Katie Solomon

Clostridium difficile associated diarrhoea in hospitalised patients: onset in the community and hospital and role of flexible sigmoidoscopy

Objectives:Clostridium difficile associated diarrhoea (CDAD) is a hospital acquired infection in which optimal methods for diagnosis and the scale of the problem in the community remain to be determined. In hospitalised patients with CDAD, we aimed to (i) study patients in whom the onset of diarrhoea was in the community and (ii) investigate the role of bedside flexible sigmoidoscopy in diagnosis. Methods: Patients with CDAD (onset in hospital or community) were studied prospectively. In those with diarrhoea of unknown aetiology, flexible sigmoidoscopy was compared with stool assay for C difficile cytotoxin. Results: Of 136 patients with CDAD (which was associated with antibiotic exposure in 96%), diarrhoea started in the community in 38 (28%; majority in own home) and while an inpatient in 98 (72%). The majority with CDAD onset in the community had been hospitalised over the preceding 12 months (86.8% v 57.1% in the hospital onset group; p<0.001). In 56 patients with pseudomembranous colitis at sigmoidoscopy, the stool C difficile cytotoxin test was negative in 29 (52%) but toxigenic C difficile was isolated from all of nine stool samples cultured. Of patients with pseudomembranous colitis, 30.4% relapsed over the subsequent 57.7(4.2) days. Conclusions: In a significant proportion of hospitalised patients with CDAD, diarrhoea started in the community. However, the majority of these had been hospital inpatients previously when they may have acquired C difficile, with the subsequent onset of diarrhoea in the community following exposure to antibiotics. Flexible sigmoidoscopy is superior to the stool C difficile cytotoxin test in a subgroup of patients with pseudomembranous colitis. Sigmoidoscopy should therefore be considered in all hospitalised patients with diarrhoea in whom the stool test for C difficile cytotoxin and enteric pathogens is negative.

Differential Effects of Varying Concentrations of Clostridium difficile Toxin A on Epithelial Barrier Function and Expression of Cytokines

BACKGROUND Presentation after Clostridium difficile infection may depend on the level of epithelial exposure to toxins. We investigated epithelial barrier function and expression of interleukin (IL)-8 and transforming growth factor (TGF)-beta in response to varying concentrations of C. difficile toxin A. METHODS T84 cells were either preexposed or continuously exposed to C. difficile toxin A (0.01-1000 ng/mL). Barrier function was assessed by measurements of transepithelial electrical resistance. RESULTS Preexposure to < or =10 ng/mL toxin A led to an increase in the release of TGF-beta 1, but there was no change in the expression of IL-8. In contrast, after preexposure to >10 ng/mL toxin A, there was enhanced expression of IL-8, but release of TGF-beta 1 was similar to that in control monolayers. After preexposure to >10 ng/mL toxin A, there was complete and irreversible loss of electrical resistance. At lower concentrations, loss of resistance across monolayers was followed by recovery, which was enhanced by all 3 recombinant isoforms of TGF-beta. Pretreatment with recombinant isoforms of TGF-beta or coculture with TGF-beta 3-expressing colonic subepithelial myofibroblasts was also protective. CONCLUSIONS In C. difficile infection, the development and severity of colonic inflammation may depend on the exposure of intestinal epithelial cells to toxins and the expression of proinflammatory (IL-8) and protective (TGF-beta) factors.

Monocytes Are Highly Sensitive to Clostridium difficile Toxin A-Induced Apoptotic and Nonapoptotic Cell Death

ABSTRACT In this study we investigated the in vitro responses of peripheral blood mononuclear preparations and purified monocytes to Clostridium difficile toxin A. In contrast to the responses of T and B cells, exposure to toxin A led to a rapid loss of monocytes in a time- and dose-dependent fashion (the majority of cells were lost within 24 h of exposure to >100 ng of toxin per ml). Transmission electron microscopy, flow cytometry, and fluorescence microscopy after propidium iodide and Hoechst staining showed that cell death in purified preparations of monocytes following exposure to 100 and 1,000 ng of toxin A per ml occurred by apoptosis. Further studies showed that 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanine iodide aggregates were retained within toxin A-exposed monocyte mitochondria, but cytochrome c was released, suggesting that the apoptotic cascade was triggered in the absence of mitochondrial permeability transition. There was also an increase in caspase-3 activity in toxin A-stimulated monocytes. Following exposure to very high concentrations of toxin A (30 μg/ml), monocyte cell death was predominantly of the necrotic type, with rapid extracellular release of lactate dehydrogenase. These studies demonstrated that C. difficile toxin A has a cell-specific effect, in which monocytes exhibit greater susceptibility than lymphocytes and their death is induced in a concentration-dependent manner.

Mortality in patients with Clostridium difficile infection correlates with host pro-inflammatory and humoral immune responses

Host anti-toxin immune responses play important roles in Clostridium difficile disease and outcome. The relationship between host immune and inflammatory responses during severe C. difficile infection (CDI) and the risk of mortality has yet to be defined. We aimed to investigate the host systemic IgG anti-toxin immune responses, the in vitro cytotoxicity of the infecting C. difficile ribotyped strain, and the host inflammatory markers and their relationship to CDI disease severity and risk of mortality. Inflammatory markers, co-morbidities and CDI outcomes were recorded in a prospective cohort of 150 CDI cases. Serum anti-cytotoxin A (TcdA) and anti-TcdB IgG titres were measured by ELISA and the infecting C. difficile isolate was ribotyped and the in vitro cytotoxin titre assessed. A low median anti-TcdA IgG titre was significantly associated with 30-day all-cause mortality (P<0.05). Ribotype 027 isolates were significantly more toxinogenic than other ribotypes (P<0.00001). High cytotoxin titres correlated with increased inflammatory markers but also higher anti-TcdA and -TcdB (P<0.05) IgG responses resulting in a lower risk of mortality. On multivariate analysis, predictors of mortality were peak white cell count >20 × 10(9) l(-1) [odds ratio (OR) 11.53; 95 % confidence interval (CI) 2.38-55.92], creatinine concentration >133 µmol l(-1) (OR 6.54; 95 % CI 1.47-29.07), Horns index >3 (OR 4.09; 95 % CI 0.76-22.18) and low anti-TcdA IgG (OR 0.97; 95 % CI 0.95-0.99), but not ribotype, cytotoxin titre or anti-TcdB IgG. Thus, host pro-inflammatory and humoral responses correlate with the cytotoxin titre of the infecting strain and effective anti-toxin immune responses reduce the risk of mortality.

PCR ribotype prevalence and molecular basis of macrolide–lincosamide–streptogramin B (MLSB) and fluoroquinolone resistance in Irish clinical Clostridium difficile isolates

BACKGROUND Antimicrobial use is recognized as a risk factor for Clostridium difficile infection (CDI) and outbreaks. We studied the relationship between PCR ribotype, antimicrobial susceptibility and the genetic basis of resistance in response to exposure to antimicrobial agents. METHODS C. difficile isolates were cultured from 133 CDI patients for whom recent antimicrobial drug exposure had been recorded. Isolates were ribotyped by PCR and assessed for their susceptibility to the macrolide-lincosamide-streptogramin B (MLS(B)) group of compounds (erythromycin and clindamycin) and fluoroquinolone antimicrobials (ciprofloxacin, levofloxacin and moxifloxacin). Where relevant, the genetic basis of resistance was determined. RESULTS Prevalent ribotypes (including 027, 001 and 106) exhibited significantly greater antimicrobial resistance compared with ribotypes 078 and 014, among others. Clindamycin-resistant ribotype 078 was detected for the first time. Ribotypes 027 and 001 were more likely to exhibit MLS(B) resistance, a feature that was associated with the erm(B) gene. Exposure to MLS(B) or fluoroquinolone antimicrobial compounds in the 8 weeks prior to the onset of infection was not associated with specific genetic markers of resistance. Single amino acid substitutions in the A and B subunits of DNA gyrase were noted and were ribotype specific and linked to resistance to moxifloxacin. CONCLUSIONS Resistance to MLS(B) and fluoroquinolone antimicrobial compounds is common among prevalent ribotypes of C. difficile. The genetic basis for antimicrobial resistance appears to be ribotype specific and conserved in the absence of recent antimicrobial selection pressure.

The host immune response to Clostridium difficile infection

Clostridium difficile infection (CDI) is the most common infectious cause of healthcare-acquired diarrhoea. Outcomes of C. difficile colonization are varied, from asymptomatic carriage to fulminant colitis and death, due in part to the interplay between the pathogenic virulence factors of the bacterium and the counteractive immune responses of the host. Secreted toxins A and B are the major virulence factors of C. difficile and induce a profound inflammatory response by intoxicating intestinal epithelial cells causing proinflammatory cytokine release. Host cell necrosis, vascular permeability and neutrophil infiltration lead to an elevated white cell count, profuse diarrhoea and in severe cases, dehydration, hypoalbuminaemia and toxic megacolon. Other bacterial virulence factors, including surface layer proteins and flagella proteins, are detected by host cell surface signal molecules that trigger downstream cell-mediated immune pathways. Human studies have identified a role for serum and faecal immunoglobulin levels in protection from disease, but the recent development of a mouse model of CDI has enabled studies into the precise molecular interactions that trigger the immune response during infection. Key effector molecules have been identified that can drive towards a protective anti-inflammatory response or a damaging proinflammatory response. The limitations of current antimicrobial therapies for CDI have led to the development of both active and passive immunotherapies, none of which have, as yet been formally approved for CDI. However, recent advances in our understanding of the molecular basis of host immune protection against CDI may provide an exciting opportunity for novel therapeutic developments in the future.

Germination efficiency of clinical Clostridium difficile spores and correlation with ribotype, disease severity and therapy failure

Spore germination is an important part of the pathogenesis of Clostridium difficile infection (CDI). Spores are resistant to antibiotics, including those therapeutically administered for CDI and strains with a high germination rate are significantly more likely to be implicated in recurrent CDI. The role of germination efficiency in cases of refractory CDI where first-line therapy fails remains unclear. We investigated spore germination efficiencies of clinical C. difficile isolates by measuring drop in OD600 and colony forming efficiency. Ribotype 027 isolates exhibited significantly higher germination efficiencies in the presence of 0.1 % (w/v) sodium taurocholate (51.66 ± 8.75 %; 95 % confidence interval (CI) 47.37-55.95 %) than ribotype 106 (41.91 ± 8.35 %; 95 % CI 37.82-46 %) (P<0.05) and ribotype 078 (42.07 ± 8.57 %, 95 % CI 37.22-46.92 %) (P<0.05). Spore outgrowth rates were comparable between the ribotype groups but the exponential phase occurred approximately 4 h later in the absence of sodium taurocholate. Spore germination efficiencies for isolates implicated in severe CDI were significantly higher (49.68 ± 10.00 %, 95 % CI 47.06-52.30 %) than non-severe CDI (40.92 ± 9.29 %, 95 % CI 37.48-44.36 %); P<0.01. Germination efficiencies were also significantly higher in recurrent CDI or when metronidazole therapy failed than when therapy was successful [(49.00 ± 10.49 %, 95 % CI 46.25-51.75 %) versus (41.42 ± 9.43 %, 95 % CI 37.93-44.91 %); P<0.01]. This study suggests an important link between C. difficile spore germination, CDI pathogenesis and response to treatment; however, further work is warranted before the complex interplay between germination dynamics and CDI outcome can be fully understood.

Phylogenetic Profiles of In-House Microflora in Drains at a Food Production Facility: Comparison and Biocontrol Implications of Listeria-Positive and -Negative Bacterial Populations

ABSTRACT Listeria species experience complex interactions with other microorganisms, which may promote growth and colonization of the organism in local environments or negatively affect them. This study investigated the microbial community at a food production facility, examining interactions between Listeria and the associated microbiome. Listeria species can be transferred between zones in the production environment by individuals or equipment, and drains may act as a reservoir for the organism, reflecting the microbial flora potentially in the production environment. Drains that were colonized by Listeria species and those determined to be free of Listeria were examined. In each case, 16S rRNA gene analysis was performed using the PhyloChip platform. Some general similarities in bacterial population structure were observed when Listeria-negative and -positive drain communities were compared, with some distinct differences also noted. These included increased populations of the genera Prevotella and Janthinobacterium associated with the absence of Listeria species, whereas Enterococcus and Rhodococcus were in higher abundance in drains colonized by Listeria species. Based on these results, a selection of bacterial species were grown in coculture biofilm with a Listeria monocytogenes strain identified as having colonized a drain at the facility. Mixed-species biofilm experiments showed that Janthinobacterium inhibited attachment and subsequent biofilm formation of L. monocytogenes; however, Enterococcus gallinarum significantly increased it. The results of this study suggest the microbial community in food processing facilities can impact the colonization of Listeria species and that influencing the microbiome in favor of antilisterial species may reduce the colonization of Listeria species and limit the likelihood of product/process contamination.

Essential role of toxin A in C. difficile 027 and reference strain supernatant-mediated disruption of Caco-2 intestinal epithelial barrier function

Clostridium difficile induces mucosal inflammation via secreted toxins A and B and initial interactions between the toxins and intestinal epithelial cells (which lead to loss of barrier function) are believed to be important in disease pathogenesis. Secreted toxin‐specific antibodies may inhibit such interactions. Using the Caco‐2 epithelial cell line, we have investigated the use of an anti‐toxin A monoclonal antibody (ATAA) in providing protection against toxin A‐mediated disruption of epithelial barrier function (assessed by measurement of transepithelial electrical resistance and luminal to basolateral flux of labelled dextran). In contrast to free antibody, ATAA conjugated to sepharose beads was more effective in neutralizing the activity of purified toxin A. Sepharose bead‐conjugated ATAA was subsequently used to investigate the contribution of toxin A in epithelial injury mediated by C. difficile supernatant samples (containing toxins A, B and other products). Loss of barrier function mediated by apical application of supernatant samples of reference and epidemic 027 strains of C. difficile was abrogated by neutralization of toxin A. However, this was not the case when the supernatant samples were applied to the basal surface of epithelial monolayers. In conclusion, our studies have shown that (i) sepharose bead‐conjugated ATAA is more effective in neutralizing toxin A than free antibody and (ii) when the apical (luminal) surface of epithelial monolayers is exposed to the secretory products of reference and 027 strains of C. difficile, toxin A is required for the initial injury that leads to loss of barrier function.

Differential binding and internalization of Clostridium difficile toxin A by human peripheral blood monocytes, neutrophils and lymphocytes.

Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor® 488 (toxin A488) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface–associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin A488‐associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A488 in neutrophils was greater on ice than at 37 °C. Studies using trypan blue suggested that over 3 h at 37 °C, most of the toxin A488‐associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A488‐associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.