Butyrate enhances Clostridioides difficile sporulation in vitro.

Short-chain fatty acids (SCFAs) are products of bacterial fermentation that help maintain important gut functions such as maintenance of the intestinal barrier, cell signaling, and immune homeostasis. The main SCFAs acetate, propionate, and butyrate have demonstrated beneficial effects for the host, including its importance in alleviating infections caused by pathogens such as Clostridioides difficile. Despite the potential role of SCFAs in mitigating C. difficile infection, their direct effect on C. difficile remains unclear. Through a set of in vitro experiments, we investigated how SCFAs influence C. difficile growth, sporulation, and toxin production. Similar to previous studies, we observed that butyrate decreased growth of C. difficile strain 630 in a dose-dependent manner. The presence of butyrate also increased C. difficile sporulation, with minimal increases in toxin production. RNA-Seq analysis validated our experimental results, demonstrating increased expression of sporulation-related genes in conjunction with changes in metabolic and regulatory genes, such as a putative carbon starvation protein, CstA. Collectively, these data suggest that butyrate may induce alternative C. difficile survival pathways, modifying its growth ability and virulence to persist in the gut environment. IMPORTANCE Several studies suggest that butyrate may modulate gut infections, such as reducing inflammation caused by the healthcare-associated Clostridioides difficile. While studies in both animal models and human studies correlate high levels of butyrate with reduced C. difficile burden, the direct impact of butyrate on C. difficile remains unclear. Our study demonstrates that butyrate directly influences C. difficile by increasing its sporulation and modifying its metabolism, potentially using butyrate as a biomarker to shift survival strategies in a changing gut environment. These data point to additional therapeutic approaches to combat C. difficile in a butyrate-directed manner.

Butyrate enhances Clostridioides difficile sporulation in vitro.

Short chain fatty acids (SCFAs) are products of bacterial fermentation that help maintain important gut functions such as the intestinal barrier, signaling, and immune homeostasis. The main SCFAs acetate, propionate, and butyrate have demonstrated beneficial effects for the host, including importance in combatting infections caused by pathogens such as Clostridioides difficile . Despite the potential role of SCFAs in mitigating C. difficile infection, their direct effect on C. difficile remains unclear. Through a set of in vitro experiments, we investigated how SCFAs influence C. difficile growth, sporulation, and toxin production. Similar to previous studies, we observed that butyrate decreased growth of C. difficile strain 630 in a dose-dependent manner. The presence of butyrate also increased C. difficile sporulation, with minimal increases in toxin production. RNA-Seq analysis validated our experimental results, demonstrating increased expression of sporulation-related genes in conjunction with alternative metabolic and related C. difficile regulatory pathways, such as the carbon catabolite repressor, CcpA. Collectively, these data suggest that butyrate may signal alternative C. difficile metabolic pathways, thus modifying its growth and virulence to persist in the gut environment. IMPORTANCE: Several studies suggest that butyrate may be important in alleviating gut infections, such as reducing inflammation caused by the healthcare-associated Clostridioides difficile . While studies in both animal models and human studies correlate high levels of butyrate with reduced C. difficile burden, the direct impact of butyrate on C. difficile remains unclear. Our study demonstrates that butyrate directly influences C. difficile by increasing its sporulation and modifying its metabolism, potentially using butyrate as a biomarker to shift survival strategies in a changing gut environment. These data point to additional therapeutic approaches to combat C. difficile in a butyrate-directed manner.

Beneficial effects of fecal microbiota transplantation in recurrent Clostridioides difficile infection.

Fecal microbiota transplantation (FMT) is highly effective in preventing recurrent Clostridioides difficile infection (rCDI). However, the mechanisms underpinning its clinical efficacy are incompletely understood. Herein, we provide an overview of rCDI pathogenesis followed by a discussion of potential mechanisms of action focusing on the current understanding of trans-kingdom microbial, metabolic, immunological, and epigenetic mechanisms. We then outline the current research gaps and offer methodological recommendations for future studies to elevate the quality of research and advance knowledge translation. By combining interventional trials with multiomics technology and host and environmental factors, analyzing longitudinally collected biospecimens will generate results that can be validated with animal and other models. Collectively, this will confirm causality and improve translation, ultimately to develop targeted therapies to replace FMT.

Diversity and Prevalence of Clostridium innocuum in the Human Gut Microbiota.

Clostridia are a polyphyletic group of Gram-positive, spore-forming anaerobes in the Firmicutes phylum that significantly impact metabolism and functioning of the human gastrointestinal tract. Recently, Clostridia were divided into two separate classes, Clostridia and Erysipelotrichia, based on phenotypic and 16S rRNA gene-based differences. While Clostridia include many well-known pathogenic bacteria, Erysipelotrichia remain relatively uncharacterized, particularly regarding their role as a pathogen versus commensal. Despite wide recognition as a commensal, the erysipelotrichial species Clostridium innocuum has recently been associated with various disease states. To further understand the ecological and potential virulent role of C. innocuum, we conducted a genomic comparison across 38 C. innocuum isolates and 194 publicly available genomes. Based on colony morphology, we isolated multiple C. innocuum cultivars from the feces of healthy human volunteers (n = 5). Comparison of the 16S rRNA gene of our isolates against publicly available microbiota data sets in healthy individuals suggests a high prevalence of C. innocuum across the human population (>80%). Analysis of single nucleotide polymorphisms (SNPs) across core genes and average nucleotide identify (ANI) revealed the presence of four clades among all available genomes (n = 232 total). Investigation of carbohydrate and protein utilization pathways, including comparison against the carbohydrate-activating enzyme (CAZyme) database, demonstrated inter- and intraclade differences that were further substantiated in vitro. Collectively, these data indicate genetic variance within the C. innocuum species that may help clarify its role in human disease and health. IMPORTANCE Clostridia are a group of medically important anaerobes as both commensals and pathogens. Recently, a new class of Erysipelotrichia containing a number of reassigned clostridial species has emerged, including Clostridium innocuum. Recent studies have implicated C. innocuum as a potential causative agent of diarrhea in patients from whom Clostridioides difficile could not be isolated. Using genomic and in vitro comparison, this study sought to characterize C. innocuum in the healthy human gut. Our analyses suggest that C. innocuum is a highly prevalent and diverse species, demonstrating clade-specific differences in metabolism and potential virulence. Collectively, this study is the first investigation into a broader description of C. innocuum as a human gut inhabitant.

The role of the gut microbiome in colonization resistance and recurrent Clostridioides difficile infection.

The species composition of the human gut microbiota is related to overall health, and a healthy gut microbiome is crucial in maintaining colonization resistance against pathogens. Disruption of gut microbiome composition and functionality reduces colonization resistance and has been associated with several gastrointestinal and non-gastrointestinal diseases. One prime example is Clostridioides difficile infection (CDI) and subsequent recurrent infections that occur after the development of systemic antibiotic-related dysbiosis. Standard-of-care antibiotics used for both acute and recurrent infections do not address dysbiosis and often worsen the condition. Moreover, monoclonal antibodies, recommended in conjunction with standard-of-care antibiotics for the prevention of recurrent CDI in patients at high risk of recurrence, reduce recurrences but do not address the underlying dysbiosis. Fecal microbiota transplantation (FMT) is an evolving therapeutic strategy in which microbes are harvested from healthy donor stool and transplanted into the gut of a recipient to restore the gut microbiome. Although effective in the prevention of recurrent CDI, some existing challenges include screening and the standardization of stool acquisition and processing. Recent safety alerts by the US Food and Drug Administration raised concern about the possibility of transmission of multidrug-resistant organisms or severe acute respiratory syndrome coronavirus 2 via FMT. Increased knowledge that microbes are beneficial in restoring the gut microbiome has led to the clinical development of several newer biotherapeutic formulations that are more regulated than FMT, which may allow for improved restoration of the gut microbiome and prevention of CDI recurrence. This review focuses on mechanisms by which gut microbiome restoration could influence colonization resistance against the pathogen C. difficile. Plain language summary: The Role of the Gut Microbiome in Clostridioides difficile Infection Introduction: A rich and diverse gut microbiome is key to immune system regulation and colonization resistance against pathogens.A disruption in the gut microbiome composition can make the gut more vulnerable to diseases such as Clostridioides difficile infection (CDI), caused by the bacterium C. difficile.CDI management presents a therapeutic dilemma, as it is usually treated with antibiotics that can treat the infection but also can damage the microbiome.Treatment of CDI using antibiotics can further reduce microbial diversity and deplete beneficial bacteria from the gut leading to a condition called dysbiosis.Antibiotic treatment can be followed by therapies that restore the gut microbiota, boost colonization resistance, and prevent the development of antimicrobial resistance.It is important to evaluate treatment options to determine their safety and effectiveness. Methods: The researchers provided an overview of the mechanisms that the gut microbiome uses to prevent colonization of the gut by pathogens.They subsequently reviewed the efficacy and shortcomings of the following treatments for CDI: - Antibiotics- Monoclonal antibodies- Fecal microbiota transplantation (FMT) Results: Commensal intestinal bacteria prevent colonization of the gut by pathogens using mechanisms such as: - Competition for key nutrients- Production of inhibitory bile acids- Short-chain fatty acid production- Lowering the luminal pH- Production of bacteriocinsAntibiotic therapy is recommended as a standard treatment for CDI. However, patients are vulnerable to recurrent CDI after discontinuation of the therapy.Monoclonal antibodies that inactivate C. difficile toxins may be recommended along with antibiotics to prevent recurrent CDI. However, this approach does not restore the microbiome.FMT is one method of microbial restoration, where stool is harvested from a healthy donor and transplanted into a patient's colon.Although FMT has shown some efficacy in the treatment of recurrent CDI, the procedure is not standardized.Safety concerns have been raised about the possibility of transmission of multidrug-resistant pathogens via FMT. Conclusion: Treatment methods that can efficiently restore the diversity of the gut microbiome are crucial in preventing recurrence of CDI.

Clostridioides difficile: innovations in target discovery and potential for therapeutic success.

INTRODUCTION: Clostridioides difficile infection (CDI) remains a worldwide clinical problem. Increased incidence of primary infection, occurrence of hypertoxigenic ribotypes, and more frequent occurrence of drug resistant, recurrent, and non-hospital CDI, emphasizes the urgent unmet need of discovering new therapeutic targets. AREAS COVERED: We searched PubMed and Web of Science databases for articles identifying novel therapeutic targets or treatments for C. difficile from 2001 to 2021. We present an updated review on current preclinical efforts on designing inhibitory compounds against these drug targets and indicate how these could become the focus of future therapeutic approaches. We also evaluate the increasing exploitability of gut microbial-derived metabolites and host-derived therapeutics targeting VEGF-A, immune targets and pathways, ion transporters, and microRNAs as anti-C. difficile therapeutics, which have yet to reach clinical trials. Our review also highlights the therapeutic potential of re-purposing currently available agents . We conclude by considering translational hurdles and possible strategies to mitigate these problems. EXPERT OPINION: Considerable progress has been made in the development of new anti-CDI drug candidates. Nevertheless, a greater comprehension of CDI pathogenesis and host-microbe interactions is beginning to uncover potential novel therapeutic targets, which can be exploited to plug gaps in the CDI drug discovery pipeline.

Multiomics Profiling Reveals Signatures of Dysmetabolism in Urban Populations in Central India.

BACKGROUND: Non-communicable diseases (NCDs) have become a major cause of morbidity and mortality in India. Perturbation of host-microbiome interactions may be a key mechanism by which lifestyle-related risk factors such as tobacco use, alcohol consumption, and physical inactivity may influence metabolic health. There is an urgent need to identify relevant dysmetabolic traits for predicting risk of metabolic disorders, such as diabetes, among susceptible Asian Indians where NCDs are a growing epidemic. METHODS: Here, we report the first in-depth phenotypic study in which we prospectively enrolled 218 adults from urban and rural areas of Central India and used multiomic profiling to identify relationships between microbial taxa and circulating biomarkers of cardiometabolic risk. Assays included fecal microbiota analysis by 16S ribosomal RNA gene amplicon sequencing, quantification of serum short chain fatty acids by gas chromatography-mass spectrometry, and multiplex assaying of serum diabetic proteins, cytokines, chemokines, and multi-isotype antibodies. Sera was also analysed for N-glycans and immunoglobulin G Fc N-glycopeptides. RESULTS: Multiple hallmarks of dysmetabolism were identified in urbanites and young overweight adults, the majority of whom did not have a known diagnosis of diabetes. Association analyses revealed several host-microbe and metabolic associations. CONCLUSIONS: Host-microbe and metabolic interactions are differentially shaped by body weight and geographic status in Central Indians. Further exploration of these links may help create a molecular-level map for estimating risk of developing metabolic disorders and designing early interventions.

Fecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection Associates With Functional Alterations in Circulating microRNAs.

BACKGROUND AND AIMS: The molecular mechanisms underlying successful fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (rCDI) remain poorly understood. The primary objective of this study was to characterize alterations in microRNAs (miRs) following FMT for rCDI. METHODS: Sera from 2 prospective multicenter randomized controlled trials were analyzed for miRNA levels with the use of the Nanostring nCounter platform and quantitative reverse-transcription (RT) polymerase chain reaction (PCR). In addition, rCDI-FMT and toxin-treated animals and ex vivo human colonoids were used to compare intestinal tissue and circulating miRs. miR inflammatory gene targets in colonic epithelial and peripheral blood mononuclear cells were evaluated by quantitative PCR (qPCR) and 3'UTR reporter assays. Colonic epithelial cells were used for mechanistic, cytoskeleton, cell growth, and apoptosis studies. RESULTS: miRNA profiling revealed up-regulation of 64 circulating miRs 4 and 12 weeks after FMT compared with screening, of which the top 6 were validated in the discovery cohort by means of RT-qPCR. In a murine model of relapsing-CDI, RT-qPCR analyses of sera and cecal RNA extracts demonstrated suppression of these miRs, an effect reversed by FMT. In mouse colon and human colonoids, C difficile toxin B (TcdB) mediated the suppressive effects of CDI on miRs. CDI dysregulated DROSHA, an effect reversed by FMT. Correlation analyses, qPCR ,and 3'UTR reporter assays revealed that miR-23a, miR-150, miR-26b, and miR-28 target directly the 3'UTRs of IL12B, IL18, FGF21, and TNFRSF9, respectively. miR-23a and miR-150 demonstrated cytoprotective effects against TcdB. CONCLUSIONS: These results provide novel and provocative evidence that modulation of the gut microbiome via FMT induces alterations in circulating and intestinal tissue miRs. These findings contribute to a greater understanding of the molecular mechanisms underlying FMT and identify new potential targets for therapeutic intervention in rCDI.

mSphere of Influence: Translating Gut Microbiome Studies To Benefit Human Health.

Anna M. Seekatz works in the field of the gut microbiome as it related to infectious diseases. In this "mSphere of Influence" article, she reflects on how two studies, "The impact of a consortium of fermented milk strains on the gut microbiome of gnotobiotic mice and monozygotic twins" (N. P. McNulty, T. Yatsunenko, A. Hsiao, et al., Sci Transl Med 3:106ra106, 2011) and "High-throughput DNA sequence analysis reveals stable engraftment of gut microbiota following transplantation of previously frozen fecal bacteria" (M. J. Hamilton, A. R. Weingarden, T. Unno, A. Khoruts, and M. J. Sadowsky, Gut Microbes 4:125-135, 2013), shaped how she approaches interpreting microbiome studies.

Dilution as a Solution: Targeting Microbial Populations with a Simplified Dilution Strategy.

The gut microbiota is an integral part of maintaining resistance against infection by Clostridioides (Clostridium) difficile, a pathogen of increasing concern in both health care and community settings. The recent article by J. M. Auchtung, E. C. Preisner, J. Collins, A. I. Lerma, and R. A. Britton (mSphere 5:e00387-20, 2020, https://doi.org/10.1128/mSphere.00387-20) demonstrates an innovative approach to identify microbes that inhibit C. difficile by employing a dilution scheme to test different microbial mixtures in vitro and in vivo This type of approach can advance the identification and validation of specific microbes that elicit functions of interest for many conditions involving the microbiota, of which the complexity and variability can often complicate causality.

Enterobacterales Infection after Intestinal Dominance in Hospitalized Patients.

The Enterobacterales order of Gram-negative bacteria includes the common nosocomial pathogens Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, and Enterobacter species. Intestinal domination by some colonizing bacterial taxa is associated with subsequent infection, but 16S rRNA gene sequencing is too costly and slow to be used in a clinical setting. The objectives of this study were to develop a PCR-based assay that can measure Enterobacterales density, validate it against 16S rRNA gene sequencing, and measure the association between Enterobacterales dominance and subsequent infection. Two quantitative PCR (qPCR) assays that were developed to quantify the absolute and relative abundance of Enterobacterales had good correlation with 16S rRNA sequence analysis (P < 0.0001). Using both PCR assays and 16S sequencing, a matched case-control study was performed comparing rectal swabs from hospitalized patients who later developed bloodstream, urinary tract, or respiratory Enterobacterales infections (n = 95) to swabs from patients who remained uninfected (n = 189). Enterobacterales abundance measured by sequencing was high in both cases and controls (means, 31.1% and 27.5%, respectively; P = 0.322). We observed an increased risk of infection that depended on both the absolute and relative abundance of Enterobacterales as measured by qPCR assay A (P = 0.012). After adjustment for albumin levels, central venous catheter presence, and use of cephalosporins at the time of swab collection, this association still approached significance (P = 0.061). These results demonstrate that using qPCR to measure intestinal colonization dominance is feasible, indicate that hospitalized patients have high levels of Enterobacterales colonization, and suggest that both relative and absolute abundance may be associated with subsequent infection.IMPORTANCE Increasing antibiotic resistance has resulted in infections that are life-threatening and difficult to treat. Interventions that prevent these infections, particularly without using antibiotics, could save lives. Intestinal colonization by pathogens, including vancomycin-resistant Enterococcus and carbapenem-resistant Enterobacteriaceae (part of the order Enterobacterales) is associated with subsequent infection, and increased colonization density is associated with increased infection risk. Therefore, colonization offers a window of opportunity for infection prevention if (i) there are rapid and inexpensive assays to detect colonization, (ii) there are safe and effective interventions, and (iii) the risk of infection outweighs the risk of the treatment. Fecal transplants are proof of principle that manipulating the microbiome can reduce such colonization and prevent infections. This study demonstrates the feasibility of implementing rapid and inexpensive assays to quantify colonization and measures the strength of association between Enterobacterales dominance and subsequent infection. The approach described here could be a valuable tool in the prevention of antibiotic-resistant infections.

Epidemiological and Microbiome Associations Between Klebsiella pneumoniae and Vancomycin-Resistant Enterococcus Colonization in Intensive Care Unit Patients.

BACKGROUND: Prior colonization by Klebsiella pneumoniae and vancomycin-resistant Enterococci (VRE) is associated with subsequent infection, particularly in intensive care unit (ICU) populations. Screening for VRE colonization, but not K. pneumoniae, is routinely performed in some health care systems. Identification of patient factors associated with K. pneumoniae colonization could enable infection prevention. METHODS: ICU patients were screened for VRE and K. pneumoniae by rectal swab culture over 2 time periods: July-October 2014 (n = 1209) and January-May 2016 (n = 1243). Patient demographics, baseline laboratory data, comorbidities, and outcomes were analyzed. 16S rRNA gene-based analysis was performed on a subset of patients (n = 248) to identify microbiota characteristics associated with VRE and K. pneumoniae colonization. RESULTS: K. pneumoniae colonization (17.3% of patients in the 2014 cohort, 7.3% in 2016) was significantly associated with VRE colonization in multivariable analysis (P = .03 in 2016; P = .08 in 2014). VRE colonization was associated with poor underlying health, whereas K. pneumoniae colonization was associated with advanced age. The most prevalent operational taxonomic units were Escherichia coli /Shigella spp., Klebsiella, and Enterococcus, consistent with high rates of detectable K. pneumoniae and VRE by culture. Microbial community structure in noncolonized patients was significantly different from those with VRE, K. pneumoniae, or both, attributable to differences in the relative abundance of Klebsiella and Enterococcus. CONCLUSIONS: K. pneumoniae co-colonizes with VRE and is a predominant taxon in ICU patients, but colonization was not associated with significant comorbidities. Screening for K. pneumoniae and VRE simultaneously could be an efficient approach for novel infection prevention strategies.

Interleukin-22-mediated host glycosylation prevents Clostridioides difficile infection by modulating the metabolic activity of the gut microbiota.

The involvement of host immunity in the gut microbiota-mediated colonization resistance to Clostridioides difficile infection (CDI) is incompletely understood. Here, we show that interleukin (IL)-22, induced by colonization of the gut microbiota, is crucial for the prevention of CDI in human microbiota-associated (HMA) mice. IL-22 signaling in HMA mice regulated host glycosylation, which enabled the growth of succinate-consuming bacteria Phascolarctobacterium spp. within the gut microbiome. Phascolarctobacterium reduced the availability of luminal succinate, a crucial metabolite for the growth of C. difficile, and therefore prevented the growth of C. difficile. IL-22-mediated host N-glycosylation is likely impaired in patients with ulcerative colitis (UC) and renders UC-HMA mice more susceptible to CDI. Transplantation of healthy human-derived microbiota or Phascolarctobacterium reduced luminal succinate levels and restored colonization resistance in UC-HMA mice. IL-22-mediated host glycosylation thus fosters the growth of commensal bacteria that compete with C. difficile for the nutritional niche.

Introduction to the special issue for The anaerobe society of the America's 14th Biennial congress in Las Vegas.

In June 2018, the Anaerobe Society of the America's (ASA) held their 14th Biennial Congress in Las Vegas, Nevada. The Congress was attended by over 200 individuals from many different countries. The focus of the meeting was the fast-growing area of anaerobes in human and animal infectious disease, computational tools to understand basic biology and therapeutic development, the role of anaerobes in the microbiome, and clinical trials of novel bacterial-based therapies. To strengthen the community of researchers working on anaerobes, the congress held two training workshops on clinical bacteriology and anaerobes in the microbiome, several networking events, as well as a dinner which honored the lifetime achievement award given to Ellen Jo Baron. The meeting was also attended by the grandfather of anaerobic bacteriology and the founder of (ASA), Sydney Finegold, at the age of 97. In all, there was a broad diversity of research presented that showed new ways that anaerobes play a important role in health and disease.

Spatial and Temporal Analysis of the Stomach and Small-Intestinal Microbiota in Fasted Healthy Humans.

Although the microbiota in the proximal gastrointestinal (GI) tract have been implicated in health and disease, much about these microbes remains understudied compared to those in the distal GI tract. This study characterized the microbiota across multiple proximal GI sites over time in healthy individuals. As part of a study of the pharmacokinetics of oral mesalamine administration, healthy, fasted volunteers (n = 8; 10 observation periods total) were orally intubated with a four-lumen catheter with multiple aspiration ports. Samples were taken from stomach, duodenal, and multiple jejunal sites, sampling hourly (≤7 h) to measure mesalamine (administered at t = 0), pH, and 16S rRNA gene-based composition. We observed a predominance of Firmicutes across proximal GI sites, with significant variation compared to stool. The microbiota was more similar within individuals over time than between subjects, with the fecal microbiota being unique from that of the small intestine. The stomach and duodenal microbiota displayed highest intraindividual variability compared to jejunal sites, which were more stable across time. We observed significant correlations in the duodenal microbial composition with changes in pH; linear mixed models identified positive correlations with multiple Streptococcus operational taxonomic units (OTUs) and negative correlations with multiple Prevotella and Pasteurellaceae OTUs. Few OTUs correlated with mesalamine concentration. The stomach and duodenal microbiota exhibited greater compositional dynamics than the jejunum. Short-term fluctuations in the duodenal microbiota were correlated with pH. Given the unique characteristics and dynamics of the proximal GI tract microbiota, it is important to consider these local environments in health and disease states.IMPORTANCE The gut microbiota are linked to a variety of gastrointestinal diseases, including inflammatory bowel disease. Despite this importance, microbiota dynamics in the upper gastrointestinal tract are understudied. Our article seeks to understand what factors impact microbiota dynamics in the healthy human upper gut. We found that the upper gastrointestinal tract contains consistently prevalent bacterial OTUs that dominate the overall community. Microbiota variability is highest in the stomach and duodenum and correlates with pH.

Increased Relative Abundance of Klebsiella pneumoniae Carbapenemase-producing Klebsiella pneumoniae Within the Gut Microbiota Is Associated With Risk of Bloodstream Infection in Long-term Acute Care Hospital Patients.

BACKGROUND: An association between increased relative abundance of specific bacterial taxa in the intestinal microbiota and bacteremia has been reported in some high-risk patient populations. METHODS: We collected weekly rectal swab samples from patients at 1 long-term acute care hospital (LTACH) in Chicago from May 2015 to May 2016. Samples positive for Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) by polymerase chain reaction and culture underwent 16S rRNA gene sequence analysis; relative abundance of the operational taxonomic unit containing KPC-Kp was determined. Receiver operator characteristic (ROC) curves were constructed using results from the sample with highest relative abundance of KPC-Kp from each patient admission, excluding samples collected after KPC-Kp bacteremia. Cox regression analysis was performed to evaluate risk factors associated with time to achieve KPC-Kp relative abundance thresholds calculated by ROC curve analysis. RESULTS: We collected 2319 samples from 562 admissions (506 patients); KPC-Kp colonization was detected in 255 (45.4%) admissions and KPC-Kp bacteremia in 11 (4.3%). A relative abundance cutoff of 22% predicted KPC-Kp bacteremia with sensitivity 73%, specificity 72%, and relative risk 4.2 (P = .01). In a multivariable Cox regression model adjusted for age, Charlson comorbidity index, and medical devices, carbapenem receipt was associated with achieving the 22% relative abundance threshold (P = .044). CONCLUSION: Carbapenem receipt was associated with increased hazard for high relative abundance of KPC-Kp in the gut microbiota. Increased relative abundance of KPC-Kp was associated with KPC-Kp bacteremia. Whether bacteremia arose directly from bacterial translocation or indirectly from skin contamination followed by bloodstream invasion remains to be determined.

Gut Microbiota and Clinical Features Distinguish Colonization With Klebsiella pneumoniae Carbapenemase-Producing Klebsiella pneumoniae at the Time of Admission to a Long-term Acute Care Hospital.

BACKGROUND: Identification of gut microbiota features associated with antibiotic-resistant bacterial colonization may reveal new infection prevention targets. METHODS: We conducted a matched, case-control study of long-term acute care hospital (LTACH) patients to identify gut microbiota and clinical features associated with colonization by Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp), an urgent antibiotic resistance threat. Fecal or rectal swab specimens were collected and tested for KPC-Kp; 16S rRNA gene-based sequencing was performed. Comparisons were made between cases and controls in calibration and validation subsamples using microbiota similarity indices, logistic regression, and unit-weighted predictive models. RESULTS: Case (n = 32) and control (n = 99) patients had distinct fecal microbiota communities, but neither microbiota diversity nor inherent clustering into community types distinguished case and control specimens. Comparison of differentially abundant operational taxonomic units (OTUs) revealed 1 OTU associated with case status in both calibration (n = 51) and validation (n = 80) subsamples that matched the canonical KPC-Kp strain ST258. Permutation analysis using the presence or absence of OTUs and hierarchical logistic regression identified 2 OTUs (belonging to genus Desulfovibrio and family Ruminococcaceae) associated with KPC-Kp colonization. Among clinical variables, the presence of a decubitus ulcer alone was independently and consistently associated with case status. Combining the presence of the OTUs Desulfovibrio and Ruminococcaceae with decubitus ulcer increased the likelihood of KPC-Kp colonization to >38% in a unit-weighted predictive model. CONCLUSIONS: We identified microbiota and clinical features that distinguished KPC-Kp gut colonization in LTACH patients, a population particularly susceptible to KPC-Kp infection. These features may warrant further investigation as markers of risk for KPC-Kp colonization.

Presence of multiple Clostridium difficile strains at primary infection is associated with development of recurrent disease.

Recurrence of Clostridium difficile infection (CDI) places a major burden on the healthcare system. Previous studies have suggested that specific C. difficile strains, or ribotypes, are associated with severe disease and/or recurrence. However, in some patients a new strain is detected in subsequent infections, complicating longitudinal studies focused on strain differences that may contribute to disease outcome. We examined ribotype composition over time in patients who did or did not develop recurrence to examine infection with multiple C. difficile ribotypes (mixed infection), during the course of infection. Using a retrospective patient cohort, we isolated and ribotyped a median of 36 C. difficile colonies from 61 patients (105 total samples) at initial infection, recurrence (a second case of CDI within 15-56 days of initial infection), and reinfection (a second case of CDI after 56 days of initial infection). We observed mixed infection in 78.6% of samples at initial infection in patients who went on to develop recurrence compared to 18.1% of patients who did not, and mixed infection remained associated with subsequent recurrence after adjusting for gender and prior antibiotic exposure (OR 3.5, 95% CI 1.3-9.4, P = .015). In patients who were sampled longitudinally (44 consecutive events in 32 patients), the dominant ribotype changed in 31.8% of consecutive samples and the newly dominant ribotype was not detected in prior samples from that patient. Our results suggest that mixed C. difficile infection is more prevalent than previously demonstrated and potentially a marker of susceptibility to recurrence.

Restoration of short chain fatty acid and bile acid metabolism following fecal microbiota transplantation in patients with recurrent Clostridium difficile infection.

A significant proportion of individuals develop recurrent Clostridium difficile infection (CDI) following initial disease. Fecal microbiota transplantation (FMT), a highly effective treatment method for recurrent CDI, has been demonstrated to induce microbiota recovery. One of the proposed functions associated with restoration of colonization resistance against C. difficile has been recovery of bile acid metabolism. In this study, we aimed to assess recovery of short chain fatty acids (SCFAs) in addition to bile acids alongside microbial community structure in six patients with recurrent CDI following treatment with FMT over time. Using 16S rRNA gene-based sequencing, we observed marked similarity of the microbiota between recipients following FMT (n = 6, sampling up to 6 months post-FMT) and their respective donors. Sustained increases in the levels of the SCFAs butyrate, acetate, and propionate were observed post-FMT, and variable recovery over time was observed in the secondary bile acids deoxycholate and lithocholate. To correlate these changes with specific microbial taxa at an individual level, we applied a generalized estimating equation approach to model metabolite concentrations with the presence of specific members of the microbiota. Metabolites that increased following FMT were associated with bacteria classified within the Lachnospiraceae, Ruminococcaceae, and unclassified Clostridiales families. In contrast, members of these taxa were inversely associated with primary bile acids. The longitudinal aspect of this study allowed us to characterize individualized patterns of recovery, revealing variability between and within patients following FMT.