASP) from whole genomes. The strains were isolated from six different Brazilian states between 1977 and 2008 and had previously been analyzed using MLVA8, MLVA11, and MLVA16. Their whole genomes were sequenced, assembled, and subjected to MLST9 MLST21, cgMLST, and SNP analyses. All the genotypes were compared by hierarchical grouping method based on the average distances between the correlation matrices of each technique. MLST9 and MLST21 had the lowest level of resolution, both revealing only four genotypes. MLVA8, MLVA11, and MLVA16 had progressively increasing levels of resolution as more loci were analyzed, identifying 6, 16, and 44 genotypes, respectively. cgMLST showed the highest level of resolution, identifying 45 genotypes, followed by the SNP-based methods, both of which had 44 genotypes. In the assessed population, MLVA was more discriminatory than MLST and was easier and cheaper to perform. SNP techniques and cgMLST provided the highest levels of resolution and the results from the two methods were in close agreement. In conclusion, the choice of genotyping technique can strongly affect one's ability to make meaningful epidemiological conclusions but is dependent on available resources: while the VNTR based techniques are more indicated to high prevalence scenarios, the WGS methods are the ones with the best discriminative power and therefore recommended for outbreaks investigation.

Genome sequence of Staphylococcus aureus strain 11819-97, an ST80-IV European community-acquired methicillin-resistant isolate.

The European methicillin-resistant Staphylococcus aureus (MRSA) clone ST80-IV has historically dominated community-associated infections in major parts of Europe and is a lineage strongly linked to skin and soft tissue infections. Here, we report the genome sequence of an ST80-IV representative, 11819-97, isolated from a skin infection in Denmark in 1997.

Genome sequence of "Candidatus Microthrix parvicella" Bio17-1, a long-chain-fatty-acid-accumulating filamentous actinobacterium from a biological wastewater treatment plant.

"Candidatus Microthrix" bacteria are deeply branching filamentous actinobacteria which occur at the water-air interface of biological wastewater treatment plants, where they are often responsible for foaming and bulking. Here, we report the first draft genome sequence of a strain from this genus: "Candidatus Microthrix parvicella" strain Bio17-1.

Nivolumab plus ipilimumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial.

Previous studies have suggested that the gut microbiome influences the response to checkpoint inhibitors (CPIs) in patients with cancer. CBM588 is a bifidogenic live bacterial product that we postulated could augment CPI response through modulation of the gut microbiome. In this open-label, single-center study (NCT03829111), 30 treatment-naive patients with metastatic renal cell carcinoma with clear cell and/or sarcomatoid histology and intermediate- or poor-risk disease were randomized 2:1 to receive nivolumab and ipilimumab with or without daily oral CBM588, respectively. Stool metagenomic sequencing was performed at multiple timepoints. The primary endpoint to compare the relative abundance of Bifidobacterium spp. at baseline and at 12 weeks was not met, and no significant differences in Bifidobacterium spp. or Shannon index associated with the addition of CBM588 to nivolumab-ipilimumab were detected. Secondary endpoints included response rate, progression-free survival (PFS) and toxicity. PFS was significantly longer in patients receiving nivolumab-ipilimumab with CBM588 than without (12.7 months versus 2.5 months, hazard ratio 0.15, 95% confidence interval 0.05-0.47, P = 0.001). Although not statistically significant, the response rate was also higher in patients receiving CBM588 (58% versus 20%, P = 0.06). No significant difference in toxicity was observed between the study arms. The data suggest that CBM588 appears to enhance the clinical outcome in patients with metastatic renal cell carcinoma treated with nivolumab-ipilimumab. Larger studies are warranted to confirm this clinical observation and elucidate the mechanism of action and the effects on microbiome and immune compartments.

Randomized trial assessing impact of probiotic supplementation on gut microbiome and clinical outcome from targeted therapy in metastatic renal cell carcinoma.

Studies suggest a link between the gut microbiome and metastatic renal cell carcinoma (mRCC) outcomes, including evidence that mRCC patients possess a lower abundance of Bifidobacterium spp. compared to healthy adults. We sought to assess if a Bifidobacterium-containing yogurt product could modulate the gut microbiome and clinical outcome from vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF-TKIs). mRCC patients initiating VEGF-TKIs, regardless of the line of therapy, were randomized to probiotic-supplemented (two 4 oz. servings of the probiotic yogurt product daily) or probiotic-restricted arms. Stool samples were collected prior to therapy and at weeks 2, 3, 4, and 12. Microbiome composition was assessed using whole-metagenome sequencing. A total of 20 patients were randomized. Bifidobacterium animalis, the active ingredient of the probiotic supplement, reached detectable levels in all patients in the probiotic-supplemented arm versus two patients in the probiotic-restricted arm. Clinical benefit rate was similar in probiotic-supplemented versus probiotic-restricted arms (70% vs. 80%, p = 0.606). Linear discriminant analysis (LDA) effect size analysis of MetaPhIAn2 abundance data predicted 25 enriched species demonstrating an LDA score >3 in either clinical benefit or no clinical benefit. In patients with clinical benefit (vs. no clinical benefit), Barnesiella intestinihominis and Akkermansia muciniphila were significantly more abundant (p = 7.4 × 10-6 and p = 5.6 × 10-3 , respectively). This is the first prospective randomized study demonstrating modulation of the gut microbiome with a probiotic in mRCC. Probiotic supplementation successfully increased the Bifidobacterium spp. levels. Analysis of longitudinal stool specimens identified an association between B. intestinihominis, A. muciniphila, and clinical benefit with therapy. Trial Registration: NCT02944617.

Analysis of Gut Microbiome Using Explainable Machine Learning Predicts Risk of Diarrhea Associated With Tyrosine Kinase Inhibitor Neratinib: A Pilot Study.

Neratinib has great efficacy in treating HER2+ breast cancer but is associated with significant gastrointestinal toxicity. The objective of this pilot study was to understand the association of gut microbiome and neratinib-induced diarrhea. Twenty-five patients (age ≥ 60) were enrolled in a phase II trial evaluating safety and tolerability of neratinib in older adults with HER2+ breast cancer (NCT02673398). Fifty stool samples were collected from 11 patients at baseline and during treatment. 16S rRNA analysis was performed and relative abundance data were generated. Shannon's diversity was calculated to examine gut microbiome dysbiosis. An explainable tree-based approach was utilized to classify patients who might experience neratinib-related diarrhea (grade ≥ 1) based on pre-treatment baseline microbial relative abundance data. The hold-out Area Under Receiver Operating Characteristic and Area Under Precision-Recall Curves of the model were 0.88 and 0.95, respectively. Model explanations showed that patients with a larger relative abundance of Ruminiclostridium 9 and Bacteroides sp. HPS0048 may have reduced risk of neratinib-related diarrhea and was confirmed by Kruskal-Wallis test (p ≤ 0.05, uncorrected). Our machine learning model identified microbiota associated with reduced risk of neratinib-induced diarrhea and the result from this pilot study will be further verified in a larger study. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, identifier NCT02673398.

Roles of bacteriophages, plasmids and CRISPR immunity in microbial community dynamics revealed using time-series integrated meta-omics.

Viruses and plasmids (invasive mobile genetic elements (iMGEs)) have important roles in shaping microbial communities, but their dynamic interactions with CRISPR-based immunity remain unresolved. We analysed generation-resolved iMGE-host dynamics spanning one and a half years in a microbial consortium from a biological wastewater treatment plant using integrated meta-omics. We identified 31 bacterial metagenome-assembled genomes encoding complete CRISPR-Cas systems and their corresponding iMGEs. CRISPR-targeted plasmids outnumbered their bacteriophage counterparts by at least fivefold, highlighting the importance of CRISPR-mediated defence against plasmids. Linear modelling of our time-series data revealed that the variation in plasmid abundance over time explained more of the observed community dynamics than phages. Community-scale CRISPR-based plasmid-host and phage-host interaction networks revealed an increase in CRISPR-mediated interactions coinciding with a decrease in the dominant 'Candidatus Microthrix parvicella' population. Protospacers were enriched in sequences targeting genes involved in the transmission of iMGEs. Understanding the factors shaping the fitness of specific populations is necessary to devise control strategies for undesirable species and to predict or explain community-wide phenotypes.

Stool Microbiome Profiling of Patients with Metastatic Renal Cell Carcinoma Receiving Anti-PD-1 Immune Checkpoint Inhibitors.

Preclinical models and early clinical data suggest an interplay between the gut microbiome and response to immunotherapy in solid tumors including metastatic renal cell carcinoma (mRCC). We sought to characterize the stool microbiome of mRCC patients receiving a checkpoint inhibitor (CPI) and to assess treatment-related changes in microbiome composition over the course of CPI therapy. Stool was collected from 31 patients before initiation of nivolumab (77%) or nivolumab plus ipilimumab (23%) therapy, of whom 58% experienced clinical benefit. Greater microbial diversity was associated with clinical benefit from CPI therapy (p = 0.001), and multiple species were associated with clinical benefit or lack thereof. Temporal profiling of the microbiome indicated that the relative abundance of Akkermansia muciniphila increased in patients deriving clinical benefit from CPIs. This study substantiates results from previous CPI-related microbiome profiling studies in mRCC. Temporal changes in microbiome composition suggest potential utility in modulating the microbiome for more successful CPI outcomes. PATIENT SUMMARY: We compared the composition and diversity of the gut microbiome in patients receiving immunotherapy for renal cell carcinoma. We found that higher microbial diversity is associated with better treatment outcomes. Treatment response is characterized by changes in microbial species over the course of treatment.

Integration of time-series meta-omics data reveals how microbial ecosystems respond to disturbance.

The development of reliable, mixed-culture biotechnological processes hinges on understanding how microbial ecosystems respond to disturbances. Here we reveal extensive phenotypic plasticity and niche complementarity in oleaginous microbial populations from a biological wastewater treatment plant. We perform meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics) on in situ samples over 14 months at weekly intervals. Based on 1,364 de novo metagenome-assembled genomes, we uncover four distinct fundamental niche types. Throughout the time-series, we observe a major, transient shift in community structure, coinciding with substrate availability changes. Functional omics data reveals extensive variation in gene expression and substrate usage amongst community members. Ex situ bioreactor experiments confirm that responses occur within five hours of a pulse disturbance, demonstrating rapid adaptation by specific populations. Our results show that community resistance and resilience are a function of phenotypic plasticity and niche complementarity, and set the foundation for future ecological engineering efforts.

A global to local genomics analysis of Clostridioides difficile ST1/RT027 identifies cryptic transmission events in a northern Arizona healthcare network.

Clostridioides difficile is a ubiquitous, diarrhoeagenic pathogen often associated with healthcare-acquired infections that can cause a range of symptoms from mild, self-limiting disease to toxic megacolon and death. Since the early 2000s, a large proportion of C. difficile cases have been attributed to the ribotype 027 (RT027) lineage, which is associated with sequence type 1 (ST1) in the C. difficile multilocus sequence typing scheme. The spread of ST1 has been attributed, in part, to resistance to fluoroquinolones used to treat unrelated infections, which creates conditions ideal for C. difficile colonization and proliferation. In this study, we analysed 27 isolates from a healthcare network in northern Arizona, USA, and 1352 publicly available ST1 genomes to place locally sampled isolates into a global context. Whole genome, single nucleotide polymorphism analysis demonstrated that at least six separate introductions of ST1 were observed in healthcare facilities in northern Arizona over an 18-month sampling period. A reconstruction of transmission networks identified potential nosocomial transmission of isolates, which were only identified via whole genome sequence analysis. Antibiotic resistance heterogeneity was observed among ST1 genomes, including variability in resistance profiles among locally sampled ST1 isolates. To investigate why ST1 genomes are so common globally and in northern Arizona, we compared all high-quality C. difficile genomes and identified that ST1 genomes have gained and lost a number of genomic regions compared to all other C. difficile genomes; analyses of other toxigenic C. difficile sequence types demonstrate that this loss may be anomalous and could be related to niche specialization. These results suggest that a combination of antimicrobial resistance and gain and loss of specific genes may explain the prominent association of this sequence type with C. difficile infection cases worldwide. The degree of genetic variability in ST1 suggests that classifying all ST1 genomes into a quinolone-resistant hypervirulent clone category may not be appropriate. Whole genome sequencing of clinical C. difficile isolates provides a high-resolution surveillance strategy for monitoring persistence and transmission of C. difficile and for assessing the performance of infection prevention and control strategies.