The TcdE holin drives toxin secretion and virulence in Clostridioides difficile.

Clostridioides difficile is the leading cause of healthcare associated infections. The Pathogenicity Locus (PaLoc) toxins TcdA and TcdB promote host disease. These toxins lack canonical N-terminal signal sequences for translocation across the bacterial membrane, suggesting alternate mechanisms of release, which have included targeted secretion and passive release from cell lysis. While the holin TcdE has been implicated in TcdA and TcdB release, its role in vivo remains unknown. Here, we show profound reductions in toxin secretion in ΔtcdE mutants in the highly virulent strains UK1 (epidemic ribotype 027, Clade 3) and VPI10463 (ribotype 087, Clade 1). Notably, tcdE deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection by reducing acute extracellular toxin to undetectable levels, limiting mucosal damage, and enabling long-term survival, in spite of continued toxin gene expression in ΔtcdE mutants. Our findings confirm TcdE's critical functions in vivo for toxin secretion and C. difficile virulence.

Genomics of Corynebacterium striatum, an emerging multidrug-resistant pathogen of immunocompromised patients.

OBJECTIVES: Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen of immunocompromised and chronically ill patients. The objective of these studies was to provide a detailed genomic analysis of disease-causing C. striatum and determine the genomic drivers of resistance and resistance-gene transmission. METHODS: A multi-institutional and prospective pathogen genomics programme flagged seven MDR C. striatum infections occurring close in time, and specifically in immunocompromised patients with underlying respiratory diseases. Whole genome sequencing was used to identify clonal relationships among strains, genetic causes of antimicrobial resistance, and their mobilization capacity. Matrix-assisted linear desorption/ionization-time-of-flight analyses of sequenced isolates provided curated content to improve rapid clinical identification in subsequent cases. RESULTS: Epidemiological and genomic analyses identified a related cluster of three out of seven C. striatum among lung transplant patients who had common procedures and exposures at an outlying institution. Genomic analyses further elucidated drivers of the MDR phenotypes, including resistance genes mobilized by IS3504 and ISCg9a-like insertion sequences. Seven mobilizable resistance genes were localized to a common chromosomal region bounded by unpaired insertion sequences, suggesting that a single recombination event could spread resistance to aminoglycosides, macrolides, lincosamides and tetracyclines to naive strains. CONCLUSION: In-depth genomic studies of MDR C. striatum reveal its capacity for clonal spread within and across healthcare institutions and identify novel vectors that can mobilize multiple forms of drug resistance, further complicating efforts to treat infections in immunocompromised populations.

Recurrent Clostridium difficile infection associates with distinct bile acid and microbiome profiles.

BACKGROUND: The healthy microbiome protects against the development of Clostridium difficile infection (CDI), which typically develops following antibiotics. The microbiome metabolises primary to secondary bile acids, a process if disrupted by antibiotics, may be critical for the initiation of CDI. AIM: To assess the levels of primary and secondary bile acids associated with CDI and associated microbial changes. METHODS: Stool and serum were collected from patients with (i) first CDI (fCDI), (ii) recurrent CDI (rCDI) and (iii) healthy controls. 16S rRNA sequencing and bile salt metabolomics were performed. Random forest regression models were constructed to predict disease status. PICRUSt analyses were used to test for associations between predicted bacterial bile salt hydrolase (BSH) gene abundances and bile acid levels. RESULTS: Sixty patients (20 fCDI, 19 rCDI and 21 controls) were enrolled. Secondary bile acids in stool were significantly elevated in controls compared to rCDI and fCDI (P < 0.0001 and P = 0.0007 respectively). Primary bile acids in stool were significantly elevated in rCDI compared to controls (P < 0.0001) and in rCDI compared to fCDI (P = 0.02). Using random forest regression, we distinguished rCDI and fCDI patients 84.2% of the time using bile acid ratios. Stool deoxycholate to glycoursodeoxycholate ratio was the single best predictor. PICRUSt analyses found significant differences in predicted abundances of bacterial BSH genes in stool samples across the groups. CONCLUSIONS: Primary and secondary bile acid composition in stool was different in those with rCDI, fCDI and controls. The ratio of stool deoxycholate to glycoursodeoxycholate was the single best predictor of disease state and may be a potential biomarker for recurrence.

Maternal intestinal flora and wheeze in early childhood.

BACKGROUND: Increasing evidence links altered intestinal flora in infancy to eczema and asthma. No studies have investigated the influence of maternal intestinal flora on wheezing and eczema in early childhood. OBJECTIVE: To investigate the link between maternal intestinal flora during pregnancy and development of wheeze and eczema in infancy. METHODS: A total of 60 pregnant women from the Boston area gave stool samples during the third trimester of their pregnancy and answered questions during pregnancy about their own health, and about their children's health when the child was 2 and 6 months of age. Quantitative culture was performed on stool samples and measured in log(10)colony-forming units (CFU)/gram stool. Primary outcomes included infant wheeze and eczema in the first 6 months of life. Atopic wheeze, defined as wheeze and eczema, was analysed as a secondary outcome. RESULTS: In multivariate models adjusted for breastfeeding, day care attendance and maternal atopy, higher counts of maternal total aerobes (TA) and enterococci (E) were associated with increased risk of infant wheeze (TA: OR 2.32 for 1 log increase in CFU/g stool [95% CI 1.22, 4.42]; E: OR 1.57 [95% CI 1.06, 2.31]). No organisms were associated with either eczema or atopic wheeze. CONCLUSIONS AND CLINICAL RELEVANCE: In our cohort, higher maternal total aerobes and enterococci were related to increased risk of infant wheeze. Maternal intestinal flora may be an important environmental exposure in early immune system development.

THE ROLE OF SPECIFIC IgG AND COMPLEMENT IN COMBATING A PRIMARY MUCOSAL INFECTION OF THE GUT EPITHELIUM.

The role of complement and complement-fixing IgG isotypes at mucosal surfaces is ill defined. Previous data have demonstrated that survival of an infection with the attaching and effacing pathogen Citrobacter rodentium requires production of systemic and CD4+ T cell-dependent IgG. We have found that both complement and complement-fixing IgG isotypes are needed to survive a C. rodentium infection. Our results indicate that both IgG and complement C3b enter the gut lumen and bind epithelially adherent, and fecally shed C. rodentium. Furthermore, C3-deficient mice demonstrate a profound survival defect, though means to replenish C3 in systemic or mucosal sites restores the protective capacity of complement in the host. Our data provide evidence that both IgG and complement interact constructively on both sides of the epithelium to fight colonizing mucosal infections.

Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin.

BACKGROUND: Glycohemoglobin (gHb), measured as hemoglobin (Hb) A(1c) or as total gHb, provides a common means for assessing long-term glycemic control in individuals with diabetes mellitus. Genetic variants and chemically modified derivatives of Hb can profoundly affect the accuracy of these measurements, although effects vary considerably among commercially available methods. The prevalence of genetic variants such as HbS, HbC, and HbE, and chemically modified derivatives such as carbamyl-Hb among patient populations undergoing testing is not insignificant. Clinical laboratories and sites responsible for point-of-care testing of gHb need to be aware of the interferences produced in assays by these Hbs. APPROACH: We conducted a review of the literature describing the effects of variant Hbs on gHb assay methods commonly used in clinical laboratories. CONTENT: This review summarizes the documented effects of both common and uncommon Hb variants and derivatives on the measurement of gHb. Where known, we discuss mechanisms of interference on specific assays and methodologies. We specifically address effects of commonly encountered Hbs, such as carbamyl-Hb, HbS, HbC, HbE, and HbF, on assays that use cation-exchange chromatography, immunoassays, or boronate affinity methods for measuring gHb. SUMMARY: A variety of patient- and laboratory-related factors can adversely affect the measurement of gHb in patients harboring Hb variants or derivatives. Identification of the variant or derivative Hb before or during testing may allow accurate measurement of gHb by the selection of a method unaffected by the given variant or derivative. However, laboratories should make available alternative, non-Hb-based methods for assessing long-term glycemic control in individuals with HbCC, HbSS, or HbSC disease, or with other underlying disorders where the concentration of gHb does not accurately reflect long-term glycemic control.

Host-microbial symbiosis in the mammalian intestine: exploring an internal ecosystem.

The mammalian intestine contains a complex, dynamic, and spatially diversified society of nonpathogenic bacteria. Very little is known about the factors that help establish host-microbial symbiosis in this open ecosystem. By introducing single genetically manipulatable components of the microflora into germfree mice, simplified model systems have been created that will allow conversations between host and microbe to be heard and understood. Other paradigms of host-microbial symbiosis suggest that these interactions will involve an exchange of biochemical signals between host and symbionts as well as among the bacteria themselves. The integration of molecular microbiology, cell biology, and gnotobiology should provide new insights about how we adapt to a microbial world and reveal the roles played by our indigenous, 'nonpathogenic' flora.

Epithelial cell growth and differentiation. III. Promoting diversity in the intestine: conversations between the microflora, epithelium, and diffuse GALT.

Homeostasis in the self-renewing mouse intestinal epithelium appears to be regulated in large part by cell nonautonomous mechanisms. The society of nonpathogenic bacteria that resides in the intestine is an important source of instructions that modify epithelial differentiation programs. The stability of this society is remarkable given its numerical, compositional, and spatial complexity, the openness of the ecosystem, and the fact that the epithelium is replaced so rapidly. The ability of components of this society to influence epithelial differentiation may represent a critical step in allowing specific groups of organisms to be assembled in specific regions of the gut. Simplified model systems have been created to define and dissect the conversations between microbe and host. These systems use inbred strains of mice that are raised under germ-free conditions and then monocontaminated with a single component of the microflora. The results suggest that a trialogue involving communications between the microflora, the epithelium, and the diffuse gut-associated lymphoid tissue (GALT) may play a key role in establishing and maintaining the spatial diversity of this remarkable ecosystem.

A model of host-microbial interactions in an open mammalian ecosystem.

The maintenance and significance of the complex populations of microbes present in the mammalian intestine are poorly understood. Comparison of conventionally housed and germ-free NMRI mice revealed that production of fucosylated glycoconjugates and an alpha1, 2-fucosyltransferase messenger RNA in the small-intestinal epithelium requires the normal microflora. Colonization of germ-free mice with Bacteroides thetaiotaomicron, a component of this flora, restored the fucosylation program, whereas an isogenic strain carrying a transposon insertion that disrupts its ability to use L-fucose as a carbon source did not. Simplified models such as this should aid the study of open microbial ecosystems.

Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

Paneth cell-rich flat adenoma of the rectum: report of a case.

A patient having familiar adenomatosis polyposis and an ileo-rectal anastomosis developed a flat mucosal lesion in the rectum. A punch biopsy revealed a villous adenoma with high-grade dysplasia. The subsequent surgical specimen indicated that the flat villous adenoma was rich in Paneth cells. Special stains included lysozyme muramidase (to visualize Paneth cells), MIB1 proliferation monoclonal antibody and single and multilabel immunohistochemistry for Paneth cells. Other methods included transmission electron microscopy and quantification with an image quantifier (Program Optilab 2.1) of lysozyme-stained Paneth cells. The subjective evaluation of hematoxylin-eosin-stained preparations demonstrated that the Paneth cells were mainly located in the lower half of the villi. Sections labeled with a specific stain (lysozyme muramidase) revealed more Paneth cells in the villi and electron microscopy showed even more in lysozyme-negative areas. Obviously some migrating dysplastic Paneth cells had retained their characteristic granules on their way towards the tip of the villi. Quantitative studies indicated that the lysozyme muramidase-positive material accounted for 41% of the adenomatous tissue. MIB1 revealed intense cell proliferation at the base of the adenoma and in the entire slopes of the villi. Despite the wide distribution of Paneth cells in intestinal metaplasia of the stomach, in the normal small intestine and in the large bowel with chronic inflammatory diseases, it is surprising that tumors arising in Paneth cells are extremely rare. The causes of the apparent natural resistance of Paneth cells to tumor development deserve to be investigated. This is the first case of Paneth cell-rich flat adenoma of the rectum in the literature.

Expression of a human alpha-1,3/4-fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production of Leb-containing glycoconjugates: a potential transgenic mouse model for studying Helicobacter pylori infection.

Helicobacter pylori is a human pathogen associated with the development of gastric and duodenal ulcers and gastric adenocarcinoma. To test the hypothesis that the human Lewis(b) blood group antigen (Le(b)) functions as a receptor for the bacteria's adhesins and mediates its attachment to gastric pit and surface mucous cells, a human alpha-1,3/4-fucosyltransferase was expressed in these cell lineages in FVB/N transgenic mice. The fucosyltransferase directed production of the Leb epitope without any apparent effect on the proliferation and differentiation programs of this lineage. Moreover, clinical isolates of H. pylori bound to these cells in transgenic mice but not in their normal littermates. Binding was blocked by pretreatment of the bacteria with soluble Le(b). This mouse model could be useful for examining the molecular pathogenesis of diseases caused by H. pylori infection. Creating novel pathways for production of specific oligosaccharides in selected cell lineages of transgenic animals represents an approach for examining the role of complex carbohydrates in regulating cellular differentiation and host-microbe interactions.

Paneth cell differentiation in the developing intestine of normal and transgenic mice.

Paneth cells represent one of the four major epithelial lineages in the mouse small intestine. It is the only lineage that migrates downward from the stem-cell zone located in the lower portion of the crypt of Lieberkühn to the crypt base. Mature Paneth cells release growth factors, digestive enzymes, and antimicrobial peptides from their apical secretory granules. Some of these factors may affect the crypt stem cell, its transit-cell descendants, differentiating villus-associated epithelial lineages, and/or the gut microflora. We used single and multilabel immunocytochemical methods to study Paneth cell differentiation during and after completion of gut morphogenesis in normal, gnotobiotic, and transgenic mice as well as in intestinal isografts. This lineage emerges coincident with cytodifferentiation of the fetal small intestinal endoderm, formation of crypts from an intervillus epithelium, and establishment of a stem-cell hierarchy. The initial differentiation program involves sequential expression of cryptdins, a phospholipase A2 (enhancing factor), and lysozyme. A dramatic increase in Paneth cell number per crypt occurs during postnatal days 14-28, when crypts proliferate by fission. Accumulation of fucosylated and sialylated glycoconjugates during this period represents the final evolution of the lineage's differentiation program. Establishment of this lineage is not dependent upon instructive interactions from the microflora. Transgenic mice containing nucleotides -6500 to +34 of the Paneth cell-specific mouse cryptdin 2 gene linked to the human growth hormone gene beginning at its nucleotide +3 inappropriately express human growth hormone in a large population of proliferating and nonproliferating cells in the intervillus epithelium up to postnatal day 5. Transgene expression subsequently becomes restricted to the Paneth cell lineage in the developing crypt. Cryptdin 2 nucleotides -6500 to +34 should be a useful marker of crypt morphogenesis and a valuable tool for conducting gain-of-function or loss-of-function experiments in Paneth cells.