Suppression of Clostridium difficile in the Gastrointestinal Tracts of Germfree Mice Inoculated with a Murine Isolate from the Family Lachnospiraceae

The indigenous microbial community of the gastrointestinal (GI) tract determines susceptibility to Clostridium difficile colonization and disease. Previous studies have demonstrated that antibiotic-treated mice challenged with C. difficile either developed rapidly lethal C. difficile infection or were stably colonized with mild disease. The GI microbial community of animals with mild disease was dominated by members of the bacterial family Lachnospiraceae, while the gut community in moribund animals had a predominance of Escherichia coli. We investigated the roles of murine Lachnospiraceae and E. coli strains in colonization resistance against C. difficile in germfree mice. Murine Lachnospiraceae and E. coli isolates were cultured from wild-type mice. The ability of each of these isolates to interfere with C. difficile colonization was tested by precolonizing germfree mice with these bacteria 4 days prior to experimental C. difficile challenge. Mice precolonized with a murine Lachnospiraceae isolate, but not those colonized with E. coli, had significantly decreased C. difficile colonization, lower intestinal cytotoxin levels and exhibited less severe clinical signs and colonic histopathology. Infection of germfree mice or mice precolonized with E. coli with C. difficile strain VPI 10463 was uniformly fatal by 48 h, but only 20% mortality was seen at 2 days in mice precolonized with the Lachnospiraceae isolate prior to challenge with VPI 10463. These findings confirm that a single component of the GI microbiota, a murine Lachnospiraceae isolate, could partially restore colonization resistance against C. difficile. Further study of the members within the Lachnospiraceae family could lead to a better understanding of mechanisms of colonization resistance against C. difficile and novel therapeutic approaches for the treatment and prevention of C. difficile infection.     

Clostridium difficile Infection: A Comprehensive Review

Clostridium difficile is one of the most important causes of healthcare acquired diarrhea. The disease spectrum caused by C. difficile infection ranges from mild, self-limited, illness to a severe, life-threatening colitis. The incidence of C. difficile associated disease has risen dramatically over the last decade, leading to increased research interest aiming at the discovery of new virulence factors and the development of new treatment and prevention regimens. This review summarizes the pathogenesis and changing epidemiology of C. difficile associated disease, the clinical spectrum and laboratory methods to diagnose C. difficile infection, and current treatment strategies.     

Gastrointestinal Tract and the Mucosal Macrophage Reservoir in HIV Infection

The gastrointestinal tract (GIT) is a primary site for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection, replication, and dissemination. After an initial explosive phase of infection, HIV establishes latency. In addition to CD4 T cells, macrophages are readily infected, which can persist for long periods of time. Though macrophages at various systemic sites are infected, those present in the GIT constitute a major cellular reservoir due to the abundance of these cells at mucosal sites. Here, we review some of the important findings regarding what is known about the macrophage reservoir in the gut and explore potential approaches being pursued in the field to reduce this reservoir. The development of strategies that can lead to a functional cure will need to incorporate approaches that can eradicate the macrophage reservoir in the GIT.     

Influences of Dietary and Environmental Stress on Microbial Populations in the Murine Gastrointestinal Tract

Aerobic and anaerobic cultural techniques and histological methods were used in a study of the effects of environmental and dietary stress on the indigenous microbiota of the gastrointestinal tract of mice. Mice previously inoculated with Salmonella typhimurium were examined in a similar manner. Three strains of mice (CD-1, Ha/ICr, and C57BL) were used. Control animals previously inoculated with S. typhimurium had low population levels of Salmonella bacteria in the small and large bowel. Mice previously inoculated with Salmonella and then deprived of food, water, and bedding for 48 h harbored high population levels of these bacteria in their small and large bowels. Coliforms increased in numbers in the large bowel of stressed mice inoculated with Salmonella and in the jejunum-ileum and cecum of stressed mice not previously inoculated with Salmonella. Control mice had high population levels of lactobacilli inhabiting the keratinized squamous epithelium of the stomach. Stressed mice showed dramatic reductions in these populations of lactobacilli. Populations of fusiform-shaped bacteria associated with the mucosal epithelium of the cecum and colon in control mice were reduced in stressed mice as determined by microscope examination of histological sections. Total anaerobic counts were similar, however, in both stressed and control animals. Environmental and dietary stress markedly alter the gastrointestinal microbiota in mice. Therefore, such stressful conditions profoundly affect the factors that regulate the localization and population levels of microorganisms in the stomach and intestines.     

The Bristol Stool Scale and Its Relationship to Clostridium difficile Infection

The Bristol stool form scale classifies the relative density of stool samples. In a prospective cohort study, we investigated the associations between stool density, C. difficile assay positivity, hospital-onset C. difficile infection, complications, and severity of C. difficile. We describe associations between the Bristol score, assay positivity, and clinical C. difficile infection.     

Role of Gamma Interferon in Controlling Murine Chlamydial Genital Tract Infection

Earlier investigations have not shown an important role for gamma interferon (IFN-gamma) in the early clearance of chlamydial infection from the murine female genital tract. In a model using a human genital isolate of Chlamydia trachomatis in IFN-gamma and IFN-gamma receptor knockout mice, we were able to demonstrate a major role for IFN-gamma in mediating control of infection throughout the course of infection.     

Survey of Incidence of Clostridium difficile Infection in Canadian Hospitals and Diagnostic Approaches

A questionnaire relating to Clostridium difficile disease incidence and diagnostic practices was sent to 380 Canadian hospitals (all with >50 beds). The national questionnaire response rate was 63%. In-house testing was performed in 17.6, 61.5, and 74.2% of the hospitals with <300, 300 to 500, and >500 beds, respectively. The average test positivity rates were 17.2, 15.3, and 13.2% for hospitals with <300, 300 to 500, and >500 beds, respectively. The average disease incidences were 23.5, 30.8, and 40.3 cases per 100,000 patient days in the hospitals with <300, 300 to 500, and >500 beds, respectively. In the 81 hospitals where in-house testing was performed, cytotoxin testing utilizing tissue culture was most common (44.4%), followed by enzyme-linked immunosorbent assay (38.3%), culture for toxigenic C. difficile (32.1%), and latex agglutination (13.6%). The clinical criteria for C. difficile testing were variable, with 85% of hospitals indicating that a test was done automatically if ordered by a doctor. Our results show that C. difficile-associated diarrhea is a major problem in hospitals with ≥200 beds. Despite a lower disease incidence in smaller hospitals, there was a higher diagnostic test positivity rate. This may reflect the preference of smaller hospitals for culture and latex agglutination tests.     

Contribution of Adenosine A2B Receptors in Clostridium difficile Intoxication and Infection

Clostridium difficile toxins A (TcdA) and B (TcdB) induce a pronounced systemic and intestinal inflammatory response. A_2B adenosine receptors (A_2BARs) are the predominant adenosine receptors in the intestinal epithelium. We investigated whether A_2BARs are upregulated in human intestinal cells by TcdA or TcdB and whether blockade of A_2BARs can ameliorate C. difficile TcdA-induced enteritis and alter the outcome of C. difficile infection (CDI). Adenosine receptor subtype (A₁, A_2A, A_2B, and A₃) mRNAs were assayed in HCT-8 cells. Ileal loops from wild-type rabbits and mice and A_2BAR^−/− mice were treated with TcdA, with or without the selective A_2BAR antagonist ATL692 or PSB1115. A murine model of CDI was used to determine the effect of A_2BAR deletion or blockade with the orally available agent ATL801, on clinical outcome, histopathology and intestinal interleukin-6 (IL-6) expression from infection. TcdA and TcdB upregulated A_2BAR gene expression in HCT-8 cells. ATL692 decreased TcdA-induced secretion and epithelial injury in rabbit ileum. Deletion of A_2BARs reduced secretion and histopathology in TcdA-challenged mouse ileum. Deletion or blockade of A_2BARs reduced histopathology, IL-6 expression, weight loss, diarrhea, and mortality in C. difficile-infected mice. A_2BARs mediate C. difficile toxin-induced enteritis and disease. Inhibition of A_2BAR activation may be a potential strategy to limit morbidity and mortality from CDI.     

Economic Evaluation of Laboratory Testing Strategies for Hospital-Associated Clostridium difficile Infection

Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in health care settings, and for patients presumed to have CDI, their isolation while awaiting laboratory results is costly. Newer rapid tests for CDI may reduce this burden, but the economic consequences of different testing algorithms remain unexplored. We used decision analysis from the hospital perspective to compare multiple CDI testing algorithms for adult inpatients with suspected CDI, assuming patient management according to laboratory results. CDI testing strategies included combinations of on-demand PCR (odPCR), batch PCR, lateral-flow diagnostics, plate-reader enzyme immunoassay, and direct tissue culture cytotoxicity. In the reference scenario, algorithms incorporating rapid testing were cost-effective relative to nonrapid algorithms. For every 10,000 symptomatic adults, relative to a strategy of treating nobody, lateral-flow glutamate dehydrogenase (GDH)/odPCR generated 831 true-positive results and cost $1,600 per additional true-positive case treated. Stand-alone odPCR was more effective and more expensive, identifying 174 additional true-positive cases at $6,900 per additional case treated. All other testing strategies were dominated by (i.e., more costly and less effective than) stand-alone odPCR or odPCR preceded by lateral-flow screening. A cost-benefit analysis (including estimated costs of missed cases) favored stand-alone odPCR in most settings but favored odPCR preceded by lateral-flow testing if a missed CDI case resulted in less than $5,000 of extended hospital stay costs and <2 transmissions, if lateral-flow GDH diagnostic sensitivity was >93%, or if the symptomatic carrier proportion among the toxigenic culture-positive cases was >80%. These results can aid guideline developers and laboratory directors who are considering rapid testing algorithms for diagnosing CDI.     

Intestinal Dysbiosis and Depletion of Butyrogenic Bacteria in Clostridium difficile Infection and Nosocomial Diarrhea

Clostridium difficile infection (CDI) causes nearly half a million cases of diarrhea and colitis in the United States each year. Although the importance of the gut microbiota in C. difficile pathogenesis is well recognized, components of the human gut flora critical for colonization resistance are not known. Culture-independent high-density Roche 454 pyrosequencing was used to survey the distal gut microbiota for 39 individuals with CDI, 36 subjects with C. difficile-negative nosocomial diarrhea (CDN), and 40 healthy control subjects. A total of 526,071 partial 16S rRNA sequence reads of the V1 to V3 regions were aligned with 16S databases, identifying 3,531 bacterial phylotypes from 115 fecal samples. Genomic analysis revealed significant alterations of organism lineages in both the CDI and CDN groups, which were accompanied by marked decreases in microbial diversity and species richness driven primarily by a paucity of phylotypes within the Firmicutes phylum. Normally abundant gut commensal organisms, including the Ruminococcaceae and Lachnospiraceae families and butyrate-producing C2 to C4 anaerobic fermenters, were significantly depleted in the CDI and CDN groups. These data demonstrate associations between the depletion of Ruminococcaceae, Lachnospiraceae, and butyrogenic bacteria in the gut microbiota and nosocomial diarrhea, including C. difficile infection. Mechanistic studies focusing on the functional roles of these organisms in diarrheal diseases and resistance against C. difficile colonization are warranted.     

Nonspecific Binding of Clostridium difficile Toxin A to Murine Immunoglobulins Occurs via the Fab Component

Clostridium difficile toxin A binds nonspecifically to a mouse monoclonal antibody (MAb) immunoglobulin G3 λ chain [IgG3(λ)], through the Fab component. This binding, which is retained even after boiling the MAb, is temperature dependent, with more toxin bound at 4 than 37°C (P = 0.0024). The nonspecific binding was decreased by incubation of the IgG3 λ MAb with α- or β-galactosidase (P = 0.0001 and 0.029, respectively), indicating that toxin A binds to a carbohydrate moiety on the Fab. However, binding was not blocked by the Bandeiraea simplicifolia lectin BS-1, indicating that a terminal α-galactose may not be involved. Binding was also not affected by competitive assays with Lewis X antigen. The dependence on carbohydrate moieties in nonspecific binding was also shown for two other MAbs, IgA(κ) and IgM(λ), with demonstration of a significant reduction in binding with α-galactosidase (P = 0.0001 and 0.0002, respectively) but not β-galactosidase (P = 0.27 and 0.25, respectively).     

Fecal microbiota transplantation for Clostridium difficile infection in Taiwan: Establishment and implementation

Clostridium difficile infection (CDI) remains a major public health issue, and fecal microbiota transplantation (FMT) has become one of the standard therapies for recurrent or refractory CDI. When compared to medical therapies, such as metronidazole or vancomycin, FMT has a high rate of treatment response with acceptable safety and efficiency. Following promulgation of the amendments in September 2018 in Taiwan, FMT has been indicated for recurrent or refractory CDI. The Taiwan Microbiota Consortium contributed to the Taiwan FMT Expert Consensus, which established basic norms and stipulated essential principles, including the indications for transplantation, eligible locations and personnel, donor screening policies, fecal sample handling, and post-FMT follow-up. However, establishing an eligible FMT team in a qualified hospital remains a clinical challenge, and the requirement for facilities and well-screened donors impedes the implementation of FMT. In this review, we aim to provide domestic FMT teams with explicit instructions to facilitate realization and increase the practice of FMT. Based on the Taiwan FMT Expert Consensus and current regulations, we performed a literature review and integrated the experiences of Taiwanese multidisciplinary experts into this article. The content intends to offer clinicians up-to-date evidence and highlight the essential points of FMT.     

Ultrasensitive Detection and Quantification of Toxins for Optimized Diagnosis of Clostridium difficile Infection

Recently developed ultrasensitive and quantitative methods for detection of Clostridium difficile toxins provide new tools for diagnosis and, potentially, for management of C. difficile infection (CDI). Compared to methods that detect toxigenic organism, ultrasensitive toxin detection may allow diagnosis of CDI with increased clinical specificity, without sacrificing clinical sensitivity; measurement of toxin levels may also provide information relevant to disease prognosis. This minireview provides an overview of these new toxin detection technologies and considers what these new tools might add to the field.     

Synergistic Effect in Viral-Bacterial Infection: Combined Infection of the Murine Respiratory Tract with Sendai Virus and Pasteurella pneumotropica

Synergism was demonstrated between Sendai virus and Pasteurella pneumotropica in the respiratory tract of mice showing no evidence of previous infection with either agent. Mice aerosol challenged with P. pneumotropica invariably eliminated the viable organism from their lungs within 72 h. In contrast, intrapulmonary killing was delayed in animals previously infected with Sendai virus. Maximum synergism was observed when virus infection preceded bacterial challenge by 6 days. At this time, a mortality rate of 37% was observed as compared with 0, 10, 20, and 10%, respectively, in those animals in which the virus infection preceded bacterial challenge by 1, 3, 9, and 12 days. Previous immunization with Sendai virus completely prevented virus infection and thus the synergistic effect. Synergism with endogenous flora was also noted. Six days after virus infection an endogenous Pasteurella sp. began to proliferate in the bronchopulmonary tissues. Up to 10(4) colony-forming units per lung were recovered but no animals died of the endogenous Pasteurella infection.     

Algorithm Combining Toxin Immunoassay and Stool Culture for Diagnosis of Clostridium difficile Infection

Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.Clostridium difficile is an important nosocomial pathogen, causing antimicrobial-associated diarrhea and pseudomembranous colitis. Toxins A (TcdA) and B (TcdB) mediate the pathogenesis of C. difficile infection (CDI), and toxin detection is an important part of diagnosis. A cytotoxicity neutralization assay (CNA) is the reference method for toxin detection, but it is expensive and time-consuming and requires tissue culture facilities (34, 35). Most laboratories now use a commercial enzyme immunoassay (EIA) to detect TcdA and/or TcdB, with the benefits of rapid turnaround time and ease of use (3, 21, 22, 23, 26, 27, 33, 35). The putative >90% sensitivity of toxin EIAs is not often realized in practice, but EIA is the only toxin detection method available to many routine medical laboratories. The demand for EIA kits detecting both TcdA and TcdB has increased due to increased worldwide prevalence of TcdA-negative, TcdB-positive (TcdA− TcdB+) strains (1, 12, 24, 29, 32).A two-step algorithm, based upon EIA-based detection of species-specific antigen glutamate dehydrogenase (GDH-Ag) and toxin detection via CNA, was suggested to have improved sensitivity and specificity in the detection of toxigenic C. difficile (34). However, the GDH-Ag assay detects both nontoxigenic and toxigenic strains, and the aforementioned shortcomings of the CNA assay make it unavailable to many routine laboratories.Bacteriologic culture can be time-consuming, but it is more straightforward and sensitive than CNA for the detection of toxigenic C. difficile. Furthermore, it provides isolates for characterization, yielding information about CDI epidemiology and antimicrobial susceptibility (11, 28, 36). We evaluated the combination of bacteriologic culture and EIA-based detection of TcdA and TcdB as a new strategy for diagnosis of CDI, especially in areas where TcdA− TcdB+ strains are prevalent.