Microbiota-Based Therapies for Clostridium difficile and Antibiotic-Resistant Enteric Infections.

Bacterial pathogens are increasingly antibiotic resistant, and development of clinically effective antibiotics is lagging. Curing infections increasingly requires antimicrobials that are broader spectrum, more toxic, and more expensive, and mortality attributable to antibiotic-resistant pathogens is rising. The commensal microbiota, comprising microbes that colonize the mammalian gastrointestinal tract, can provide high levels of resistance to infection, and the contributions of specific bacterial species to resistance are being discovered and characterized. Microbiota-mediated mechanisms of colonization resistance and pathogen clearance include bactericidal activity, nutrient depletion, immune activation, and manipulation of the gut's chemical environment. Current research is focusing on development of microbiota-based therapies to reduce intestinal colonization with antibiotic-resistant pathogens, with the goal of reducing pathogen transmission and systemic dissemination.

Imaging Manifestations of Chest Trauma.

Trauma is the leading cause of death among individuals under 40 years of age, and pulmonary trauma is common in high-impact injuries. Unlike most other organs, the lung is elastic and distensible, with a physiologic capacity to withstand significant changes in contour and volume. The most common types of lung parenchymal injury are contusions, lacerations, and hematomas, each having characteristic imaging appearances. A less common type of lung injury is herniation. Chest radiography is often the first-line imaging modality performed in the assessment of the acutely injured patient, although there are inherent limitations in the use of this modality in trauma. CT images are more accurate for the assessment of the nature and extent of pulmonary injury than the single-view anteroposterior chest radiograph that is typically obtained in the trauma bay. However, the primary limitations of CT concern the need to transport the patient to the CT scanner and a longer processing time. The American Association for the Surgery of Trauma has established the most widely used grading scale to describe lung injury, which serves to communicate severity, guide management, and provide useful prognostic factors in a systematic fashion. The authors provide an in-depth exploration of the most common types of pulmonary parenchymal, pleural, and airway injuries. Injury grading, patient management, and potential complications of pulmonary injury are also discussed. ©RSNA, 2021.

Bile acid sensitivity and in vivo virulence of clinical Clostridium difficile isolates.

Clostridium difficile is an anaerobic bacterium that causes diarrheal illnesses. Disease onset is linked with exposure to oral antibiotics and consequent depletion of secondary bile acids. Here we investigate the relationship between in vitro secondary bile acid tolerance and in vivo disease scores of diverse C. difficile strains in mice.

Loss of Microbiota-Mediated Colonization Resistance to Clostridium difficile Infection With Oral Vancomycin Compared With Metronidazole.

Antibiotic administration disrupts the intestinal microbiota, increasing susceptibility to pathogens such as Clostridium difficile. Metronidazole or oral vancomycin can cure C. difficile infection, and administration of these agents to prevent C. difficile infection in high-risk patients, although not sanctioned by Infectious Disease Society of America guidelines, has been considered. The relative impacts of metronidazole and vancomycin on the intestinal microbiota and colonization resistance are unknown. We investigated the effect of brief treatment with metronidazole and/or oral vancomycin on susceptibility to C. difficile, vancomycin-resistant Enterococcus, carbapenem-resistant Klebsiella pneumoniae, and Escherichia coli infection in mice. Although metronidazole resulted in transient loss of colonization resistance, oral vancomycin markedly disrupted the microbiota, leading to prolonged loss of colonization resistance to C. difficile infection and dense colonization by vancomycin-resistant Enterococcus, K. pneumoniae, and E. coli. Our results demonstrate that vancomycin, and to a lesser extent metronidazole, are associated with marked intestinal microbiota destruction and greater risk of colonization by nosocomial pathogens.

Utility of MRI in the Characterization of Indeterminate Small Renal Lesions Previously Seen on Screening CT Scans of Potential Renal Donor Patients.

OBJECTIVE: The purpose of this study was to determine whether MRI could more confidently characterize indeterminate small renal lesions (< 15 mm) previously seen on CT scans of potential renal donor patients and whether such characterization could impact surgical management and donor candidate status. MATERIALS AND METHODS: After dedicated contrast-enhanced renal CT examinations of a population of renal donor patients identified indeterminate small renal lesions (< 15 mm), dedicated renal MRI examinations were performed for 55 of those patients. Two radiologists used consensus reading of established MRI characteristics to characterize indeterminate small lesions as simple cysts, hemorrhagic cysts, angiomyolipomas, or solid renal masses. RESULTS: A total of 94 indeterminate small renal lesions were detected on CT. MRI was able to confidently diagnose 93 of those lesions, including 83 cysts, eight hemorrhagic cysts, and two angiomyolipomas. MRI directly affected the surgical management of four of the patients (7%). CONCLUSION: For potential renal donor patients, MRI can be an effective means of characterizing lesions that are deemed to be too small to characterize by CT. MRI can also potentially alter the surgical management and donor status of this group of patients.

Cloning and characterization of voltage-gated calcium channel alpha1 subunits in Xenopus laevis during development.

Voltage-gated calcium channels play a critical role in regulating the Ca2+ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore-forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that CaV1.2, CaV2.1, CaV2.2, and CaV3.2 are expressed during neurula stages throughout the neural tube. These, along with CaV1.3 and CaV2.3, beginning at early tail bud stages, and CaV3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development.

A Machine Learning-based Approach for Collaborative Non-Adherence Detection during Opioid Abuse Surveillance using a Wearable Biosensor.

Wearable biosensors can be used to monitor opioid use, a problem of dire societal consequence given the current opioid epidemic in the US. Such surveillance can prompt interventions that promote behavioral change. The effectiveness of biosensor-based monitoring is threatened by the potential of a patient's collaborative non-adherence (CNA) to the monitoring. We define CNA as the process of giving one's biosensor to someone else when surveillance is ongoing. The principal aim of this paper is to leverage accelerometer and blood volume pulse (BVP) measurements from a wearable biosensor and use machine-learning for the novel problem of CNA detection in opioid surveillance. We use accelerometer and BVP data collected from 11 patients who were brought to a hospital Emergency Department while undergoing naloxone treatment following an opioid overdose. We then used the data collected to build a personalized classifier for individual patients that capture the uniqueness of their blood volume pulse and triaxial accelerometer readings. In order to evaluate our detection approach, we simulate the presence (and absence) of CNA by replacing (or not replacing) snippets of the biosensor readings of one patient with another. Overall, we achieved an average detection accuracy of 90.96% when the collaborator was one of the other 10 patients in our dataset, and 86.78% when the collaborator was from a set of 14 users whose data had never been seen by our classifiers before.

Pathogenicity Locus, Core Genome, and Accessory Gene Contributions to Clostridium difficile Virulence.

Clostridium difficile is a spore-forming anaerobic bacterium that causes colitis in patients with disrupted colonic microbiota. While some individuals are asymptomatic C. difficile carriers, symptomatic disease ranges from mild diarrhea to potentially lethal toxic megacolon. The wide disease spectrum has been attributed to the infected host's age, underlying diseases, immune status, and microbiome composition. However, strain-specific differences in C. difficile virulence have also been implicated in determining colitis severity. Because patients infected with C. difficile are unique in terms of medical history, microbiome composition, and immune competence, determining the relative contribution of C. difficile virulence to disease severity has been challenging, and conclusions regarding the virulence of specific strains have been inconsistent. To address this, we used a mouse model to test 33 clinical C. difficile strains isolated from patients with disease severities ranging from asymptomatic carriage to severe colitis, and we determined their relative in vivo virulence in genetically identical, antibiotic-pretreated mice. We found that murine infections with C. difficile clade 2 strains (including multilocus sequence type 1/ribotype 027) were associated with higher lethality and that C. difficile strains associated with greater human disease severity caused more severe disease in mice. While toxin production was not strongly correlated with in vivo colonic pathology, the ability of C. difficile strains to grow in the presence of secondary bile acids was associated with greater disease severity. Whole-genome sequencing and identification of core and accessory genes identified a subset of accessory genes that distinguish high-virulence from lower-virulence C. difficile strains.IMPORTANCEClostridium difficile is an important cause of hospital-associated intestinal infections, and recent years have seen an increase in the number and severity of cases in the United States. A patient's antibiotic history, immune status, and medical comorbidities determine, in part, the severity of C. difficile infection. The relative virulence of different clinical C. difficile strains, although postulated to determine disease severity in patients, has been more difficult to consistently associate with mild versus severe colitis. We tested 33 distinct clinical C. difficile isolates for their ability to cause disease in genetically identical mice and found that C. difficile strains belonging to clade 2 were associated with higher mortality. Differences in survival were not attributed to differences in toxin production but likely resulted from the distinct gene content in the various clinical isolates.

Characterization of HIV diversity, phylodynamics and drug resistance in Washington, DC.

BACKGROUND: Washington DC has a high burden of HIV with a 2.0% HIV prevalence. The city is a national and international hub potentially containing a broad diversity of HIV variants; yet few sequences from DC are available on GenBank to assess the evolutionary history of HIV in the US capital. Towards this general goal, here we analyze extensive sequence data and investigate HIV diversity, phylodynamics, and drug resistant mutations (DRM) in DC. METHODS: Molecular HIV-1 sequences were collected from participants infected through 2015 as part of the DC Cohort, a longitudinal observational study of HIV+ patients receiving care at 13 DC clinics. Sequences were paired with Cohort demographic, risk, and clinical data and analyzed using maximum likelihood, Bayesian and coalescent approaches of phylogenetic, network and population genetic inference. We analyzed 601 sequences from 223 participants for int (~864 bp) and 2,810 sequences from 1,659 participants for PR/RT (~1497 bp). RESULTS: Ninety-nine and 94% of the int and PR/RT sequences, respectively, were identified as subtype B, with 14 non-B subtypes also detected. Phylodynamic analyses of US born infected individuals showed that HIV population size varied little over time with no significant decline in diversity. Phylogenetic analyses grouped 13.5% of the int sequences into 14 clusters of 2 or 3 sequences, and 39.0% of the PR/RT sequences into 203 clusters of 2-32 sequences. Network analyses grouped 3.6% of the int sequences into 4 clusters of 2 sequences, and 10.6% of the PR/RT sequences into 76 clusters of 2-7 sequences. All network clusters were detected in our phylogenetic analyses. Higher proportions of clustered sequences were found in zip codes where HIV prevalence is highest (r = 0.607; P<0.00001). We detected a high prevalence of DRM for both int (17.1%) and PR/RT (39.1%), but only 8 int and 12 PR/RT amino acids were identified as under adaptive selection. We observed a significant (P<0.0001) association between main risk factors (men who have sex with men and heterosexuals) and genotypes in the five well-supported clusters with sufficient sample size for testing. DISCUSSION: Pairing molecular data with clinical and demographic data provided novel insights into HIV population dynamics in Washington, DC. Identification of populations and geographic locations where clustering occurs can inform and complement active surveillance efforts to interrupt HIV transmission.

Innate Immune Defenses Mediated by Two ILC Subsets Are Critical for Protection against Acute Clostridium difficile Infection.

Infection with the opportunistic enteric pathogen Clostridium difficile is an increasingly common clinical complication that follows antibiotic treatment-induced gut microbiota perturbation. Innate lymphoid cells (ILCs) are early responders to enteric pathogens; however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we challenged C57BL/6, Rag1(-/-) (which lack T and B cells), and Rag2(-/-)Il2rg(-/-) (Ragγc(-/-)) mice (which additionally lack ILCs) with C. difficile. In contrast to Rag1(-/-) mice, ILC-deficient Ragγc(-/-) mice rapidly succumbed to infection. Rag1(-/-) but not Ragγc(-/-) mice upregulate expression of ILC1- or ILC3-associated proteins following C. difficile infection. Protection against infection was restored by transferring ILCs into Ragγc(-/-) mice. While ILC3s made a minor contribution to resistance, loss of IFN-γ or T-bet-expressing ILC1s in Rag1(-/-) mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile.

The role of voltage-gated calcium channels in neurotransmitter phenotype specification: Coexpression and functional analysis in Xenopus laevis.

Calcium activity has been implicated in many neurodevelopmental events, including the specification of neurotransmitter phenotypes. Higher levels of calcium activity lead to an increased number of inhibitory neural phenotypes, whereas lower levels of calcium activity lead to excitatory neural phenotypes. Voltage-gated calcium channels (VGCCs) allow for rapid calcium entry and are expressed during early neural stages, making them likely regulators of activity-dependent neurotransmitter phenotype specification. To test this hypothesis, multiplex fluorescent in situ hybridization was used to characterize the coexpression of eight VGCC α1 subunits with the excitatory and inhibitory neural markers xVGlut1 and xVIAAT in Xenopus laevis embryos. VGCC coexpression was higher with xVGlut1 than xVIAAT, especially in the hindbrain, spinal cord, and cranial nerves. Calcium activity was also analyzed on a single-cell level, and spike frequency was correlated with the expression of VGCC α1 subunits in cell culture. Cells expressing Cav 2.1 and Cav 2.2 displayed increased calcium spiking compared with cells not expressing this marker. The VGCC antagonist diltiazem and agonist (-)BayK 8644 were used to manipulate calcium activity. Diltiazem exposure increased the number of glutamatergic cells and decreased the number of γ-aminobutyric acid (GABA)ergic cells, whereas (-)BayK 8644 exposure decreased the number of glutamatergic cells without having an effect on the number of GABAergic cells. Given that the expression and functional manipulation of VGCCs are correlated with neurotransmitter phenotype in some, but not all, experiments, VGCCs likely act in combination with a variety of other signaling factors to determine neuronal phenotype specification.

Dissection, culture, and analysis of Xenopus laevis embryonic retinal tissue.

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation(1-16). The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates (12,14-18). While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells (7,19-23). For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues (8,19-22,24-33). Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level (5,8,21,24,27-30,33-39). Xenopus laevis, a classic model system for the study of early neural development (19,27,29,31-32,40-42), serves as a particularly suitable system for retinal primary cell culture (10,38,43-45). Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction (25,38,43). In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products (10,24,44-45). However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Geographic variation in venom allelic composition and diets of the widespread predatory marine gastropod Conus ebraeus.

BACKGROUND: Members of the predatory gastropod genus Conus use a venom comprised of a cocktail of peptide neurotoxins, termed conotoxins or conopeptides, to paralyze prey and conotoxin gene family members diversify via strong positive selection. Because Conus venoms are used primarily to subdue prey, the evolution of venoms is likely affected by predator-prey interactions. METHODOLOGY/PRINCIPAL FINDINGS: To identify the selective forces that drive the differentiation of venoms within species of Conus, we examined the distribution of alleles of a polymorphic O-superfamily conotoxin locus of Conus ebraeus at Okinawa, Guam and Hawaii. Previous analyses of mitochondrial cytochrome oxidase I gene sequences suggest that populations of C. ebraeus, a worm-eating Conus, are not structured genetically in the western and central Pacific. Nonetheless, because the sample size from Guam was relatively low, we obtained additional data from this location and reexamined patterns of genetic variation at the mitochondrial gene at Okinawa, Guam and Hawaii. We also utilized a DNA-based approach to identify prey items of individuals of C. ebraeus from Guam and compared this information to published data on diets at Okinawa and Hawaii. Our results show that conotoxin allelic frequencies differ significantly among all three locations, with strongest differentiation at Hawaii. We also confirm previous inferences that C. ebraeus exhibits no genetic differentiation between Okinawa, Guam and Hawaii at the mitochondrial locus. Finally, DNA-based analyses show that eunicid polychaetes comprise the majority of the prey items of C. ebraeus at Guam; while this results compares well with observed diet of this species at Okinawa, C. ebraeus preys predominantly on nereid polychaetes at Hawaii. CONCLUSIONS/SIGNIFICANCE: These results imply that strong selection pressures affect conotoxin allelic frequencies. Based on the dietary information, the selection may derive from geographic variation in dietary specialization and local coevolutionary arms races between Conus and their prey.