# An Automatic Analysis System for High-Throughput _Clostridium Diffic_ Toxin Activity Screening
## 1 Introduction
### Clinical Definition and Motivation  
_C. difficile_ is a Gram-positive anaerobic bacterium that is a major cause of hospital-acquired infections [1; 2; 3]. The incidence of CDI has been rising in hospitals and long-term health care facilities during the past several years (see Figure 2) [7]. Additionally, infections are becoming more severe with more complications, deaths, and higher healthcare-associated costs [2]. This increased morbidity and mortality requires a change not only in management and treatment, but also in early diagnosis. Infection by _C. difficile_ usually occurs when patients are placed on broad-spectrum antibiotics for an unrelated condition, altering the microbiome in the gastrointestinal tract and allowing this opportunistic pathogen to thrive. CDI causes a range of symptoms from diarrhea and abdominal pain to pseudomenbanous colitis, toxic megacolon, and death [8; 9]. Even though antibiotics are a main  
Figure 1: Cell-rounding assay to identify anti-toxin inhibitors of TcdB. (**a**) Fluorescence image of human foreskin fibroblast (HFF) cells before toxin challenge. Adherent HFF cells are stained with cytosolic cell tracker dye. (**b**) Fluorescence image of HFFs after challenge with TcdB. Adherent HFFs round upon the addition of toxin, indicating actin cytoskeletal dysfunction following intracellular toxin activity against host Rho/Rac GTPases. Figure 2: Increases in _C. difficile_ infection over time. Graph includes patients aged 65 years and older who were either hospitalized with CDI or who acquired _C. difficile_ during a hospital stay. Rates for 1996–1999 were based on U.S. Census Bureau civilian population estimates adjusted for the net underenumeration in the 1990 census. Rates for 2000–2009 were calculated using U.S. Census Bureau 2000-based postcensal civilian population estimates [7]. risk factor for the development of CDI, the antibiotics vancomycin, metronidazoleazole, and fidaxomicin are the gold standard and first-line treatments for CDI. Initial cure rates with these antibiotics range from 72.7-81.1%, and 15-25% of patients will have recurrent infection [10; 11]. Further, after the first recurrence, patients have approximately a 50% chance of subsequent recurrences [11]. In addition to rising rates of infection and recurrence, antibiotic use contributes to increasing antibiotic resistance and precludes the recovery of a healthy microbiome. An alternative or complementary treatment strategy for CDI is to target the exotoxin mediators of clinical disease with anti-toxin strategies [12; 13]. CDI is mediated by two large multi-subunit Clostridial toxins: TcdA and TcdB. Epidemiologic studies show that only _C. difficile_ expressing toxin are able to cause clinical disease [14; 15]. Each toxin contains a glucosyltransferasean domain (GTD), a cysteine protease domain (CPD), a translocation domain, and a receptor-binding domain [1; 16]. The toxins are taken up into host enterocytes by endocytosis, where acidification of the endosome mediates conformational changes that allow the translocation domain to extrude the GTD and CPD into the host cell cytosol [16]. The cytosolic, mammalian-specific sugar 1D-myo-inositol hexakisphosphate (\(IP_{6}\)) activates the CPD to cleave off the GTD, where it exerts its toxigenic effects via monoglucylation of members of the Rho/Rac family of small GTPases [16]. Glucosylation of Rho/Rac GTPases causes actin cytoskeletal disruption in the host cell that can be seen by a characteristic cellular shrinking and rounding morphology [1] (see Figure 1). Due to the essential function of the toxins in mediating disease, strategies that neutralize toxin activity could treat CDI as a monotherapy or in combination with antibiotics. Efforts to identify small-molecule inhibitors of the CPD and GTD domains have demonstrated the feasibility of this strategy to effectively treat CDI _in situ_ and with _in vivo_ animal models [12; 17; 18; 19; 20; 21]. However, these proof-of-concept inhibitors have yet to advance to clinical trials.