Clostridium difficile (C. difficile) is a bacterium that causes diarrhea and more serious intestinal conditions such as colitis. Symptoms include Watery ... Nausea Abdominal pain or tenderness You might get C. difficile disease if you have an illness that ...
... C diff antigen; GDH Formal name: Clostridium difficile Culture; C. difficile Toxin, A and B; C. difficile Cytotoxin Assay; Glutamate Dehydrogenase Test Related tests: Stool Culture ; O&P At a Glance Test Sample The ...
... Pediatrician Health Issues Conditions Abdominal ADHD Allergies & Asthma Autism Cancer Chest & Lungs Chronic Conditions Cleft & Craniofacial Developmental ... absolutely necessary. Last Updated 11/21/2015 Source Immunizations ＆ Infectious Diseases: An Informed Parent's Guide (Copyright © 2006 ...
Heinlen, Latisha; Ballard, Jimmy D.
Clostridium difficile is the leading cause of hospital-acquired diarrhea in Europe and North America and is a serious re-emerging pathogen. Recent outbreaks have led to increasing morbidity and mortality and have been associated with a new strain (BI/NAP1/027) of C. difficile that produces more toxin than historical strains. With the increasing incidence of C. difficile infection, clinicians have also seen a change in the epidemiology with increased infections in previously low-risk populations. This chapter highlights the current knowledge on C. difficile virulence, human disease, epidemic outbreaks, and optimal treatment strategies. PMID:20697257
Leuzzi, Rosanna; Adamo, Roberto; Scarselli, Maria
Clostridium difficile infection (CDI) is recognized as a major cause of nosocomial diseases ranging from antibiotic related diarrhea to fulminant colitis. Emergence during the last 2 decades of C. difficile strains associated with high incidence, severity and lethal outcomes has increased the challenges for CDI treatment. A limited number of drugs have proven to be effective against CDI and concerns about antibiotic resistance as well as recurring disease solicited the search for novel therapeutic strategies. Active vaccination provides the attractive opportunity to prevent CDI, and intense research in recent years led to development of experimental vaccines, 3 of which are currently under clinical evaluation. This review summarizes recent achievements and remaining challenges in the field of C. difficile vaccines, and discusses future perspectives in view of newly-identified candidate antigens.
Wilcox, Mark H
The Advisory Committee on Antimicrobial Resistance and Healthcare Associated Infection (ARHAI) was created at the height of the incidence of Clostridium difficile infection (CDI). This article describes the role of ARHAI in the evaluation of laboratory testing for CDI, a related consultation on the legal requirements for manufacturers of in vitro diagnostic medical devices, a CDI healthcare bundle and surveillance of CDI in children.
Hargreaves, Katherine R.; Clokie, Martha R. J.
Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using “whole-phages” are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. PMID:24808893
Seekatz, Anna M.; Young, Vincent B.
Clostridium difficile infection (CDI) is the leading health care–associated illness. Both human and animal models have demonstrated the importance of the gut microbiota’s capability of providing colonization resistance against C. difficile. Risk factors for disease development include antibiotic use, which disrupts the gut microbiota, leading to the loss of colonization resistance and subsequent CDI. Identification of the specific microbes capable of restoring this function remains elusive. Future studies directed at how microbial communities influence the metabolic environment may help elucidate the role of the microbiota in disease development. These findings will improve current biotherapeutics for patients with CDI, particularly those with recurrent disease. PMID:25036699
Duggan, Sean T
Fidaxomicin is a first-in-class macrocyclic antibacterial that primarily demonstrates activity against species of clostridia, predominantly Clostridium difficile, while having limited or no activity against normal faecal microflora. Fidaxomicin is minimally absorbed following oral administration and is excreted almost solely in the faeces. Fidaxomicin displayed a high level of antibacterial activity against C. difficile in vitro, with a minimum inhibitory concentration required to inhibit 90% of C. difficile strains of 0.125-0.5 μg/mL, and was ≈2- to 8-fold more active than vancomycin or metronidazole. Fidaxomicin demonstrated a prolonged postantibiotic effect against C. difficile relative to vancomycin and metronidazole. In two randomized, double-blind, phase III trials, oral fidaxomicin 200 mg every 12 hours for 10 days was no less effective than oral vancomycin 125 mg every 6 hours for 10 days in the treatment of C. difficile infection, based on noninferiority analyses of clinical cure rates (primary endpoint). Fidaxomicin therapy was associated with a significantly lower rate of recurrence, as well as a significantly higher rate of global cure (i.e. sustained clinical response; resolution of diarrhoea without recurrence) compared with vancomycin therapy in the two clinical trials. Fidaxomicin was generally well tolerated in patients with C. difficile infection, with a tolerability profile generally similar to that of vancomycin.
Putsathit, Papanin; Kiratisin, Pattarachai; Ngamwongsatit, Puriya; Riley, Thomas V
Clostridium difficile is the aetiological agent in ca. 20% of cases of antimicrobial-associated diarrhoea in hospitalised adults. Diseases caused by this organism range from mild diarrhoea to occasional fatal pseudomembranous colitis. The epidemiology of C. difficile infection (CDI) has changed notably in the past decade, following epidemics in the early 2000s of PCR ribotype (RT) 027 infection in North America and Europe, where there was an increase in disease severity and mortality. Another major event has been the emergence of RT 078, initially as the predominant ribotype in production animals in the USA and Europe, and then in humans in Europe. Although there have been numerous investigations of the epidemiology of CDI in North America and Europe, limited studies have been undertaken elsewhere, particularly in Asia. Antimicrobial exposure remains the major risk factor for CDI. Given the high prevalence of indiscriminate and inappropriate use of antimicrobials in Asia, it is conceivable that CDI is relatively common among humans and animals. This review describes the level of knowledge in Thailand regarding C. difficile detection methods, prevalence and antimicrobial susceptibility profile, as well as the clinical features of, treatment options for and outcomes of the disease. In addition, antimicrobial usage in livestock in Thailand will be reviewed. A literature search yielded 18 studies mentioning C. difficile in Thailand, a greater number than from any other Asian country. It is possible that the situation in Thailand in relation to CDI may mirror the situation in other developing Asians countries.
Knoop, F C; Owens, M; Crocker, I C
Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978. Since clinical and pathological features of C. difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens. The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions. PMID:8358706
The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America from the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer t...
Bujanda, Luis; Cosme, Angel
Clostridium difficile is the most frequent cause of nosocomial diarrhea and is a significant cause of morbidity among hospitalized patients. The inflammation is produced as a result of a non-specific response to toxins. In the last few years, a hypervirulent strain, NAP1/BI/027, has been reported. Symptoms usually consist of abdominal pain and diarrhea. The diagnosis should be suspected in any patient who develops diarrhea during antibiotic therapy or 6-8 weeks after treatment. Diagnosis should be confirmed by the detection of CD toxin in stool and by colonoscopy in special situations. The treatment of choice is metronidazole or vancomycin. In some patients who do not respond to this therapy or who have complications, subtotal colectomy may be required. Relapse is frequent and must be distinguished from reinfection. Prevention and control in healthcare settings requires careful attention.
Al-Jashaami, Layth S.
Since the discovery of Clostridium difficile infection (CDI) in the 1970s, there has been an increase in the incidence, severity, and recurrence rate of the disease. We reviewed the recent CDI literature in PubMed published before February 28, 2016 that focused on advances in therapy. Despite a large number of studies describing methods for diagnosing the disease, there is currently no definitive test that identifies this infection with certainty, which complicates therapy. Recommended therapy for CDI includes oral metronidazole for mild cases and oral vancomycin or fidaxomicin for moderate to severe cases, each given for 10 to 14 days. For infection with spore-forming C difficile, this length of treatment may be insufficient to lead to cure; however, continuing antibiotics for longer periods of time may unfavorably alter the microbiome, preventing recovery. Treatment with metronidazole has been associated with an increasing failure rate, and the only clear recommended form of metronidazole for treatment of CDI is the intravenous formulation for patients unable to take oral medications. For vancomycin or fidaxomicin treatment of first CDI recurrences, the drug used in the initial bout can be repeated. For second or future recurrences, vancomycin can be given in pulsed or tapered doses. New modalities of treatment, such as bacteriotherapy and immunotherapy, show promise for the treatment of recurrent CDI. PMID:27917075
Abt, Michael C; McKenney, Peter T; Pamer, Eric G
Clostridium difficile is a major cause of intestinal infection and diarrhoea in individuals following antibiotic treatment. Recent studies have begun to elucidate the mechanisms that induce spore formation and germination and have determined the roles of C. difficile toxins in disease pathogenesis. Exciting progress has also been made in defining the role of the microbiome, specific commensal bacterial species and host immunity in defence against infection with C. difficile. This Review will summarize the recent discoveries and developments in our understanding of C. difficile infection and pathogenesis.
Na, Xi; Kelly, Ciaran
Clostridium difficile infection (CDI) is one of the most prevalent nosocomial infections. A dramatic increase in the incidence and severity of CDI has been noted in the past decade. Current recommendations suggest metronidazole as first-line therapy in mild to moderately severe CDI and oral vancomycin in individuals with severe CDI, or when metronidazole fails or is contradicted. Alterations of the colonic microbiota, usually caused by antimicrobial therapy, seem to play a critical role in CDI pathogenesis. Probiotics are live microorganisms that confer a health benefit to the host, and have been used in CDI. Although a wide variety of probiotics have been studied, the exact role of probiotics in preventing and treating CDI is not clear. In this study, we reviewed the current literature and recommendations on the most commonly studied protiotic agents (Saccharomyces boulardii, Lactobacillus species, and probiotic mixtures) used to prevent or treat CDI. Lactobacillus-containing probiotic mixtures and S. boulardii may be effective in the prevention of CDI in high-risk antibiotic recipients but this finding is based on small, individual studies, and further, larger, well-controlled studies are needed to confirm preliminary positive findings and to better delineate the efficacy of probiotics in CDI prevention or treatment. PMID:21992956
Cooper, Christopher C; Jump, Robin L P; Chopra, Teena
Clostridium difficile is one of the foremost nosocomial pathogens. Preventing infection is particularly challenging. Effective prevention efforts typically require a multifaceted bundled approach. A variety of infection control procedures may be advantageous, including strict hand decontamination with soap and water, contact precautions, and using chlorine-containing decontamination agents. Additionally, risk factor reduction can help reduce the burden of disease. The risk factor modification is principally accomplished though antibiotic stewardship programs. Unfortunately, most of the current evidence for prevention is in acute care settings. This review focuses on preventative approaches to reduce the incidence of Clostridium difficile infection in healthcare settings.
Foglia, Ginamarie; Shah, Siddhi; Luxemburger, Christine; Pietrobon, Patricia J Freda
Clostridium difficile has become the most frequent hospital-acquired infection in North America and the EU. C. difficile infection (CDI) is present worldwide and disease awareness is increasing. In the US, EU, and Canada, in addition to hospital diagnosed disease, CDI has also been reported with increasing frequency in the community. Hypervirulent strains have increased the morbidity and mortality associated with CDI. Current treatment options are suboptimal. Of all patients treated for CDI, 20% relapse and 65% of those experiencing a second relapse become chronic cases. An association between increased serum levels of IgG antibody against toxin A and asymptomatic carriage of C. difficile provides a rationale for vaccine development. Sanofi Pasteur's C. difficile candidate vaccine is being developed for the prevention of primary disease. The target population is adults at risk of CDI, those with planned hospitalization, long-term care/nursing home residents, and adults with co-morbidities requiring frequent/prolonged antibiotic use.
Jump, Robin LP
Clostridium difficile infection, the most frequent cause of nosocomial diarrhea, disproportionately affects older adults. The two most important risk factors for developing C. difficile infection are antimicrobial exposure and age >65 years old. Risk factors specific to older adults are frequent interactions with healthcare systems and age-related changes in physiology, including immune senescence and changes to the gut microbiome. Metronidazole and oral vancomcyin are the mainstays of conventional treatment for C. difficile infection. Alternative therapies include fidaxomicin, a narrow-spectrum macrocyclic antibiotic, and fectal bacteriotherapy, which offers an excellent therapeutic outcome. Strategies to prevent C. difficile infections include enhanced infection control measures and reducing inappropriate antimicrobial use through stewardship. PMID:24955106
Monteiro, Mario A; Ma, Zuchao; Bertolo, Lisa; Jiao, Yuening; Arroyo, Luis; Hodgins, Douglas; Mallozzi, Michael; Vedantam, Gayatri; Sagermann, Martin; Sundsmo, John; Chow, Herbert
Clostridium difficile is responsible for thousands of deaths each year and a vaccine would be welcomed, especially one that would disrupt bacterial maintenance, colonization and persistence in carriers and convalescent patients. Structural explorations at the University of Guelph (ON, Canada) discovered that C. difficile may express three phosphorylated polysaccharides, named PSI, PSII and PSIII; this review captures our recent efforts to create vaccines based on these glycans, especially PSII, the common antigen that has precipitated immediate attention. The authors describe the design and immunogenicity of vaccines composed of raw polysaccharides and conjugates thereof. So far, it has been observed that anti-PSII antibodies can be raised in farm animals, mice and hamster models; humans and horses carry anti-PSII IgA and IgG antibodies from natural exposure to C. difficile, respectively; phosphate is an indispensable immunogenic epitope and vaccine-induced PSII antibodies recognize PSII on C. difficile outer surface.
Best, Emma L.; Freeman, Jane; Wilcox, Mark H.
Models of Clostridium difficile infection (C. difficile) have been used extensively for Clostridium difficile (C. difficile) research. The hamster model of C. difficile infection has been most extensively employed for the study of C. difficile and this has been used in many different areas of research, including the induction of C. difficile, the testing of new treatments, population dynamics and characterization of virulence. Investigations using in vitro models for C. difficile introduced the concept of colonization resistance, evaluated the role of antibiotics in C. difficile development, explored population dynamics and have been useful in the evaluation of C. difficile treatments. Experiments using models have major advantages over clinical studies and have been indispensible in furthering C. difficile research. It is important for future study programs to carefully consider the approach to use and therefore be better placed to inform the design and interpretation of clinical studies. PMID:22555466
Banaszkiewicz, Aleksandra; Pituch, Hanna
Inflammatory bowel disease (IBD) is a chronic, immune-mediated disease of the gastrointestinal tract that develops in genetically susceptible individuals. Questions about the role of infections in the development and exacerbations of inflammatory bowel disease remain unanswered. Among numerous bacteria that have been linked to IBD, the most frequently associated is Clostridium difficile. Clinical symptoms of C. difficile infection and an exacerbation of inflammatory bowel disease are often indistinguishable. In cases of diarrhea in patients with IBD and C. difficile infection, antibiotic treatment is recommended. This review attempts to summarize C. difficile infection's epidemiology and clinical features and describes current evidence on treatment of C. difficile infection in children with IBD.
AD N o GENETIC ENGINEERING OF CLOSTRIDIUM DIFFICILE TOXIN A VACCINE 0 C%" ANNUAL REPORT ! Lycurgus L. Muldrow Joe Johnson July 14, 1988 Supported by...17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number) FIELD GROUP SUB-GROUP Clostridium difficile Vaccine ...development of vaccines . Improvement of vaccine biotechnology in the area of recombinant DNA studies using Clostridium difficile toxin A as the model, is
Toxinotyping is a PCR-restriction fragment length polymorphism (RFLP)-based method for differentiation of Clostridium difficile strains according to the changes in the pathogenicity locus (PaLoc), a region coding for toxins A and B. Toxinotypes are a heterogenous group of strains that are important in the development of molecular diagnostic tests and vaccines and are a good basis for C. difficile phylogenetic studies. Here we describe an overview of the 34 currently known toxinotypes (I to XXXIV) and some changes in nomenclature. PMID:26511734
Brazier, J S
According to the UK media and popular press, Clostridium difficile is now a fully fledged member of that notorious but ill-defined group of microorganisms portrayed to the general public as superbugs. Following the trail blazed by methicillin-resistant Staphylococcus aureus (MRSA), C. difficile has made the transition from being an obscure anaerobic bacterium, mainly of interest to specialist anaerobic microbiologists, to that of an infamous superbug responsible for outbreaks of hospital-acquired infection that commonly result in serious disease and death. This review tracks the rise in scientific knowledge and public awareness of this organism.
Rupnik, Maja; Janezic, Sandra
Toxinotyping is a PCR-restriction fragment length polymorphism (RFLP)-based method for differentiation of Clostridium difficile strains according to the changes in the pathogenicity locus (PaLoc), a region coding for toxins A and B. Toxinotypes are a heterogenous group of strains that are important in the development of molecular diagnostic tests and vaccines and are a good basis for C. difficile phylogenetic studies. Here we describe an overview of the 34 currently known toxinotypes (I to XXXIV) and some changes in nomenclature.
Tariq, Raseen; Khanna, Sahil
Clostridium difficile was first identified in 1978 as a diarrhea-causing bacterium in humans. In the last three decades, C. difficile infection (CDI) has reached an epidemic state, both in health care and community settings worldwide. There has been substantial progress in the field of CDI, including identification of novel risk factors, presence of CDI in individuals not considered at risk previously, and treatment options including new drugs, monoclonal antibodies, and fecal microbiota transplantation. This review discusses epidemiology, novel and traditional risk factors, and updates in management for CDI.
Kumar, Manish; Adhikari, Sudip; Hurdle, Julian G.
The nitroheterocyclic classes of drugs have a long history of use in treating anaerobic infections, as exemplified by metronidazole as a first-line treatment for mild-to-moderate Clostridium difficile infection (CDI). Since direct comparisons of the three major classes of nitroheterocyclic drugs (i.e. nitroimidazole, nitazoxanide and nitrofurans) and nitrosating agents against C. difficile are under-examined, in this study their actions against C. difficile were compared. Results show that whilst transient resistance occurs to metronidazole and nitazoxanide, stable resistance arises to nitrofurans upon serial passage. All compounds killed C. difficile at high concentrations in addition to the host defence nitrosating agent S-nitrosoglutathione (GSNO). This suggests that GSNO killing of C. difficile contributes to its efficacy in murine CDI. Although nitric oxide production could not be detected for the nitroheterocyclic drugs, the cellular response to metronidazole and nitrofurans has some overlap with the response to GSNO, causing significant upregulation of the hybrid-cluster protein Hcp that responds to nitrosative stress. These findings provide new insights into the action of nitroheterocyclic drugs against C. difficile. PMID:25129314
Boetzkes, Alexander; Felkel, Katharina Wiebke; Zeiser, Johannes; Jochim, Nelli; Just, Ingo; Pich, Andreas
Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.
Hsu, Chien-Hui; Jeng, Yung-Ming; Ni, Yen-Hsuan
Crohn disease is a chronic inflammatory disorder, which is rare in pediatric patients. The definite etiology and mechanism to induce an acute exacerbation of Crohn disease remains mostly unknown. The authors report on a 14-year-old girl with Crohn disease who has acute gastrointestinal symptoms caused by toxin A-producing Clostridium difficile, which mimicked a flare-up of Crohn disease. There was no preceding antibiotic prescription before the episode. The disease activity did not improve after steroid treatment, which is unusual for Crohn disease. However, all symptoms were dramatically relieved after eradication of C difficile, and led to a symptom-free period for more than 3 years. This case report aims to address the unusual presentation of a usual pathogen, C difficile, in a pediatric patient with Crohn disease.
Rebeaud, Fabien; Bachmann, Martin F
Clostridium difficile infection is a major cause of nosocomial disease in Western countries. The recent emergence of hypervirulent strains resistant to most antibiotics correlates with increasing disease incidence, severity and lethal outcomes. Current treatments rely on metronidazol and vancomycin, but the limited ability of these antibiotics to cure infection and prevent relapse highlights the need for new strategies. A better knowledge of the molecular mechanisms of the disease, the host immune response and identification of key virulence factors of Clostridium difficile now permits the development of new products specifically targeting the pathogen. Immune-based strategies relying on active vaccination or passive administration of antibody products are the focus of intense research and, today, the efficacy of monoclonal antibodies and of two vaccines are evaluated clinically. This review presents recent data, discusses the different strategies and highlights the challenges linked to the development of immunization strategies against this emerging threat.
Ercis, Serpil; Ergin, Alper; Hasçelik, Gülşen
This study was aimed to detect the presence of Clostridium difficile toxin in the stool samples of patients with antibiotic-associated diarrhea or pseudomembranous colitis, and to relate its presence with the clinical findings of the patients. Between January 1997-April 2003, a total of 726 stool samples were investigated for C. difficile toxin A and/or B by enzyme immunoassay. Of them, 68 (9.4%) were found positive for C. difficile toxin (62 were toxin A, 6 were toxin B). C. difficile associated diarrhea were found to be related mostly with the use of beta-lactam/beta-lactamase inhibitor combinations (32/68), followed by aminoglycosides (12/68), and cephalosporins (8/68). The ages of the patients were between 1-86 years old (mean: 43.3 years), and 36 (52.9%) of them had an underlying conditions. Chronic obstructive pulmonary disease and chronic renal failure were the underlying disease in 18, malignancy in 11, and others (diabetes, hepatitis, transplantation, multiple sclerosis) in 7 of the patients. In conclusion, toxin detection and knowledge of the risk factors are the beneficial guidelines for the diagnosis of C. difficile associated diarrhea in the routine setting.
Harvey, Roger B; Norman, Keri N; Andrews, Kathleen; Hume, Michael E; Scanlan, Charles M; Callaway, Todd R; Anderson, Robin C; Nisbet, David J
The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America from the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer to human beings. The objective of the present study was to determine the prevalence of toxigenic C. difficile in chickens and retail poultry meat in Texas. Seven C. difficile isolates were detected in fecal samples of 300 (2.3%) broiler chickens. Three cultivation procedures were evaluated for isolation of C. difficile from poultry meat and detected 1/32 (3.1%), 2/32 (6.2%), and 4/32 (12.5%) for the three procedures, respectively. Chicken and poultry meat isolates were characterized as toxinotype V and pulsed-field gel electrophoresis gel type-NAP7 or NAP7-variant. Susceptibilities to 11 antimicrobial agents in the current study suggested somewhat reduced resistance than reported for other meat or animal toxinotype V isolates.
Hookman, Perry; Barkin, Jamie S
A new, hypervirulent strain of Clostridium difficile, called NAP1/BI/027, has been implicated in C. difficile outbreaks associated with increased morbidity and mortality since the early 2000s. The epidemic strain is resistant to fluoroquinolones in vitro, which was infrequent prior to 2001. The name of this strain reflects its characteristics, demonstrated by different typing methods: pulsed-field gel electrophoresis (NAP1), restriction endonuclease analysis (BI) and polymerase chain reaction (027). In 2004 and 2005, the US Centers for Disease Control and Prevention (CDC) emphasized that the risk of C. difficile-associated diarrhea (CDAD) is increased, not only by the usual factors, including antibiotic exposure, but also gastrointestinal surgery/manipulation, prolonged length of stay in a healthcare setting, serious underlying illness, immune-compromising conditions, and aging. Patients on proton pump inhibitors (PPIs) have an elevated risk, as do peripartum women and heart transplant recipients. Before 2002, toxic megacolon in C. difficile-associated colitis (CDAC), was rare, but its incidence has increased dramatically. Up to two-thirds of hospitalized patients may be infected with C. difficile. Asymptomatic carriers admitted to healthcare facilities can transmit the organism to other susceptible patients, thereby becoming vectors. Fulminant colitis is reported more frequently during outbreaks of C. difficile infection in patients with inflammatory bowel disease (IBD). C. difficile infection with IBD carries a higher mortality than without underlying IBD. This article reviews the latest information on C. difficile infection, including presentation, vulnerable hosts and choice of antibiotics, alternative therapies, and probiotics and immunotherapy. We review contact precautions for patients with known or suspected C. difficile-associated disease. Healthcare institutions require accurate and rapid diagnosis for early detection of possible outbreaks, to initiate
Cohen, Nathaniel A; Ben Ami, Ronen; Guzner-Gur, Hanan; Santo, Moshe E; Halpern, Zamir; Maharshak, Nitsan
Clostridium difficile-associated diarrhea is a problem most hospital-based physicians will face in their career. This review aims to refresh current knowledge with regard to Clostridium difficile infection and bring physicians up to date with the latest developments in the growing field of fecal microbiota transplantation, the benefits it offers, and the promise this and other developments hold for the future.
Diab, S S; Songer, G; Uzal, F A
Clostridium difficile is considered one of the most important causes of diarrhea and enterocolitis in horses. Foals and adult horses are equally susceptible to the infection. The highly resistant spore of C. difficile is the infectious unit of transmission, which occurs primarily via the fecal-oral route, with sources of infection including equine feces, contaminated soil, animal hospitals, and feces of other animals. Two major risk factors for the development of C. difficile associated disease (CDAD) in adult horses are hospitalization and antimicrobial treatment, although sporadically, cases of CDAD can occur in horses that have not received antimicrobials or been hospitalized. The most common antibiotics associated with CDAD in horses are erythromycin, trimethoprim/sulfonamides, β-lactam antimicrobials, clindamycin, rifampicin, and gentamicin. Clinical signs and intestinal lesions of CDAD infection are not specific and they cannot be used to distinguish infections by C. difficile from infections by other agents, such as Clostridium perfringens or Salmonella sp. The distribution of lesions throughout the intestinal tract seems to be age-dependent. Small intestine is invariably affected, and colon and cecum may or may not have lesions in foals<1-month old. Naturally acquired disease in older foals and adult horses has a more aboral distribution, affecting colon and sometimes cecum, but rarely the small intestine. Detection of toxin A, toxin B or both in intestinal contents or feces is considered the most reliable diagnostic criterion for CDAD in horses. Isolation of toxigenic strains of C. difficile from horses with intestinal disease is highly suggestive of CDAD. A better understanding of pathogenesis, reservoirs of infection, and vaccines and other methods of control is needed. Also further studies are recommended to investigate other possible predisposing factors and/or etiological agents of enteric diseases of horses.
Mehlich, Agnieszka; Górska, Sabina; Gamian, Andrzej; Myc, Andrzej
Clostridium difficile pathogen is a cause of the most frequent nosocomial infection, which is antibiotic-associated diarrhea. Antibiotic treatment causes disruption of the microbiome balance, which makes the gut a friendly environment for the pathogen. It leads to pseudomembranous colitis, toxic megacolon and even death. Clostridium difficile infection (CDI) is particularly dangerous to elderly patients, leading to the highest mortality rate. C. difficile is equipped with many virulence factors such as toxin A and B, binary toxin CDT, flagellum, S-layer proteins, Cwp66 and GroEL proteins, protease Cwp84, fibronectin-binding protein and the ability to form biofilm and spores. Problems with anti-CDI therapy prompt researchers and clinicians to seek alternative ways of therapy. Identification of immunological epitopes in outer layer proteins and the use of them as antigens for anti-CDI vaccines would be a rational approach to prevent the disease, but unfortunately such vaccines are not available yet. In this article we review the course of the disease, virulence and risk factors. We summarize briefly epidemiological data and the latest achievements in CDI treatment.
Alcalá-Hernández, Luis; Mena-Ribas, Ana; Niubó-Bosh, Jordi; Marín-Arriaza, Mercedes
Clostridium difficile is the leading cause of nosocomial diarrhoea in developed countries, and is one of the main aetiologic agents of community diarrhea. The eruption of the hypervirulent strain BI/NAP1/027 has given rise to an increase in the morbidity and mortality of C.difficile infection (CDI). This document aims to review the main clinical pictures of CDI and the laboratory diagnosis, including sampling, transport and storage of specimens, specimen processing, diagnostic procedures, antimicrobial susceptibility testing, and molecular characterisation of the isolates. The main purpose of the article is to develop a practical document that provides answers to the main questions that arise in the laboratory diagnosis of CDI.
Marsh, Jane W; Curry, Scott R
Metronidazole and vancomycin remain the front-line therapies for most Clostridium difficile infections (CDI). However, recurrent CDI occurs in ∼ 25% of patients, causing significant morbidity and mortality and healthcare costs. For this population, traditional antibiotic therapies fail and new treatment options are greatly needed. The US Food and Drug Administration recently approved fidaxomicin for CDI treatment. This narrow-spectrum antibiotic preserves the normal gut microbiota and shows promise as a treatment for severe and recurrent CDI. Monoclonal antibodies and vaccines directed against toxin are currently in clinical trials and represent alternative, non-antibiotic therapies. Less traditional therapeutic interventions include bacteriotherapy with non-toxigenic C. difficile and fecal transplant. This commentary will provide an overview of current and forthcoming CDI therapies.
Ivarsson, Mattias E; Leroux, Jean-Christophe; Castagner, Bastien
Significant progress has been made by industry and academia in the past two years to address the medical threats posed by Clostridium difficile infection. These developments provide an excellent example of how patient need has driven a surge of innovation in drug discovery. Indeed, only two drugs were approved for the infection in the past 30 years but there are 13 treatment candidates in clinical trials today. What makes the latter number even more remarkable is the diversity in the strategies represented (antibiotics, microbiota supplements, vaccines, antibiotic quenchers and passive immunization). In this review, we provide a snapshot of the current stage of these breakthroughs and argue that there is still room for further innovation in treating C. difficile infection.
Leber, Amy L.
The detection and diagnosis of Clostridium difficile infection in pediatric populations have some unique considerations in comparison to testing in adults. The testing methodologies, including toxigenic culture, cell cytotoxicity, antigen detection, and, more recently, molecular testing, are the same in all age groups. However, limited data exist on the specific performance characteristics in children. In this review, we focus on the challenges of testing in pediatric populations and assess the available data on test performance in these populations. Additionally, a review of the existing guidance for testing is provided. PMID:26912759
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Starley, Brad; Galagan, Jack Carl; Yabes, Joseph Michael; Evans, Sara
Background and Aims. Studies have shown effects of diet on gut microbiota. We aimed to identify foods associated with recurrent Clostridium difficile infection (CDI). Methods. In this cross-sectional survey, consecutive patients diagnosed with CDI were identified by electronic medical records. Colitis symptoms and positive Clostridium difficile assay were confirmed. Health-care onset-health-care facility associated CDI was excluded. Food surveys were mailed to 411 patients. Survey responses served as the primary outcome measure. Spearman's rank correlation identified risk factors for CDI recurrence. Results. Surveys were returned by 68 patients. Nineteen patients experienced CDI recurrence. Compared to patients without CDI recurrence, patients with CDI recurrence had more antibiotics prescribed preceding their infection (p = 0.003). Greater numbers of the latter also listed tea (p = 0.002), coffee (p = 0.013), and eggs (p = 0.013), on their 24-hour food recall. Logistic regression identified tea as the only food risk factor for CDI recurrence (adjusted OR: 5.71; 95% CI: 1.26–25.89). Conclusion. The present results indicate a possible association between tea and CDI recurrence. Additional studies are needed to characterize and confirm this association. PMID:27651790
Hell, M; Permoser, M; Chmelizek, G; Kern, J M; Maass, M; Huhulescu, S; Indra, A; Allerberger, F
Clostridium difficile is considered to be a leading cause of hospital-acquired diarrhea. C. difficile (CDI) infection shows a high rate of recurrence. There would have to be a predominantly monoclonal mechanism of CDI within individual patients in order for molecular epidemiologic tools such as polymerase chain reaction (PCR) ribotyping to be useful in outbreak investigation or differentiation between infection relapse versus re-infection. It was the aim of our study to determine whether CDI is of monoclonal or of polyclonal genesis. Between December 2009 and June 2010, 11 patients with nosocomial CDI were chosen arbitrarily. Five individual colonies of C. difficile were picked from each of the primary culture plates. Of 55 isolates gained, 47 were available for PCR ribotyping (eight isolates failed attempts to re-culture). Among these 47 isolates, eight different PCR ribotypes were identified. Only one of the 11 patients had a stool sample that yielded more than one ribotype (PCR ribotypes 438 and 232); this 67-year-old female cancer patient was already suffering from recurring diarrhea prior to the fatal episode of colitis which was subsequently investigated. We conclude that polyclonal infections may occasionally occur in patients with CDI. Our findings of predominantly monoclonal origin of CDI within patients suggest that molecular epidemiologic investigations can be used reliably for outbreak investigations or discrimination between relapse and re-infection.
Lessa, Fernanda C.; Gould, Carolyn V.; McDonald, L. Clifford
The dramatic changes in the epidemiology of Clostridium difficile infection (CDI) during recent years, with increases in incidence and severity of disease in several countries, have made CDI a global public health challenge. Increases in CDI incidence have been largely attributed to the emergence of a previously rare and more virulent strain, BI/NAP1/027. Increased toxin production and high-level resistance to fluoroquinolones have made this strain a very successful pathogen in healthcare settings. In addition, populations previously thought to be at low risk are now being identified as having severe CDI. Recent genetic analysis suggests that C. difficile has a highly fluid genome with multiple mechanisms to modify its content and functionality, which can make C. difficile adaptable to environmental changes and potentially lead to the emergence of more virulent strains. In the face of these changes in the epidemiology and microbiology of CDI, surveillance systems are necessary to monitor trends and inform public health actions. PMID:22752867
The laboratory diagnosis of Clostridium difficile-associated disease (CDAD) is based on culture and toxin detection in fecal specimens. Culture is performed on a commercially available selective media. C. difficile colony morphology is typical when viewed under a dissecting microscope. Definitive identification is best obtained by gas liquid chromatography. Culture is very sensitive but, when used alone without toxin testing, it leads to low specificity and misdiagnosis of CDAD when high rates of asymptomatic carriage exist. Toxin detection by a tissue culture cytotoxin assay followed by neutralisation with specific antiserum is often considered the standard. However, this approach lacks sensitivity and has not detected up to 30% of patients with confirmed CDAD. Multiple enzyme immunoassays (EIAs) have been introduced by various manufacturers for the detection of toxin A alone or for both toxins A and B. Some of these are designed to give results in less than 1 h. Comparative studies of EIA kits reported that the sensitivity and specificity are slightly lower than cytotoxin assays. Toxigenic culture tests C. difficile isolates for toxin production: colonies isolated on selective media are tested for in-vitro toxin production either by a cytotoxicity assay or by direct EIA. It has higher sensitivity than the cytotoxicity assay and equivalent specificity. In the routine laboratory, culture and toxin detection should be performed on every specimen and, in culture-positive and fecal toxin-negative cases, toxigenic cultures should be performed on isolated colonies.
Elliott, Briony; Androga, Grace O; Knight, Daniel R; Riley, Thomas V
Over the recent decades, Clostridium difficile infection (CDI) has emerged as a global public health threat. Despite growing attention, C. difficile remains a poorly understood pathogen, however, the exquisite sensitivity offered by next generation sequencing (NGS) technology has enabled analysis of the genome of C. difficile, giving us access to massive genomic data on factors such as virulence, evolution, and genetic relatedness within C. difficile groups. NGS has also demonstrated excellence in investigations of outbreaks and disease transmission, in both small and large-scale applications. This review summarizes the molecular epidemiology, evolution, and phylogeny of C. difficile, one of the most important pathogens worldwide in the current antibiotic resistance era.
Clostridium difficile is a bacterium that can spread by water. It often causes acute gastrointestinal illness in older adults who are hospttalized and/or receiving antibiotics; however, community associated infections affecting otherwise healthy individuals have become more ...
Clostridium difficile is the leading cause of nosocomially acquired intestinal infection in the United States, affecting virtually all cases of pseudomembranous colitis and up to 20% of cases of antibiotic-associated diarrhea. Even after receiving antibiotic treatment with either metronidazole or vancomycin, 20% of patients will have recurrent Clostridium difficile diarrhea. An innovative approach to the problem involves the introduction of competing, nonpathogenic (probiotic) organisms into the intestinal tract to restore microbial balance. The theoretical premise behind this approach is that the protective intestinal microflora is damaged by antibiotic treatment; the initial antibiotic exposure thus leaves the host susceptible to colonization and subsequent infection by Clostridium difficile. A so-called "second-hit" to the intestinal microflora occurs when the infected host is treated with flagyl or vancomycin, further destroying susceptible bacterial flora. Probiotic agents, such as Lactobacillus GG and Saccharomyces boulardii, have been studied for the treatment of Clostridium difficile. We are currently running a prospective, randomized, placebo-controlled trial of Lactobacillus GG in combination with standard antibiotics for the treatment of Clostridium difficile infection. Although it is too early to draw statistically significant conclusions, two patterns seem to be emerging: Lactobacillus GG is effective in reducing the 3-wk recurrence rate of Clostridium difficile, and patients feel better when taking Lactobacillus GG, as compared with the placebo, with early disappearance of abdominal cramps and diarrhea. In conclusion, the use of probiotics for the treatment of primary and recurrent Clostridium difficile diarrhea looks promising. Patients seem to have less recurrent Clostridium difficile diarrhea and early symptomatic improvement when using the probiotic Lactobacillus GG.
Vanjak, Dominique; Girault, Guillaume; Branger, Catherine; Rufat, Pierre; Valla, Dominique-Charles; Fantin, Bruno
During 2001, Clostridium difficile infection was observed in 23 patients hospitalized in a hepatology ward (attack rate, 0.9%). Since strain typing ruled out a clonal dissemination, we performed a case-control study. In addition to antibiotic use as a risk factor, the C. difficile infection rate was higher among patients with autoimmune hepatitis (P<.01).
Austin, Matthew; Mellow, Mark; Tierney, William M
In recent years, Clostridium difficile infections have become more frequent, more severe, more refractory to standard treatment, and more likely to recur. Current antibiotic treatment regimens for Clostridium difficile infection alter the normal gut flora, which provide colonization resistance against Clostridium difficile. Over the past few years, there has been a marked increase in the knowledge of the gut microbiota and its role in health maintenance and disease causation. This has, fortuitously, coincided with the use of a unique microbial replacement therapy, fecal microbiota transplantation, in the treatment of patients with multiple recurrent Clostridium difficile infections. We briefly review current knowledge of the gut microbiota's functions. We then review the indications for use of fecal microbiota transplantation in Clostridium difficile infection, the techniques employed, and results of treatment. Fecal microbiota transplantation has been shown to be efficacious for patients with multiply recurrent Clostridium difficile infections (reported cure rates of 90%), with an excellent short-term safety profile, and has been included in the American College of Gastroenterology treatment guidelines for this troublesome disease.
Liubakka, Alyssa; Vaughn, Byron P
Clostridium difficile infection (CDI) is a major source of morbidity and mortality for hospitalized patients. Although most patients have a clinical response to existing antimicrobial therapies, recurrent infection develops in up to 30% of patients. Fecal microbiota transplant is a novel approach to this complex problem, with an efficacy rate of nearly 90% in the setting of multiple recurrent CDI. This review covers the current epidemiology of CDI (including toxigenic and nontoxigenic strains, risk factors for infection, and recurrent infection), methods of diagnosis, existing first-line therapies in CDI, the role of fecal microbiota transplant for multiple recurrent CDIs, and the potential use of fecal microbial transplant for patients with severe or refractory infection.
Martinez, Fernando J; Leffler, Daniel A; Kelly, Ciaran P
The incidence and severity of Clostridium difficile infection (CDI) have increased dramatically over the past decade. Its treatment, however, has largely remained the same with the exception of oral vancomycin use as a first-line agent in severe disease. From 1999 to 2004, 20,642 deaths were attributed to CDI in the United States, almost 7 times the rate of all other intestinal infections combined. Worldwide, several major CDI outbreaks have occurred, and many of these were associated with the NAP1 strain. This ‘epidemic’ strain has contributed to the rising incidence and mortality of CDI. The purpose of this article is to review the current management, treatment, infection control, and prevention strategies that are needed to combat this increasingly morbid disease. PMID:22826646
Dupont, Herbert L
Clostridium difficile infection (CDI) is increasing in frequency and severity in and out of the hospital, with a high probability of recurrence after treatment. The recent literature on CDI was reviewed using PubMed to include recent publications dealing with diagnosis and therapy. Real-time polymerase chain reaction is a sensitive and useful diagnostic test for CDI but there are growing concerns of false-positive test results if the rate of CDI is low in the patient population providing samples and/or if the population being studied commonly includes people with C difficile colonization. Recommended therapy of CDI includes oral metronidazole for milder cases of CDI and oral vancomycin or fidaxomicin for more severe cases, each given for 10 days. Colectomy is being performed more frequently in patients with fulminant CDI. For treatment of first recurrences the drug used in the first bout can be used again and for second recurrences longer courses of vancomycin often are given in a tapered dose or intermittently to allow gut flora reconstitution, or other treatments including fidaxomicin may be used. Bacteriotherapy with fecal transplantation is playing an increasing role in therapy of recurrent cases. Metagenomic studies of patients with CDI during successful therapy are needed to determine how best to protect the flora from assaults from antibacterial drugs and to develop optimal therapeutic approaches. Immunotherapy and immunoprophylaxis offer opportunities to prevent CDI, to speed up recovery from CDI, and to eliminate recurrent infection. Humanized monoclonal antitoxin antibodies and active immunization with vaccines against C difficile or its toxins are both in development and appear to be of potential value.
Stevenson, Emma; Minton, Nigel P; Kuehne, Sarah A
Clostridium difficile is widely publicised as a problem in the health-care system. Disruption of the normal gut microbiota by antibiotic therapy allows C. difficile to colonise the colon. On colonisation, C. difficile produces two toxins that lead to disease, with symptoms ranging from mild-to-severe diarrhoea, to fulminant and often fatal pseudomembranous colitis (PMC). How C. difficile establishes initial colonisation of the host is an area of active investigation. Recently there has been increased research into the role of C. difficile flagella in colonisation and adherence. Novel research has also elucidated a more complex role of flagella in C. difficile virulence pertaining to the regulation of toxin gene expression. This review focuses on new insights into the specific role of C. difficile flagella in colonisation and toxin gene expression.
Monteiro, Mario A
Clostridium difficile vaccines composed of surface polysaccharides (PSs) have the potential to simultaneously control infection and colonization levels in humans. Hot water-phenol treatment of C. difficile biomass can extricate water-soluble PS-I and PS-II; and water- and phenol-soluble PS-III. C. difficile vaccines based on PS-II have attracted the most attention due its facile purification and ubiquitous expression by C. difficile ribotypes. Anti PS-II antibodies recognize both C. difficile vegetative cell and sporulating preparations and confer protection against C. difficile infection in a mouse model. The design of such an efficacious C. difficile PS-II conjugate vaccine is described here.
Freeman, J.; Bauer, M. P.; Baines, S. D.; Corver, J.; Fawley, W. N.; Goorhuis, B.; Kuijper, E. J.; Wilcox, M. H.
Summary: The epidemiology of Clostridium difficile infection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data. There have been clear changes in the clinical presentation, response to treatment, and outcome of CDI. These changes have been driven to a major degree by the emergence and epidemic spread of a novel strain, known as PCR ribotype 027 (sometimes referred to as BI/NAP1/027). We review the evidence for the changing epidemiology, clinical virulence and outcome of treatment of CDI, and the similarities and differences between data from various countries and continents. Community-acquired CDI has also emerged, although the evidence for this as a distinct new entity is less clear. There are new data on the etiology of and potential risk factors for CDI; controversial issues include specific antimicrobial agents, gastric acid suppressants, potential animal and food sources of C. difficile, and the effect of the use of alcohol-based hand hygiene agents. PMID:20610822
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Wenzler, Eric; Mulugeta, Surafel G.; Danziger, Larry H.
Clostridium difficile remains a major public health threat and continues to contribute to excess morbidity, mortality and healthcare costs. Antimicrobial stewardship programs have demonstrated success in combating C. difficile, primarily through antibiotic restrictive strategies. As the incidence and prevalence of C. difficile associate disease continues to increase both in the hospital and community setting, additional stewardship approaches are needed. This manuscript reviews stewardship interventions that have been successful against C. difficile associated disease and proposes future tactics that antimicrobial stewardship programs may employ to develop a more global approach to combat this difficult pathogen. PMID:27025621
Rineh, Ardeshir; Kelso, Michael J; Vatansever, Fatma; Tegos, George P; Hamblin, Michael R
The Gram-positive anaerobic bacterium Clostridium difficile produces toxins A and B, which can cause a spectrum of diseases from pseudomembranous colitis to C. difficile-associated diarrhea. A limited number of C. difficile strains also produce a binary toxin that exhibits ADP ribosyltransferase activity. Here, the structure and the mechanism of action of these toxins as well as their role in disease are reviewed. Nosocomial C. difficile infection is often contracted in hospital when patients treated with antibiotics suffer a disturbance in normal gut microflora. C. difficile spores can persist on dry, inanimate surface for months. Metronidazole and oral vancomycin are clinically used for treatment of C. difficile infection but clinical failure and concern about promotion of resistance are motivating the search for novel non-antibiotic therapeutics. Methods for controlling both toxins and spores, replacing gut microflora by probiotics or fecal transplant, and killing bacteria in the anaerobic gut by photodynamic therapy are discussed. PMID:24410618
Piacenti, Frank J; Leuthner, Kimberly D
Antimicrobial stewardship programs are essential to health care institutions to promote the appropriate use of antibiotics not only to decrease antimicrobial resistance but to prevent the spread and infection of Clostridium difficile. Clostridium difficile-associated diarrhea is increasing rapidly in the United States and is now considered a major public health problem that poses an immediate threat to the health of patients prescribed antibiotics, more so than antimicrobial resistance. Clostridium difficile-associated disease is the result of collateral damage to the normal bacterial flora of the human body, which is an inevitable consequence of any antibiotic use. Antimicrobial stewardship programs such as audit with feedback and antibiotic restriction are designed to help limit Clostridium difficile infections and other hospital-associated organisms by optimizing antimicrobial selection, dosing, de-escalation, and duration of therapy. These programs also incorporate implementation of hospital-wide guidelines, staff education, enforcement of infection-control policies, and the use of electronic medical records when possible to help control antibiotic use. This article reviews the literature on how antimicrobial stewardship programs impact Clostridium difficile rates and discusses experiences in designing, implementing, monitoring, and follow-through of such programs.
Chumbler, Nicole M.; Rutherford, Stacey A.; Zhang, Zhifen; Farrow, Melissa A.; Lisher, John P.; Farquhar, Erik; Giedroc, David P.; Spiller, Benjamin W.; Melnyk, Roman A.; Lacy, D. Borden
Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics. PMID:27571750
Lyerly, D M; Krivan, H C; Wilkins, T D
Clostridium difficile is the etiologic agent of pseudomembranous colitis, a severe, sometimes fatal disease that occurs in adults undergoing antimicrobial therapy. The disease, ironically, has been most effectively treated with antibiotics, although some of the newer methods of treatment such as the replacement of the bowel flora may prove more beneficial for patients who continue to relapse with pseudomembranous colitis. The organism produces two potent exotoxins designated toxin A and toxin B. Toxin A is an enterotoxin believed to be responsible for the diarrhea and mucosal tissue damage which occur during the disease. Toxin B is an extremely potent cytotoxin, but its role in the disease has not been as well studied. There appears to be a cascade of events which result in the expression of the activity of these toxins, and these events, ranging from the recognition of a trisaccharide receptor by toxin A to the synergistic action of the toxins and their possible dissemination in the body, are discussed in this review. The advantages and disadvantages of the various assays, including tissue culture assay, enzyme immunoassay, and latex agglutination, currently used in the clinical diagnosis of the disease also are discussed. PMID:3144429
He, Xiangyun; Wang, Jufang; Steele, Jennifer; Sun, Xingmin; Nie, Weijia; Tzipori, Saul; Feng, Hanping
We describe a novel ultrasensitive cell-based immunocytotoxicity assay for detecting less then 1 pg/ml of Clostridium difficile toxins in porcine clinical samples. The assay is simple to perform with a turnaround time of approximately 3 hours and capable of detecting less then 1 pg/ml of toxin A. Using this assay, we were able to detect the presence of C. difficile toxins in the fecal and serum specimens of experimentally infected piglets. PMID:19393695
Napolitano, Lena M; Edmiston, Charles E
Clostridium difficile infections are the leading cause of health care-associated infectious diarrhea, posing a significant risk for both medical and surgical patients. Because of the significant morbidity and mortality associated with C difficile infections, knowledge of the epidemiology of C difficile in combination with a high index of suspicion and susceptible patient populations (including surgical, postcolectomy, and inflammatory bowel disease patients) is warranted. C difficile infections present with a wide spectrum of disease, ranging from mild diarrhea to fulminant colitis or small bowel enteritis and recurrent C difficile infections. Early implementation of medical and operative treatment strategies for C difficile infections is imperative for optimal patient outcomes. National and international guidelines recommend early operative consultation and total abdominal colectomy with end ileostomy and preservation of rectum. Diverting loop ileostomy and colonic lavage followed by intravenous metronidazole and intracolonic vancomycin administered via the efferent limb of the ileostomy should be considered as an alternative to total colectomy in selected patients. New and emerging strategies for C difficile infection treatment include monoclonal antibodies, vaccines, probiotics, biotherapeutics, and new antibiotics. A successful C difficile prevention and eradication program requires a multidisciplinary approach that includes early disease recognition, implementation of guidelines for monitoring adherence to environmental control, judicious hand hygiene, evidence-based treatment and management strategies, and a focused antibiotic stewardship program. Surgeons are an important part of the clinical team in the management of C difficile infection prevention and treatment.
Zheng, Yi; Luo, Yun; Lv, Yinxiang; Huang, Chen; Sheng, Qinsong; Zhao, Peng; Ye, Julian; Jiang, Weiqin; Liu, Lulu; Song, Xiaojun; Tong, Zhou; Chen, Wenbin; Lin, Jianjiang; Tang, Yi-Wei; Jin, Dazhi; Fang, Weijia
The entire process of Clostridium difficile colonization to infection develops in large intestine. However, the real colonization pattern of C. difficile in preoperative colorectal cancer patients has not been studied. In this study, 33 C. difficile strains (16.1%) were isolated from stool samples of 205 preoperative colorectal cancer patients. C. difficile colonization rates in lymph node metastasis patients (22.3%) were significantly higher than lymph node negative patients (10.8%) (OR=2.314, 95%CI=1.023-5.235, P =0.025). Meanwhile, patients positive for stool occult blood had lower C. difficile colonization rates than negative patients (11.5% vs. 24.0%, OR=0.300, 95%CI=0.131-0.685, P =0.019). A total of 16 sequence types were revealed by multilocus sequence typing. Minimum spanning tree and time-space cluster analysis indicated that all C. difficile isolates were epidemiologically unrelated. Antibiotic susceptibility testing showed all isolates were susceptible to vancomycin and metronidazole. The results suggested that the prevalence of C. difficile colonization is high in preoperative colorectal cancer patients, and the colonization is not acquired in the hospital. Since lymph node metastasis colorectal cancer patients inevitably require adjuvant chemotherapy and C. difficile infection may halt the ongoing treatment, the call for sustained monitoring of C. difficile in those patients is apparently urgent.
Ojha, Suvash Chandra; Chankhamhaengdecha, Surang; Singhakaew, Sombat; Ounjai, Puey; Janvilisri, Tavan
Spores are a potent agent for Clostridium difficile transmission. Therefore, factors inhibiting spores have been of continued interest. In the present study, we investigated the influence of microwave irradiation in addition to conductive heating for C. difficile spore inactivation in aqueous suspension. The spores of 15 C. difficile isolates from different host origins were exposed to conductive heating and microwave irradiation. The complete inhibition of spore viability at 10(7) CFU/ml was encountered following microwave treatment at 800 W for 60 s, but was not observed in the conductive-heated spores at the same time-temperature exposure. The distinct patterns of ultrastructural alterations following microwave and conductive heat treatment were observed and the degree of damages by microwave was in the exposure time-dependent manner. Microwave would therefore be a simple and time-efficient tool to inactivate C. difficile spores, thus reducing the risk of C. difficile transmission.
Péchiné, Séverine; Hennequin, Claire; Boursier, Céline; Hoys, Sandra; Collignon, Anne
Clostridium difficile is a pathogen which is responsible for diarrhea and colitis, particularly after treatment with antibiotics. Clinical signs are mainly due to two toxins, TcdA and TcdB. However, the first step of pathogenesis is the colonization process. We evaluated C. difficile surface proteins as vaccine antigens in the hamster model to prevent intestinal colonization. This vaccination induced a partial protection of hamsters against death after a C. difficile challenge. A proteomic analysis of animal sera allowed us to identify proteins which could be responsible for the protection observed. Among these proteins, we identified the GroEL heat shock protein. To confirm the role of the specific GroEL antibodies in the delayed C. difficile colonization of hamsters, we performed an immunization assay in a mouse model. After intranasal immunization with the recombinant protein GroEL, we observed a lower C. difficile intestinal colonization in the immunized group as compared to the control group.
Mathur, Harsh; Rea, Mary C; Cotter, Paul D; Ross, R Paul; Hill, Colin
Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibiotic-associated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. Recently, advancements in next generation sequencing technology (NGS) have highlighted the extent of damage to the gut microbiota caused by broad-spectrum antibiotics, often resulting in C. difficile infection (CDI). Currently the treatment of choice for CDI involves the use of metronidazole and vancomycin. However, recurrence and relapse of CDI, even after rounds of metronidazole/vancomycin administration is a problem that must be addressed. The efficacy of alternative antibiotics such as fidaxomicin, rifaximin, nitazoxanide, ramoplanin and tigecycline, as well as faecal microbiota transplantation has been assessed and some have yielded positive outcomes against C. difficile. Some bacteriocins have also shown promising effects against C. difficile in recent years. In light of this, the potential for emerging treatment options and efficacy of anti-C. difficile vaccines are discussed in this review.
Sears, Cynthia L.; Oliva-Hemker, Maria
Abstract: Children with inflammatory bowel disease (IBD) are disproportionately susceptible to Clostridium difficile infection (CDI) and the incidence is increasing. There has also been growing recognition of asymptomatic C. difficile colonization in pediatric IBD, which can sometimes be very difficult to distinguish from symptomatic C. difficile–associated disease in this population. In this study, we discuss the current knowledge of C. difficile infection in children with IBD, reviewing epidemiology, risk factors, and outcomes that often differ from the adult IBD population, and discuss the complexities and dilemmas of diagnosing and treating CDI in pediatric IBD. PMID:26689599
Gens, Krista D; Elshaboury, Ramy H; Holt, Jessica S
Due to the increased incidence and recurrence of Clostridium difficile infection, health care providers are seeking new and alternative treatments to the standard antibiotic therapy. The objective of this article is to present a review on the background, microbiologic efficacy, clinical efficacy, and safety of fecal microbiota transplantation and to provide an overview of emerging treatment options currently under investigation. Emerging treatment options discussed include the use of monoclonal antibodies directed against toxins A and B, C difficile vaccination, and transplantation of nontoxigenic C difficile strains.
Péchiné, Séverine; Janoir, Claire; Boureau, Hélène; Gleizes, Aude; Tsapis, Nicolas; Hoys, Sandra; Fattal, Elias; Collignon, Anne
Clostridium difficile pathogenesis is mainly due to toxins A and B. However, the first step of pathogenesis is the colonization process. We evaluated C. difficile surface proteins as vaccine antigens to diminish intestinal colonization in a human flora-associated mouse model. First, we used the flagellar cap protein FliD of C. difficile, in order to test several immunization routes: intranasal, rectal, and intragastric. The rectal route, which is the most efficient, was used to vaccine groups of mice with different antigen combinations. After immunizations, the mice were challenged with the toxigenic C. difficile and a significant statistical difference between the control group and the immunized groups was observed in the colonization levels of C. difficile.
Androga, Grace O; McGovern, Alan M; Elliott, Briony; Chang, Barbara J; Perkins, Timothy T; Foster, Niki F; Riley, Thomas V
Clostridium difficile PCR ribotype 033 (RT033) is found in the gastrointestinal tracts of production animals and, occasionally, humans. The illumigene C. difficile assay (Meridian Bioscience, Inc.) failed to detect any of 52 C. difficile RT033 isolates, while all strains signaled positive for the binary toxin genes but were reported as negative for C. difficile by the Xpert C. difficile/Epi assay (Cepheid).
Wang, Qiong; Euler, Chad W.; Delaune, Aurelia
As a consequence of excessive antibiotic therapies in hospitalized patients, Clostridium difficile, a Gram-positive anaerobic spore-forming intestinal pathogen, is the leading cause of hospital-acquired diarrhea and colitis. Drug treatments for these diseases are often complicated by antibiotic-resistant strains and a high frequency of treatment failures and relapse; therefore, novel nonantibiotic approaches may prove to be more effective. In this study, we recombinantly expressed a prophage lysin identified from a C. difficile strain, CD630, which we named PlyCD. PlyCD was found to have lytic activity against specific C. difficile strains. However, the recombinantly expressed catalytic domain of this protein, PlyCD1–174, displayed significantly greater lytic activity (>4-log kill) and a broader lytic spectrum against C. difficile strains while still retaining a high degree of specificity toward C. difficile versus commensal clostridia and other bacterial species. Our data also indicated that noneffective doses of vancomycin and PlyCD1–174 when combined in vitro could be significantly more bactericidal against C. difficile. In an ex vivo treatment model of mouse colon infection, we found that PlyCD1–174 functioned in the presence of intestinal contents, significantly decreasing colonizing C. difficile compared to controls. Together, these data suggest that PlyCD1–174 has potential as a novel therapeutic for clinical application against C. difficile infection, either alone or in combination with other preexisting treatments to improve their efficacy. PMID:26392484
Ghose, Chandrabali; Eugenis, Ioannis; Edwards, Adrianne N; Sun, Xingmin; McBride, Shonna M; Ho, David D
Clostridium difficile is a spore-forming, anaerobic, Gram-positive organism that is the leading cause of antibiotic-associated infectious diarrhea, commonly known as C. difficile infection (CDI). C. difficile spores play an important role in the pathogenesis of CDI. Spore proteins, especially those that are surface-bound may play an essential role in the germination, colonization and persistence of C. difficile in the human gut. In our current study, we report the identification of two surface-bound spore proteins, CdeC and CdeM that may be utilized as immunization candidates against C. difficile. These spore proteins are immunogenic in mice and are able to protect mice against challenge with C. difficile UK1, a clinically-relevant 027/B1/NAP1 strain. These spore proteins are also able to afford high levels of protection against challenge with C. difficile 630Δerm in golden Syrian hamsters. This unprecedented study shows the vaccination potential of C. difficile spore exosporium proteins.
Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate (TCCFB) followed by alcohol shock and isolation on tryp...
Dubberke, Erik R.
Prevention of Clostridium difficile infection (CDI) has become extremely important because of increases in CDI incidence and severity. Unfortunately CDI prevention efforts are hampered by lack of data to support optimal prevention methods, especially for endemic CDI. Studies are needed to define optimal prevention practices and to investigate novel prevention methods. PMID:20929366
The incidence and severity of disease associated with toxigenic Clostridium difficile (Cd) have increased in hospitals in North America from the emergence of newer, more virulent strains of Cd. Toxigenic Cd has been isolated from food animals and retail meat with potential implications of transfer ...
Pokusaeva, Karina; Carpenter, Robert
Clostridium difficile is a leading cause of antibiotic-associated diarrhea and the etiologic agent responsible for C. difficile infection. Toxin A (TcdA) and toxin B (TcdB) are nearly indispensable virulence factors for Clostridium difficile pathogenesis. Given the toxin-centric mechanism by which C. difficile pathogenesis occurs, the selective sequestration with neutralization of TcdA and TcdB by nonantibiotic agents represents a novel mode of action to prevent or treat C. difficile-associated disease. In this preclinical study, we used quantitative enzyme immunoassays to determine the extent by which a novel drug, calcium aluminosilicate uniform particle size nonswelling M-1 (CAS UPSN M-1), is capable of sequestering TcdA and TcdB in vitro. The following major findings were derived from the present study. First, we show that CAS UPSN M-1 efficiently sequestered both TcdA and TcdB to undetectable levels. Second, we show that CAS UPSN M-1's affinity for TcdA is greater than its affinity for TcdB. Last, we show that CAS UPSN M-1 exhibited limited binding affinity for nontarget proteins. Taken together, these results suggest that ingestion of calcium aluminosilicate might protect gastrointestinal tissues from antibiotic- or chemotherapy-induced C. difficile infection by neutralizing the cytotoxic and proinflammatory effects of luminal TcdA and TcdB. PMID:26149988
Piekarska, Marta; Wandałowicz, Alicja D; Miigoć, Henryka
Clostridium difficile is the most common cause of an antibiotic-associated diarrhoea. Frequency of Clostridium difficile infections (CDI) increased in the last decade. This study presents current preventive measure i.e. hand washing, disposable gloves. Additionally, the article presents diagnostic methods: detection glutamine dehydrogenase (GDH), toxins A and B, cytotoxicity neutralization test, polymerase chain reaction methods (PCR) i.e. nucleic acid amplification test (NAAT) and stool culture. Moreover available methods of treatment were presented depending on severity of CDI e.i. metronidazole, vancomycin, fidaxomicin, rifaximin. Furthermore, the review provides information about alternative methods of treatment in view of new hypervirulent strains of C. difficile and increasing resistance to commonly used antibiotics, including: fuscid acid, bacitracin, probiotics, non-toxigenic strains, immunoglobulins, monoclonal antibodies, vaccines, toxins binders and fecal transplant.
van Leeuwen, Pim T.; van der Peet, Jasper M.; Bikker, Floris J.; Hoogenkamp, Michel A.; Oliveira Paiva, Ana M.; Kostidis, Sarantos; Mayboroda, Oleg A.
ABSTRACT The facultative anaerobic polymorphic fungus Candida albicans and the strictly anaerobic Gram-positive bacterium Clostridium difficile are two opportunistic pathogens residing in the human gut. While a few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, the nature of the interactions between these two microbes has not been studied thus far. In the current study, both chemical and physical interactions between C. albicans and C. difficile were investigated. In the presence of C. albicans, C. difficile was able to grow under aerobic, normally toxic, conditions. This phenomenon was neither linked to adherence of bacteria to hyphae nor to biofilm formation by C. albicans. Conditioned medium of C. difficile inhibited hyphal growth of C. albicans, which is an important virulence factor of the fungus. In addition, it induced hypha-to-yeast conversion. p-Cresol, a fermentation product of tyrosine produced by C. difficile, also induced morphological effects and was identified as an active component of the conditioned medium. This study shows that in the presence of C. albicans, C. difficile can persist and grow under aerobic conditions. Furthermore, p-cresol, produced by C. difficile, is involved in inhibiting hypha formation of C. albicans, directly affecting the biofilm formation and virulence of C. albicans. This study is the first detailed characterization of the interactions between these two gut pathogens. IMPORTANCE Candida albicans and Clostridium difficile are two opportunistic pathogens that reside in the human gut. A few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, but none have shown the interaction(s) that these two organisms may or may not have with each other. In this study, we used a wide range of different techniques to better understand this interaction at a macroscopic and microscopic level. We found that in the presence of C. albicans, C. difficile
Hogendorf, Wouter F J; Gisch, Nicolas; Schwudke, Dominik; Heine, Holger; Bols, Mikael; Pedersen, Christian Marcus
The emergence of hypervirulent resistant strains have made Clostridium difficile a notorious nosocomial pathogen and has resulted in a renewed interest in preventive strategies, such as vaccines based on (synthetic) cell wall antigens. Recently, the structure of the lipoteichoic acid (LTA) of this species has been elucidated. Additionally, this LTA was found to induce the formation of protective antibodies against C. difficile in rabbits and mice. The LTA from C. difficile is isolated as a microheterogenous mixture, differing in size and composition, impeding any structure-activity relationship studies. To ensure reliable biological results, pure and well-defined synthetic samples are required. In this work the total synthesis of LTAs from C. difficile with defined chain length is described and the initial biological results are presented.
Polage, Christopher R.; Chin, David L.; Leslie, Jhansi L.; Tang, Jevon; Cohen, Stuart H.; Solnick, Jay V.
Clostridium difficile testing is shifting from toxin detection to C. difficile detection. Yet, up to 60% of patients with C. difficile by culture test negative for toxins and it is unclear if they are infected or carriers. We reviewed medical records for 7,046 inpatients with a C. difficile toxin test from 2005–2009 to determine the duration of diarrhea and rate of complications and mortality among toxin-positive (toxin+) and toxin− patients. Overall, toxin− patients had less severe diarrhea, fewer diarrhea days and lower mortality (P<0.001, all comparisons) than toxin+ patients. One toxin− patient (n=1/6,121; 0.02%) was diagnosed with pseudomembranous colitis but there were no complications such as megacolon or colectomy for fulminant CDI among toxin− patients. These data suggest that C. difficile-attributable complications are rare among patients testing negative for C. difficile toxins and more studies are needed to evaluate the clinical significance of C. difficile detection in toxin− patients. PMID:23009731
Popoff, M R; Dodin, A
Neuraminidase production was investigated in 57 Clostridium butyricum strains, 16 Clostridium beijerinckii strains, and 25 Clostridium difficile strains. Neuraminidase activity was found only in C. butyricum strains originating from one human newborn with neonatal necrotizing enterocolitis, two newborns with hemorrhagic colitis, one infected placenta, and one adult with peritonitis, It was concluded that neuraminidase was not a major virulence factor in C. butyricum strains. PMID:4056013
The aging society and the advanced of supportive treatment means that large numbers of elderly patients with risk factors for C difficile enterocolitis will continue to receive care in intensive care unit. Antibiotic resistance and older, sicker patients means that combination antibiotic therapy will become a trend in clinical setting. Age, several co-morbidities, and gastrointestinal surgery appear to be specific risk factors for C difficile infection. Diarrhea which is the only symptom in hospitalized patient should drive us to rethink about the possibility of C difficile infection especially in the elderly patient. Prudent use of antibiotic, infection control are strategies to prevent C difficile infection in clinical setting. Elderly patients who undergo gastrointestinal surgery have an increased rate of C difficile infection because of commonly used nasogastric tube. Gastrointestinal surgical patients typically have preoperative bowel preparation, receive oral preoperative antibiotics that are poorly absorbed, impaired bowel motility secondary to ileus, receive systemic preoperative antibiotics prophylaxis, and have variable lengths of no oral caloric intake during the preoperative period. The continued imprudent use of prolonged postoperative systemic antibiotics for presumed preventive purposes, particularly among the elderly and patients who have nasogastric tubes or other enteric tubes, appears to be a recipe for preventable infections with C difficile.
Background Manuka honey originates from the manuka tree (Leptospermum scoparium) and its antimicrobial effect has been attributed to a property referred to as Unique Manuka Factor that is absent in other types of honey. Antibacterial activity of Manuka honey has been documented for several bacterial pathogens, however there is no information on Clostridium difficile, an important nosocomial pathogen. In this study we investigated susceptibility of C. difficile to Manuka honey and whether the activity is bactericidal or bacteriostatic. Methods Three C. difficile strains were subjected to the broth dilution method to determine minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for Manuka honey. The agar well diffusion method was also used to investigate sensitivity of the C. difficile strains to Manuka honey. Results The MIC values of the three C. difficile strains were the same (6.25% v/v). Similarly, MBC values of the three C. difficile strains were the same (6.25% v/v). The activity of Manuka honey against all three C. difficile strains was bactericidal. A dose–response relationship was observed between the concentrations of Manuka honey and zones of inhibition formed by the C. difficile strains, in which increasing concentrations of Manuka honey resulted in increasing size of zone of inhibition formed. Maximum zone of inhibition was observed at 50% (v/v) Manuka honey and the growth inhibition persisted over 7 days. Conclusion C. difficile is appreciably susceptible to Manuka honey and this may offer an effective way of treating infections caused by the organism. PMID:23651562
Jöbstl, M; Heuberger, S; Indra, A; Nepf, R; Köfer, J; Wagner, M
Prevalence of Clostridium difficile was examined in Austrian ground meat samples and bactofugates, following an evaluation of enrichment broths. Bactofugation is a centrifugation procedure used at sensitive dairies to lower the concentration of spores in raw milk before heat treatment. Among the five enrichment broths tested, C. difficile moxalactam norfloxacin boullion (CDMN) was the only one that allowed recovery of C. difficile from artificially spiked meat samples. Use of Tween 80 as a detergent in the enrichment of artificially contaminated bactofugates improved recovery of C. difficile. Following the enrichment procedures (meat without the use of TWEEN 80), one hundred ground meat samples and fifty bactofugates were enriched for 10-15days in CDMN and presumed positive colonies were isolated and identified by Gram staining, observation of colony fluorescence and ID 32 A ribotyping. Subsequently PCR ribotyping, PCR-based identification of toxin genes (tcdA, tcdB) and antimicrobial drug susceptibility testing to metronidazole, vancomycin, clindamycin and moxifloxacin were performed. C. difficile was isolated from three (3%) of the one hundred retail ground meat samples. Two C. difficile isolates of the same rare ribotype AI-57 were toxin gene-negative and sensitive to all antibiotics tested. One isolate was assignable to one of the most prevalent clinical ribotypes isolated in Austria and harboured the tcdA and tcdB genes. This isolate was also resistant to clindamycin and moxifloxacin. None of the fifty bactofugates tested were positive for C. difficile. The presence of an isolate of human origin could indicate contamination by human shedders during food processing rather than evidencing zoonotic potential. Bactofugates, although constituting concentrated spore suspensions, were not contaminated with C. difficile spores. This finding excludes raw milk as a major source of food contamination. In conclusion, C. difficile recovery rates found in our study were
Engevik, Melinda A.; Yacyshyn, Mary Beth; Engevik, Kristen A.; Wang, Jiang; Darien, Benjamin; Hassett, Daniel J.; Yacyshyn, Bruce R.
The majority of antibiotic-induced diarrhea is caused by Clostridium difficile (C. difficile). Hospitalizations for C. difficile infection (CDI) have tripled in the last decade, emphasizing the need to better understand how the organism colonizes the intestine and maintain infection. The mucus provides an interface for bacterial-host interactions and changes in intestinal mucus have been linked host health. To assess mucus production and composition in healthy and CDI patients, the main mucins MUC1 and MUC2 and mucus oligosaccharides were examined. Compared with healthy subjects, CDI patients demonstrated decreased MUC2 with no changes in surface MUC1. Although MUC1 did not change at the level of the epithelia, MUC1 was the primary constituent of secreted mucus in CDI patients. CDI mucus also exhibited decreased N-acetylgalactosamine (GalNAc), increased N-acetylglucosamine (GlcNAc), and increased terminal galactose residues. Increased galactose in CDI specimens is of particular interest since terminal galactose sugars are known as C. difficile toxin A receptor in animals. In vitro, C. difficile is capable of metabolizing fucose, mannose, galactose, GlcNAc, and GalNAc for growth under healthy stool conditions (low Na+ concentration, pH 6.0). Injection of C. difficile into human intestinal organoids (HIOs) demonstrated that C. difficile alone is sufficient to reduce MUC2 production but is not capable of altering host mucus oligosaccharide composition. We also demonstrate that C. difficile binds preferentially to mucus extracted from CDI patients compared with healthy subjects. Our results provide insight into a mechanism of C. difficile colonization and may provide novel target(s) for the development of alternative therapeutic agents. PMID:25552581
Russello, Giuseppe; Brovarone, Flavia; Bardaro, Marcellino; Carretto, Edoardo
Clostridium difficile associated diseases (CDADs) or C. difficile infections (CDIs) are increasing in incidence, severity and mortality. Among patients with CDIs, those with recurrent disease are less responsive to traditional therapies with commonly used drugs, such as metronidazole and vancomycin. Faecal microbiota transplantation is an old therapeutic procedure that has been recently proposed as a safe and effective treatment for CDI patients non-responsive to antibiotic therapy. In this paper we discuss the microbiological procedures that should be performed on faecal microbiota donors.
Guerreiro, Isabelle; Achonu, Camille; Volkening, Grace; MacFarlane, Sam; McCreight, Liz; Egan, Cathy; Robertson, Jennifer; Garber, Gary
In 2008, Clostridium difficile rates were increasing in Ontario, Canada, and in response, hospitals were mandated by the Ontario Ministry of Health to publicly report their C difficile infection (CDI) rates. In order to assist hospitals which had ongoing CDI outbreaks, a process of an external infection control resource team (ICRT) was introduced. This article describes the function and process of the ICRT, managed by Public Health Ontario, and reviews the lessons learned over the first 5 years of operation. These lessons may assist other hospitals in managing their own infection prevention and control outbreak.
Ghose, Chandrabali; Kelly, Ciarán P
Clostridium difficile is a spore-forming anaerobic gram-positive organism that is the leading cause of antibiotic-associated nosocomial infectious diarrhea in the Western world. This article describes the evolving epidemiology of C difficile infection (CDI) in the twenty-first century, evaluates the importance of vaccines against the disease, and defines the roles of both innate and adaptive host immune responses in CDI. The effects of passive immunotherapy and active vaccination against CDI in both humans and animals are also discussed.
Zhang, Lihua; Dong, Danfeng; Jiang, Cen; Li, Zhen; Wang, Xuefeng; Peng, Yibing
Clostridium difficile is well recognized as the common pathogen of nosocomial diarrhea, meanwhile, asymptomatic colonization with C. difficile in part of the population has also drawn public attention. Although gut microbiota is known to play an important role in the pathogenesis of C. difficile infection (CDI), whether there is any alteration of gut microbial composition in asymptomatic C. difficile carriers hasn't been clearly described. The purpose of this study was to explore the differences in gut microbiome among CDI patients, asymptomatic C. difficile carriers and healthy individuals. We performed fecal microbiota analysis on the samples of eight CDI patients, eight asymptomatic C. difficile carriers and nine healthy subjects using 16S rRNA gene pyrosequencing. CDI patients and asymptomatic carriers showed reduced microbial richness and diversity compared with healthy subjects, accompanied with a paucity of phylum Bacteroidetes and Firmicutes as well as an overabundance of Proteobacteria. Some normally commensal bacteria, especially butyrate producers, were significantly depleted in CDI patients and asymptomatic carriers. Furthermore, the differences observed in microbial community structure between CDI patients and asymptomatic carriers suggested that the gut microbiota may be a potential factor of disease state for CDI. Our study demonstrates the characterization and diversity of gut microbiota in CDI and asymptomatic C. difficile colonization, which will provide new ideas for surveillance of the disease state and development of microbiota-targeted agents for CDI prevention and treatment.
Kociolek, Larry K; Gerding, Dale N
This Review summarizes the latest advances in the treatment and prevention of Clostridium difficile infection (CDI), which is now the most common health-care-associated infection in the USA. As traditional, standard CDI antibiotic therapies (metronidazole and vancomycin) are limited by their broad spectrum and further perturbation of the intestinal microbiota, which result in unacceptably high recurrence rates, novel therapeutic strategies for CDI are needed. Emerging CDI therapies are focused on limiting further perturbation of the intestinal microbiota and/or restoring the microbiota to its pre-morbid state, reducing colonization of the intestinal tract by toxigenic strains of C. difficile and bolstering the host immune response against C. difficile toxins. Fidaxomicin is associated with reduced CDI recurrences, and other emerging narrow-spectrum CDI antibiotic therapies might eventually demonstrate a similar benefit. Prevention of intestinal colonization of toxigenic strains of C. difficile can be achieved through restoration of the intestinal microbiota with faecal microbiota transplantation, as well as by colonizing the gut with nontoxigenic C. difficile strains. Finally, emerging immunological therapies, including monoclonal antibodies and vaccines against C. difficile toxins, might protect against CDI and subsequent CDI recurrences. The available clinical data for these emerging therapies, and their relative advantages and disadvantages, are described.
Lin, Cynthia J.; Wade, Timothy J.; Hilborn, Elizabeth D.
Clostridium difficile is a bacterium that can spread by water. It often causes acute gastrointestinal illness in older adults who are hospitalized and/or receiving antibiotics; however, community-associated infections affecting otherwise healthy individuals have become more commonly reported. A case-crossover study was used to assess emergency room (ER) and outpatient visits for C. difficile infection following flood events in Massachusetts from 2003 through 2007. Exposure status was based on whether or not a flood occurred prior to the case/control date during the following risk periods: 0–6 days, 7–13 days, 14–20 days, and 21–27 days. Fixed-effects logistic regression was used to estimate the risk of diagnosis with C. difficile infection following a flood. There were 129 flood events and 1575 diagnoses of C. difficile infection. Among working age adults (19–64 years), ER and outpatient visits for C. difficile infection were elevated during the 7–13 days following a flood (Odds Ratio, OR = 1.69; 95% Confidence Interval, CI: 0.84, 3.37). This association was more substantial among males (OR = 3.21; 95% CI: 1.01–10.19). Associations during other risk periods were not observed (p < 0.05). Although we were unable to differentiate community-associated versus nosocomial infections, a potential increase in C. difficile infections should be considered as more flooding is projected due to climate change. PMID:26090609
Nawrocki, Kathryn L.; Edwards, Adrianne N.; Daou, Nadine; Bouillaut, Laurent
ABSTRACT Clostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile. In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile. The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile. Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficile. IMPORTANCE This study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile. We also demonstrate that CodY regulates known effectors of
Könönen, Eija; Rasinperä, Marja; Virolainen, Anni; Mentula, Silja; Lyytikäinen, Outi
Due to increased interest directed to Clostridium difficile-associated infections, a questionnaire survey of laboratory diagnostics of toxin-producing C. difficile was conducted in Finland in June 2006. Different aspects pertaining to C. difficile diagnosis, such as requests and criteria used for testing, methods used for its detection, yearly changes in diagnostics since 1996, and the total number of investigations positive for C. difficile in 2005, were asked in the questionnaire, which was sent to 32 clinical microbiology laboratories, including all hospital-affiliated and the relevant private clinical microbiology laboratories in Finland. The situation was updated by phone and email correspondence in September 2008. In June 2006, 28 (88%) laboratories responded to the questionnaire survey; 24 of them reported routinely testing requested stool specimens for C. difficile. Main laboratory methods included toxin detection (21/24; 88%) and/or anaerobic culture (19/24; 79%). In June 2006, 18 (86%) of the 21 laboratories detecting toxins directly from feces, from the isolate, or both used methods for both toxin A (TcdA) and B (TcdB), whereas only one laboratory did so in 1996. By September 2008, all of the 23 laboratories performing diagnostics for C. difficile used methods for both TcdA and TcdB. In 2006, the number of specimens processed per 100,000 population varied remarkably between different hospital districts. In conclusion, culturing C. difficile is common and there has been a favorable shift in toxin detection practice in Finnish clinical microbiology laboratories. However, the variability in diagnostic activity reported in 2006 creates a challenge for national monitoring of the epidemiology of C. difficile and related diseases.
Kovacs-Simon, Andrea; Leuzzi, Rosanna; Kasendra, Magdalena; Minton, Nigel; Titball, Richard W; Michell, Stephen L
Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease.
Aljarallah, Khalid M.
Clostridium difficile-associated disease continues to be one of the leading health concerns worldwide. C. difficile is considered as a causative agent of nosocomial diarrhea that causes serious infection, which may result in death. The incidences of C. difficile infection (CDI) in developed countries have become increasingly high which may be attributed to the emergence of newer epidemic strains, extensive use of antibiotics, and limited alternative therapies. The available treatment options against CDI are expensive and promote resistance. Therefore, there is urgent need for new approaches to meet these challenges. This review discusses the current understanding of CDI, the existing clinical treatment strategies and future potential options as antidifficile agents based on the available published works. PMID:28293151
Rao, Krishna; Young, Vincent B
This article discusses the use of fecal microbiota transplantation (FMT) for the treatment of recurrent Clostridium difficile infection (CDI). The disruption of the normal gut microbiota is central to the pathogenesis of CDI, and disruption persists in recurrent disease. The use of FMT for recurrent CDI is characterized by a high response rate and short term safety is excellent, although the long-term effects of FMT are as yet unknown.
Alasmari, Faisal; Seiler, Sondra M.; Hink, Tiffany; Burnham, Carey-Ann D.; Dubberke, Erik R.
Background. Clostridium difficile infection (CDI) incidence has increased dramatically over the last decade. Recent studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We sought to identify the prevalence and risk factors for asymptomatic C. difficile carriage on admission to the hospital. Methods. Patients admitted to Barnes-Jewish Hospital without diarrhea were enrolled from June 2010 through October 2011. Demographic information and healthcare and medication exposures 90 days prior to admission were collected. Stool specimens or rectal swabs were collected within 48 hours of admission and stored at −30°C until cultured. Clostridium difficile isolates were typed and compared with isolates from patients with CDI. Results. A stool/swab specimen was obtained for 259 enrolled subjects on admission. Two hundred four (79%) were not colonized, 40 (15%) had toxigenic C. difficile (TCD), and 15 (6%) had nontoxigenic C. difficile. There were no differences between TCD-colonized and -uncolonized subjects for age (mean, 56 vs 58 years; P = .46), comorbidities, admission from another healthcare facility (33% vs 24%; P = .23), or recent hospitalization (50% vs 50%; P = .43). There were no differences in antimicrobial exposures in the 90 days prior to admission (55% vs 56%; P = .91). Asymptomatic carriers were colonized with strains similar to strains from patients with CDI, but the relative proportions were different. Conclusions. There was a high prevalence of TCD colonization on admission. In contrast to past studies, TCD colonization was not associated with recent antimicrobial or healthcare exposures. Additional investigation is needed to determine the role of asymptomatic TCD carriers on hospital-onset CDI incidence. PMID:24755858
Minton, Nigel; Carter, Glen; Herbert, Mike; O'keeffe, Triona; Purdy, Des; Elmore, Mike; Ostrowski, Anna; Pennington, Oliver; Davis, Ian
Clostridum difficile is a major cause of healthcare-associated disease in the western world, and is particularly prominent in the elderly. Its incidence is rising concomitant with increasing longevity. More effective countermeasures are required. However, the pathogenesis of C. difficile infection is poorly understood. The lack of effective genetic tools is a principal reason for this ignorance. For many years, the only tools available for the transfer of genes into C. difficile have been conjugative transposons, such as Tn916, delivered via filter mating from Bacillus subtilis donors. They insert into a preferred site within the genome. Therefore, they may not be employed for classical mutagenesis studies, but can be employed to modulate gene function through the delivery of antisense RNA. Attempts to develop transformation procedures have so far met with little success. However, in recent years the situation has been dramatically improved through the demonstration of efficient conjugative transfer of both replication-proficient and replication-deficient plasmids from Escherichia coli donors. This efficient transfer can only be achieved in certain strains through negation of the indigenous restriction barrier, and is generally most effective when the plasmid employed is based on the replicon of the C. difficile plasmid, pCD6.
obligate anaerobic, spore forming rod bacteria , remains one of the major causes of health-care associated diarrhea1,2. From recognition in the 1970...from other bacteria ). This study had some limitations. Mild or asymptomatic cases of C. difficile may have not been tested or diagnosed. Data
Silva, Rodrigo Otávio Silveira; D'Elia, Mirella Lauria; Tostes Teixeira, Erika Procópio; Pereira, Pedro Lúcio Lithg; de Magalhães Soares, Danielle Ferreira; Cavalcanti, Álvaro Roberto; Kocuvan, Aleksander; Rupnik, Maja; Santos, André Luiz Quagliatto; Junior, Carlos Augusto Oliveira; Lobato, Francisco Carlos Faria
Despite some case reports, the importance of Clostridium perfringens and Clostridium difficile for wild carnivores remains unclear. Thus, the objective of this study was to identify C. perfringens and C. difficile strains in stool samples from wild carnivore species in Brazil. A total of 34 stool samples were collected and subjected to C. perfringens and C. difficile isolation. Suggestive colonies of C. perfringens were then analyzed for genes encoding the major C. perfringens toxins (alpha, beta, epsilon and iota) and the beta-2 toxin (cpb2), enterotoxin (cpe) and NetB (netb) genes. C. difficile strains were analyzed by multiplex-PCR for toxins A (tcdA) and B (tcdB) and a binary toxin gene (cdtB) and also submitted to a PCR ribotyping. Unthawed aliquots of samples positive for C. difficile isolation were subjected to the detection of A/B toxins by a cytotoxicity assay (CTA). C. perfringens was isolated from 26 samples (76.5%), all of which were genotyped as type A. The netb gene was not detected, whereas the cpb2 and cpe genes were found in nine and three C. perfringens strains, respectively. C. difficile was isolated from two (5.9%) samples. A non-toxigenic strain was recovered from a non-diarrheic maned wolf (Chrysocyon brachyurus). Conversely, a toxigenic strain was found in the sample of a diarrheic ocelot (Leopardus pardallis); an unthawed stool sample was also positive for A/B toxins by CTA, indicating a diagnosis of C. difficile-associated diarrhea in this animal. The present work suggests that wild carnivore species could carry C. difficile strains and that they could be susceptible to C. difficile infection.
Yan, Weiwei; Shin, Kang-Soon; Wang, Shih-Jon; Xiang, Hua; Divers, Thomas; McDonough, Sean; Bowman, James; Rowlands, Anne; Akey, Bruce; Mohamed, Hussni
Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 107 C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses. PMID:24136208
Mathur, Harsh; Rea, Mary C; Cotter, Paul D; Ross, R Paul; Hill, Colin
Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibiotic-associated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. Recently, advancements in next generation sequencing technology (NGS) have highlighted the extent of damage to the gut microbiota caused by broad-spectrum antibiotics, often resulting in C. difficile infection (CDI). Currently the treatment of choice for CDI involves the use of metronidazole and vancomycin. However, recurrence and relapse of CDI, even after rounds of metronidazole/vancomycin administration is a problem that must be addressed. The efficacy of alternative antibiotics such as fidaxomicin, rifaximin, nitazoxanide, ramoplanin and tigecycline, as well as faecal microbiota transplantation has been assessed and some have yielded positive outcomes against C. difficile. Some bacteriocins have also shown promising effects against C. difficile in recent years. In light of this, the potential for emerging treatment options and efficacy of anti-C. difficile vaccines are discussed in this review. PMID:25564777
Leuzzi, Rosanna; Spencer, Janice; Buckley, Anthony; Brettoni, Cecilia; Martinelli, Manuele; Tulli, Lorenza; Marchi, Sara; Luzzi, Enrico; Irvine, June; Candlish, Denise; Veggi, Daniele; Pansegrau, Werner; Fiaschi, Luigi; Savino, Silvana; Swennen, Erwin; Cakici, Osman; Oviedo-Orta, Ernesto; Giraldi, Monica; Baudner, Barbara; D'Urzo, Nunzia; Maione, Domenico; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia
Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection. PMID:23716610
Kirk, Joseph A; Fagan, Robert P
Clostridium difficile infection has increased in incidence and severity over the past decade, and poses a unique threat to human health. However, genetic manipulation of C. difficile remains in its infancy and the bacterium remains relatively poorly characterised. Low-efficiency conjugation is currently the only available method for transfer of plasmid DNA into C. difficile. This is practically limiting and has slowed progress in understanding this important pathogen. Conjugation efficiency varies widely between strains, with important clinically relevant strains such as R20291 being particularly refractory to plasmid transfer. Here we present an optimised conjugation method in which the recipient C. difficile is heat treated prior to conjugation. This significantly improves conjugation efficiency in all C. difficile strains tested including R20291. Conjugation efficiency was also affected by the choice of media on which conjugations were performed, with standard BHI media giving most transconjugant recovery. Using our optimised method greatly increased the ease with which the chromosome of R20291 could be precisely manipulated by homologous recombination. Our method improves on current conjugation protocols and will help speed genetic manipulation of strains otherwise difficult to work with.
Pye, Hayley; Kohoutova, Darina; Mosse, Charles A.; Yahioglu, Gokhan; Stamati, Ioanna; Deonarain, Mahendra; Battah, Sinan; Ready, Derren; Allan, Elaine; Mullany, Peter; Lovat, Laurence B.
Background Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections. Methods PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29. Results Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm2) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT. Conclusion This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon. PMID:26313448
Leuzzi, Rosanna; Spencer, Janice; Buckley, Anthony; Brettoni, Cecilia; Martinelli, Manuele; Tulli, Lorenza; Marchi, Sara; Luzzi, Enrico; Irvine, June; Candlish, Denise; Veggi, Daniele; Pansegrau, Werner; Fiaschi, Luigi; Savino, Silvana; Swennen, Erwin; Cakici, Osman; Oviedo-Orta, Ernesto; Giraldi, Monica; Baudner, Barbara; D'Urzo, Nunzia; Maione, Domenico; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia; Douce, Gillian R; Scarselli, Maria
Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.
Silva, Rodrigo Otávio Silveira; Rupnik, Maja; Diniz, Amanda Nádia; Vilela, Eduardo Garcia; Lobato, Francisco Carlos Faria
Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies about C. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficile ribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen.
Riccobono, E.; Di Pilato, V.; Della Malva, N.; Meini, S.; Ciraolo, F.; Torricelli, F.
Clostridium difficile, belonging to ribotype 018 (RT018), is one of the most prevalent genotypes circulating in hospital settings in Italy. Here, we report the draft genome of C. difficile CD8-15 belonging to RT018, isolated from a patient with fatal C. difficile-associated infection. PMID:27587821
Pokrywka, Marian; Feigel, Jody; Douglas, Barbara; Grossberger, Susan; Hensler, Amelia; Hensler, Amelia; Weber, David
Prevention strategies for Clostridium difficile infection traditionally have addressed barrier precautions, environmental disinfection, and health care worker hand hygiene. When applied as a bundle, this approach has been used widely as an evidence-based strategy to prevent hospital-acquired C. difficile infection. Expanding the bundle to include patient hand hygiene is a nurse-driven approach to prevent C. difficile transmission.
Li, Wei; Wang, Hongfei; Lei, Cheng; Ying, Tianlei; Tan, Xiangshi
Clostridium difficile is a human pathogen that causes severe antibiotic-associated Clostridium difficile infection (CDI). Herein the MnSODcd from C. difficile was cloned, expressed in Escherichia Coli,and characterized by X-ray crystallography, UV/Vis and EPR spectroscopy, and activity assay, et al. The crystal structure of MnSODcd (2.32 Å) reveals a manganese coordination geometry of distorted trigonal bipyramidal, with His111, His197 and Asp193 providing the equatorial ligands and with His56 and a hydroxide or water forming the axial ligands. The catalytic activity of MnSODcd (8,600 U/mg) can be effectively inhibited by 2-methoxyestradiol with an IC50 of 75 μM. The affinity investigation between 2-methoxyestradiol and MnSODcd by ITC indicated a binding constant of 8.6 μM with enthalpy changes (ΔH = -4.08 ± 0.03 kcal/mol, ΔS = 9.53 ± 0.02 cal/mol/deg). An inhibitory mechanism of MnSODcd by 2-methoxyestradiol was probed and proposed based on molecular docking models and gel filtration analysis. The 2-methoxyestradiol may bind MnSODcd to interfere with the cross-linking between the two active sites of the dimer enzyme, compromising the SOD activity. These results provide valuable insight into the rational design of MnSODcd inhibitors for potential therapeutics for CDI.
Kim, Mi Na; Koh, Seong-Joon; Kim, Jung Mogg; Im, Jong Pil; Jung, Hyun Chae; Kim, Joo Sung
AIM: To investigate the effect of Clostridium difficile (C. difficile) infection in an interleukin 10-deficient (IL-10-/-) mouse model of inflammatory bowel disease. METHODS: Bone marrow-derived dendritic cells isolated from wild type (WT) and IL-10-/-mice were stimulated for 4 h with C. difficile toxin A (200 μg/mL), and gene expression of interferon (IFN)-γ, IL-12 and IL-23 was determined by real-time reverse transcription polymerase chain reaction. WT and IL-10-/- mice (n = 20 each) were exposed to an antibiotic cocktail for three days and then were injected with clindamycin (i.p.). Mice (n = 10 WT, 10 IL-10-/-) were then challenged with oral administration of C. difficile (1 × 105 colony forming units of strain VPI 10463). Animals were monitored daily for 7 d for signs of colitis. Colonic tissue samples were evaluated for cytokine gene expression and histopathologic analysis. RESULTS: C. difficile toxin A treatment induced IFN-γ gene expression to a level that was significantly higher in BDMCs from IL-10-/- compared to those from WT mice (P < 0.05). However, expression of IL-12 and IL-23 was not different among the groups. Following C. difficile administration, mice developed diarrhea and lost weight within 2-3 d. Weight loss was significantly greater in IL-10-/- compared to WT mice (P < 0.05). C. difficile infection induced histopathologic features typical of colitis in both IL-10-/- and WT mice. The histopathologic severity score was significantly higher in the IL-10-/- than in WT mice (mean ± standard error; 5.50 ± 0.53 vs 2.44 ± 0.46; P < 0.05). This was accompanied by a significantly greater increase in IFN-γ gene expression in colonic tissues from IL-10-/- than from WT mice challenged with C. difficile (P < 0.05). CONCLUSION: These results indicate that colitis is more severe after C. difficile infection in IL-10-/-mice, and that IFN-γ expression is involved in this process. PMID:25493020
Yun, B; Oh, S; Griffiths, M W
Clostridium difficile is a spore-forming, toxin-producing, anaerobic bacterium that colonizes the human gastrointestinal tract. This pathogen causes antibiotic-associated diarrhea and colitis in animals and humans. Antibiotic-associated diseases may be treated with probiotics, and interest is increasing in such uses of probiotics. This study investigated the effect of Lactobacillus strains on the quorum-sensing signals and toxin production of C. difficile. In addition, an in vivo experiment was designed to assess whether Lactobacillus acidophilus GP1B is able to control C. difficile-associated disease. Autoinducer-2 activity was measured for C. difficile using the Vibrio harveyi coupled bioluminescent assay. Cell extract (10μg/mL) of L. acidophilus GP1B exhibited the highest inhibitory activity among 5 to 40μg/mL cell-extract concentrations. Real-time PCR data indicated decreased transcriptional levels in luxS, tcdA, tcdB, and txeR genes in the presence of 10μg/mL of cell extract of L. acidophilus GP1B. Survival rates at 5d for mice given the pathogen alone with L. acidophilus GP1B cell extract or L. acidophilus GP1B were 10, 70, and 80%, respectively. In addition, the lactic acid-produced L. acidophilus GP1B exhibits an inhibitory effect against the growth of C. difficile. Both the L. acidophilus GP1B and GP1B cell extract have significant antipathogenic effects on C. difficile.
Testore, G P; Nardi, F; Babudieri, S; Giuliano, M; Di Rosa, R; Panichi, G
The possibility that the small intestine may represent a reservoir for Clostridium difficile was studied, using segments of human jejunum collected at necropsy. Our results (three of 100 specimens positive for C difficile culture) support the hypothesis that C difficile can be found in human jejunum and that it adheres to the normal mucosa as a resident bacterium. These findings suggest that gastrointestinal disease caused by C difficile has an endogenous origin. PMID:3745477
Zhao, Song; Ghose-Paul, Chandrabali; Zhang, Keshan; Tzipori, Saul; Sun, Xingmin
Clostridium difficile (C. difficile) causes over 500,000 infections per year in the US, with an estimated 15,000 deaths and an estimated cost of $1–3 billion. Moreover, a continual rise in the incidence of severe C. difficile infection (CDI) has been observed worldwide. Currently, standard treatment for CDI is the administration of antibiotics. While effective, these treatments do not prevent and may contribute to a disease recurrence rate of 15–35%. Prevention of recurrence is one of the most challenging aspects in the field. A better knowledge of the molecular mechanisms of the disease, the host immune response and identification of key virulence factors of C. difficilenow permits the development of immune-based therapies. Antibodies specific for C. difficile toxins have been shown to effectively treat CDI and prevent disease relapse in animal models and in humans. Vaccination has been recognized as the most cost-effective treatment/prevention for CDI. This review will summarize CDI transmission, epidemiology, major virulent factors and highlights the rational and the development of immune-based approaches against this remerging threat. PMID:25668664
Alam, M Jahangir; Anu, Ananna; Walk, Seth T; Garey, Kevin W
As Clostridium difficile spores are resistant to many household cleaning products, the potential for community household contamination is high. The purpose of this study was to assess the prevalence of toxigenic C. difficile from environmental sources from a large urban area. Three to 5 household items or environmental dust was collected from 30 houses in Houston, Texas. A total of 127 environmental samples were collected from shoe bottoms (n = 63), bathroom surfaces (n = 15), house floor dusts (n = 12), or other household surfaces (n = 37). Forty one of 127 samples (32.3%) grew C. difficile. All 41 isolates were positive for toxin A and B genes and no isolate was positive for binary toxin genes. Shoe bottom swab samples had the highest percent of positive samples (25/63; 39.7%) followed by bathroom/toilet surfaces (5/15; 33.3%), house floor dust (4/12; 33.3%), and other surface swabs (7/37; 18.9%). Strains were grouped into 25 different ribotypes, the most prevalent type was 001 (5 strains). In conclusion, a high rate of environmental contamination of C. difficile was observed from community households from a large urban area.
Silva, Rodrigo Otávio Silveira; Rupnik, Maja; Diniz, Amanda Nádia; Vilela, Eduardo Garcia; Lobato, Francisco Carlos Faria
Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies aboutC. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficileribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen. PMID:26676318
Neubauer, Heinrich; Schmoock, Gernot; Baier, Sylvia; Harlizius, Jürgen; Nienhoff, Hendrik; Brase, Katja; Zimmermann, Stefan; Seyboldt, Christian
Clostridium difficile was isolated from 147 of 201 (73%) rectal swabs of piglets from 15 farms of Lower Saxony and North Rhine-Westphalia. In 14 farms, 14 to 100% (mean, 78%) of the animals tested were culture positive. The rate of isolation was 68% postpartum, increased to 94% in animals 2 to 14 days of age, and declined to 0% for animals 49 days of age and older. There was no link between isolation and antibiotic treatment or diarrhea of piglets. Strains were assigned to 10 PCR ribotypes, and up to 4 PCR ribotypes were found to be present at the same time on a farm. The closely related PCR ribotypes 078 (55%) and 126 (20%) were most frequently recovered and were present in 13 of the 14 positive farms. The comparison of multilocus VNTR (variable number of tandem repeats) analysis (MLVA) data from this study and previously published data on human, porcine, and bovine PCR ribotype 078 isolates from 5 European countries revealed genetic differences between strains of different geographic origin and confirmed the relatedness of human and porcine C. difficile isolates. This study demonstrated that the human-pathogenic PCR ribotypes 078 and 126 are predominant in piglets in Germany. The results suggest that presence of C. difficile is correlated with animal age but not with antibiotic treatment or clinical disease. MLVA indicated that strains of the same geographical origin are often genetically related and corroborated the hypothesis of a close epidemiological connection between human and porcine C. difficile isolates. PMID:24025903
Harvey, Roger B; Norman, Keri N; Andrews, Kathleen; Norby, Bo; Hume, Michael E; Scanlan, Charles M; Hardin, Margaret D; Scott, Harvey M
The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America from the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer to human beings. The objective of the present study was to determine the prevalence of C. difficile in pork from sausage manufacturing plants and retail meat in Texas. Twenty-three C. difficile isolates were detected from 243 meat samples (9.5%) from 3 sausage-manufacturing plants and 5 retail meat outlets from 2004 to 2009. Twenty-two isolates were positive for toxins A, B, and binary toxin, and were characterized as toxinotype V, PFGE type-NAP7, or "NAP7-variant." Susceptibilities to 11 antimicrobial agents in the current study were similar to those reported previously for toxinotype V isolates, although the results suggested somewhat reduced resistance than reported for other meat, animal, or human clinical toxinotype V isolates.
Martínez-Meléndez, Adrián; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Maldonado-Garza, Héctor Jesús; Villarreal-Treviño, Licet; Garza-González, Elvira
Clostridium difficile (C. difficile) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from self-limited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI.
Jafari, Nazila V.; Songane, Mario; Stabler, Richard A.; Elawad, Mamoun; Wren, Brendan W.; Allan, Elaine
Clostridium difficile is an important nosocomial pathogen and the leading cause of antibiotic-associated diarrhea. Multilocus sequence typing indicates that C. difficile strains belong to five distinct genetic clades encompassing several PCR ribotypes (RT). Since their emergence in 2003, hypervirulent RT027 strains have been a major focus of research; in contrast, our current understanding of RT017-mediated disease pathogenesis lags far behind. In this study, we aimed to characterize host immunity to CF5 and M68, two genetically well-defined RT017 strains. Both strains engaged with host Toll-like receptor 2/6 (TLR2/6), TLR2-CD14, and TLR5 to similar extents in a model cell line. Despite this, CF5 mediated significantly greater dendritic cell (DC) interleukin-12 (IL-12), IL-27, and IL-10 immunity than M68. Both strains elicited similar IL-1β mRNA levels, and yet only M68 caused a marked increase in secretory IL-1β. A CF5 cocultured-DC cytokine milieu drove an equipotent Th1 and Th17 response, while M68 promoted greater Th17 immunity. Human gastrointestinal ex vivo cytokine responses to both strains were characterized. Taken together, our data suggest that C. difficile strains mediate overlapping and yet distinct mucosal and DC/T cell immunity. Finally, toxin-driven IL-1β release supports the hypothesis that this cytokine axis is a likely target for therapeutic intervention for C. difficile infection. PMID:25225246
Zhao, Song; Ghose-Paul, Chandrabali; Zhang, Keshan; Tzipori, Saul; Sun, Xingmin
Clostridium difficile (C. difficile) causes over 500,000 infections per year in the US, with an estimated 15,000 deaths and an estimated cost of $1-3 billion. Moreover, a continual rise in the incidence of severe C. difficile infection (CDI) has been observed worldwide. Currently, standard treatment for CDI is the administration of antibiotics. While effective, these treatments do not prevent and may contribute to a disease recurrence rate of 15-35%. Prevention of recurrence is one of the most challenging aspects in the field. A better knowledge of the molecular mechanisms of the disease, the host immune response and identification of key virulence factors of C. difficilenow permits the development of immune-based therapies. Antibodies specific for C. difficile toxins have been shown to effectively treat CDI and prevent disease relapse in animal models and in humans. Vaccination has been recognized as the most cost-effective treatment/prevention for CDI. This review will summarize CDI transmission, epidemiology, major virulent factors and highlights the rational and the development of immune-based approaches against this remerging threat.
Gerding, Dale N
We are in the midst of a resurgence of Clostridium difficile infection (CDI) in North America and Europe for which morbidity and mortality are higher than ever seen. C. difficile has risen in frequency to become the most common healthcare-associated infection pathogen, exceeding methicillin-resistant Staphylococcus aureus in many hospitals. Protection against CDI is thought to be mediated first by the normal bacterial microbiota, supplemented by an adaptive immune antibody response directed primarily at C. difficile toxins. Treatment of CDI is with antimicrobials that also further disrupt the protective bacterial microbiota leaving the patient susceptible to recurrent CDI. In addition, patients most susceptible to CDI, the advanced elderly, may already have a limited immune response and fail to increase their adaptive immune response with infection. The importance of both of these protective modalities has been demonstrated by 1) the success of fecal microbiota to restore "colonization resistance" for patients with multiple recurrences of CDI, and 2) the marked reduction in CDI recurrences with the use of intravenous monoclonal antibodies directed against toxin A and toxin B as an adjunct to antimicrobial treatment. Anti-toxin vaccines, passive monoclonal anti-toxin antibodies, and non-toxigenic C. difficile (to restore colonization resistance) are already undergoing patient clinical trials. The opportunity to prevent CDI is compelling and future research should focus on understanding the critical elements of the microbiota needed to restore colonization resistance and on development of novel immunologic strategies that include systemic and mucosal vaccines and passive immune modulators.
Martínez-Meléndez, Adrián; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Maldonado-Garza, Héctor Jesús; Villarreal-Treviño, Licet; Garza-González, Elvira
Clostridium difficile (C. difficile) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from self-limited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI. PMID:28321156
Janezic, Sandra; Potocnik, Mojca; Zidaric, Valerija; Rupnik, Maja
Clostridium difficile is one of the most important human and animal pathogens. However, the bacterium is ubiquitous and can be isolated from various sources. Here we report the prevalence and characterization of C. difficile in less studied environmental samples, puddle water (n = 104) and soil (n = 79). C. difficile was detected in 14.4% of puddle water and in 36.7% of soil samples. Environmental strains displayed antimicrobial resistance patterns comparable to already published data of human and animal isolates. A total of 480 isolates were grouped into 34 different PCR ribotypes. More than half of these (52.9%; 18 of 34) were already described in humans or animals. However, 14 PCR ribotypes were new in our PCR ribotype library and all but one were non-toxigenic. The multilocus sequence analysis of these new PCR ribotypes revealed that non-toxigenic environmental isolates are phylogenetically distinct and belong to three highly divergent clades, two of which have not been described before. Our data suggest that environment is a potential reservoir of genetically diverse population of C. difficile. PMID:27880843
Martin-Verstraete, Isabelle; Peltier, Johann; Dupuy, Bruno
The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection. PMID:27187475
Pérez-Cobas, Ana Elena; Moya, Andrés; Gosalbes, María José; Latorre, Amparo
Antibiotics strongly disrupt the human gut microbiota, which in consequence loses its colonization resistance capacity, allowing infection by opportunistic pathogens such as Clostridium difficile. This bacterium is the main cause of antibiotic-associated diarrhea and a current problem in developed countries, since its incidence and severity have increased during the last years. Furthermore, the emergence of antibiotic resistance strains has reduced the efficiency of the standard treatment with antibiotics, leading to a higher rate of relapses. Here, we review recent efforts focused on the impact of antibiotics in the gut microbiome and their relationship with C. difficile colonization, as well as, in the identification of bacteria and mechanisms involved in the protection against C. difficile infection. Since a healthy gut microbiota is able to avoid pathogen colonization, restoration of the gut microbiota seems to be the most promising approach to face C. difficile infection, especially for recurrent cases. Therefore, it would be possible to design probiotics for patients undergoing antimicrobial therapies in order to prevent or fight the expansion of the pathogen in the gut ecosystem.
Visser, Monique; Sepehrim, Shadi; Olson, Nancy; Du, Tim; Mulvey, Michael R; Alfa, Michelle J
The aim of the present study was to determine whether Clostridium difficile was present in uncooked retail ground beef and ground pork products sold in Winnipeg, Manitoba. Using an alcohol treatment protocol and inoculation of cultures on C difficile Moxalactam Norfloxacin (CDMN), toxigenic C difficile was found in 6.3% of 48 meat samples. The C difficile isolates belonged to different pulsotypes, all of which had been previously isolated from the stool of Manitoba patients with C difficile disease. Because cooking of meat will not eradicate C difficile spores, this raises a concern regarding potential foodborne transmissibility of this organism. PMID:23450202
Cecil, Jane A
Clostridium difficile was first reported as a cause of antibiotic-associated colitis in 1978. In more recent years we have witnessed disturbing trends associated with C. difficile infection (CDI). CDI has become more common, affecting populations previously considered at low risk, more severe with an associated increase in mortality, and more difficult to treat with some patients experiencing multiple relapses and a reduced responsiveness to previously effective antibiotics. These trends have been coincident with the emergence of a new hypervirulent strain responsible for several outbreaks in the last decade. Fortunately, we have also seen promising developments, particularly with regard to testing and treatment. This review discusses recent changes in the epidemiology of CDI and recent developments in the testing, treatment and prevention of CDI.
Eckmann, Christian; Lyon, Sue
26th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 9-12th April 2016, Amsterdam, The Netherlands The European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) is the annual scientific meeting of the European Society of Clinical Microbiology. ECCMID 2016, held in Amsterdam, The Netherlands, was attended by over 11,600 clinical microbiologists and infectious disease physicians from more than 120 countries. The Congress offered an essential opportunity to learn more about the diagnosis, prevention and treatment of healthcare-associated infections, especially those caused by Clostridium difficile. Recurrent C. difficile infections have an especially serious adverse impact on patients, their families and healthcare systems across Europe and around the world, and continue to be a cause for concern among ECCMID delegates and their colleagues responsible for managing vulnerable patients in acute hospitals and other healthcare facilities.
Parekh, Sejal; Dabrowski, Hannah; Petkar, Hawabibee
Previous evidence suggested a significant variation in the testing algorithms used across the United Kingdom for the diagnosis of Clostridium difficile infection (CDI) and new national guidelines were issued in 2012. The main aim of this paper was to explore if such variation in testing and reporting is still present, to compare the management of CDI cases, and to investigate if there is any significant variation in the antibiotic policies among different hospitals. Using London hospitals as a sample, results show that there is still a wide variation of testing methods and reporting used, making comparisons difficult. It is likely that the overall variability in practices would be greater at a national and, even more, at international level. The relationship between broad-spectrum antibiotics and C. difficile incidence and alternative approaches in antibiotic guidelines may require further studies. PMID:26877769
Bouillaut, Laurent; Self, William T.
Clostridium difficile, a proteolytic Gram-positive anaerobe, has emerged as a significant nosocomial pathogen. Stickland fermentation reactions are thought to be important for growth of C. difficile and appear to influence toxin production. In Stickland reactions, pairs of amino acids donate and accept electrons, generating ATP and reducing power in the process. Reduction of the electron acceptors proline and glycine requires the d-proline reductase (PR) and the glycine reductase (GR) enzyme complexes, respectively. Addition of proline in the medium increases the level of PR protein but decreases the level of GR. We report the identification of PrdR, a protein that activates transcription of the PR-encoding genes in the presence of proline and negatively regulates the GR-encoding genes. The results suggest that PrdR is a central metabolism regulator that controls preferential utilization of proline and glycine to produce energy via the Stickland reactions. PMID:23222730
Jarrad, Angie M.; Karoli, Tomislav; Debnath, Anjan; Tay, Chin Yen; Huang, Johnny X.; Kaeslin, Geraldine; Elliott, Alysha G.; Miyamoto, Yukiko; Ramu, Soumya; Kavanagh, Angela M.; Zuegg, Johannes; Eckmann, Lars; Blaskovich, Mark A.T.; Cooper, Matthew A.
Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window. PMID:26117821
DuPont, Herbert L
Clostridium difficile infection (CDI) is increasing in all regions of the world where sought. There is no gold standard for diagnosis of CDI, with available tests having limitations. Prevention of CDI will be seen with antibiotic stewardship, improved disinfection of hospitals and nursing homes, chemo- and immuno-prophylaxis and next generation probiotics. The important therapeutic agents are oral vancomycin and fidaxomicin with metronidazole being used only in mild cases or when oral therapy cannot be given. Current therapy of CDI for 10 days is associated with high rate of recurrence that may be prevented by prolonging initial therapy. Future treatment strategies will focus on drugs that inhibit C. difficile, reduce toxin activity and inflammation in the gut, and improve colonic flora diversity.
Jarrad, Angie M; Karoli, Tomislav; Debnath, Anjan; Tay, Chin Yen; Huang, Johnny X; Kaeslin, Geraldine; Elliott, Alysha G; Miyamoto, Yukiko; Ramu, Soumya; Kavanagh, Angela M; Zuegg, Johannes; Eckmann, Lars; Blaskovich, Mark A T; Cooper, Matthew A
Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window.
Bouillaut, Laurent; Dubois, Thomas; Sonenshein, Abraham L; Dupuy, Bruno
Synthesis of the major toxin proteins of the diarrheal pathogen, Clostridium difficile, is dependent on the activity of TcdR, an initiation (sigma) factor of RNA polymerase. The synthesis of TcdR and the activation of toxin gene expression are responsive to multiple components in the bacterium's nutritional environment, such as the presence of certain sugars, amino acids, and fatty acids. This review summarizes current knowledge about the mechanisms responsible for repression of toxin synthesis when glucose or branched-chain amino acids or proline are in excess and the pathways that lead to synthesis of butyrate, an activator of toxin synthesis. The regulatory proteins implicated in these mechanisms also play key roles in modulating bacterial metabolic pathways, suggesting that C. difficile pathogenesis is intimately connected to the bacterium's metabolic state.
Sinh, Preetika; Barrett, Terrence A.; Yun, Laura
The incidence of Clostridium difficile infection (CDI) has significantly increased in the last decade in the United States adding to the health care burden of the country. Patients with inflammatory bowel disease (IBD) have a higher prevalence of CDI and worse outcomes. In the past, the traditional risk factors for CDI were exposure to antibiotics and hospitalizations in elderly people. Today, it is not uncommon to diagnose CDI in a pregnant women or young adult who has no risk factors. C. difficile can be detected at the initial presentation of IBD, during a relapse or in asymptomatic carriers. It is important to keep a high index of suspicion for CDI in IBD patients and initiate prompt treatment to minimize complications. We summarize here the changing epidemiology, pathogenesis, risk factors, clinical features, and treatment of CDI in IBD. PMID:21915178
Fløe, Andreas; Leutscher, Peter
Treatment of severe Clostridium difficile infection (CDI) poses a clinical challenge. Emerging evidence supports the use of faecal microbiota transplantation (FMT). An 81-year-old man was admitted with a third recurrent episode of CDI within two months. Because of clinical deterioration with development of pancolitis in spite of two weeks of metronidazole and vanco-mycin treatment, FMT was performed using a duodenal tube. The patient recovered completely without further relapse during follow-up. FMT was shown to be an efficient adjuvant treatment of complicated CDI.
Surawicz, Christina M.
Diarrhea is a common side effect of antibiotics. Antibiotics can cause diarrhea in 5-25% of individuals who take them but its occurrence is unpredictable. Diarrhea due to antibiotics is called antibiotic-associated diarrhea (AAD). Diarrhea may be mild and resolve when antibiotics are discontinued, or it may be more severe. The most severe form of AAD is caused by overgrowth of Clostridium difficile which can cause severe diarrhea, colitis, pseudomembranous colitis, or even fatal toxic megacolon. Rates of diarrhea vary with the specific antibiotic as well as with the individual susceptibility.
Padua, David; Pothoulakis, Charalabos
Clostridium difficile is being recognized as a growing threat to many health-care systems. Epidemiology data shows that infection rates are soaring and the disease burden is increasing. Despite the efficacy of standard treatments, it is becoming evident that novel therapeutics will be required to tackle this disease. These new treatments aim to enhance the intestinal microbial barrier, activate the immune system and neutralize the toxins that mediate this disease. Many of these therapies are still in the beginning stages of investigation, however, in the next few years, more clinical data will become available to help implement many of these exciting new therapeutic approaches.
Lees, E A; Miyajima, F; Pirmohamed, M; Carrol, E D
Clostridium difficile is an important nosocomial pathogen in adults. Its significance in children is less well defined, but cases of C. difficile infection (CDI) appear to be increasingly prevalent in paediatric patients. This review aims to summarize reported Clostridium difficile carriage rates across children of different age groups, appraise the relationship between CDI and factors such as method of delivery, type of infant feed, antibiotic use, and co-morbidities, and review factors affecting the gut microbiome in children and the host immune response to C. difficile. Searches of PubMed and Google Scholar using the terms 'Clostridium difficile neonates' and 'Clostridium difficile children' were completed, and reference lists of retrieved publications screened for further papers. In total, 88 papers containing relevant data were included. There was large inter-study variation in reported C. difficile carriage rates. There was an association between CDI and recent antibiotic use, and co-morbidities such as immunosuppression and inflammatory bowel disease. C. difficile was also found in stools of children with diarrhoea attributed to other pathogens (e.g. rotavirus). The role of C. difficile in the paediatric gut remains unclear; is it an innocent bystander in diarrhoeal disease caused by other organisms, or a pathogen causing subclinical to severe symptoms? Further investigation of the development of serological and local host response to C. difficile carriage may shed new light on disease mechanisms. Work is underway on defining a framework for diagnosis and management of paediatric CDI.
Spigaglia, Patrizia; Barbanti, Fabrizio; Morandi, Matteo; Moro, Maria Luisa; Mastrantonio, Paola
A laboratory diagnosis survey of Clostridium difficile infection (CDI) was performed in Italy in 2012-2013. Questionnaires from 278 healthcare settings from 15 regions of Italy were collected and analysed. Eighty seven percent of the laboratories declared to routinely perform CDI diagnosis, 99% of them only after the clinician's request. Among the 216 laboratories providing information on the size of the hospitals in which they were located, 65 had more than 500 beds (large hospitals), while 151 had less than 500 beds (small hospitals). The average percentage of positive tests for C. difficile toxins was 12.2%. Almost half of the laboratories (42%) used immunoenzymatic assay (EIA) for Tox A/B as a stand-alone method, while only 34% used an algorithm for CDI as indicated by the European guidelines. A low percentage of laboratories performed molecular assays or C. difficile culture, 25% and 29%, respectively. Most laboratories (161/278) declared to type C. difficile strains, the majority in collaboration with a reference laboratory. Among the 103 C. difficile clinical isolates collected during the study, 31 different PCR-ribotypes were identified. PCR-ribotype 356/607 (27%) was predominant, followed by 018 (12%). These two PCR-ribotypes show 87.5% of similarity in ribotyping profile. PCR-ribotypes 027 and 078 represented 8% and 4% of the strains, respectively. Four PCR-ribotypes (027, 033, 078 and 126) were positive for the binary toxin CDT. In particular, PCR-ribotype 033 produces only CDT, and it has recently been associated with symptomatic cases. The majority of strains were multidrug resistant. In particular, all strains PCR-ribotypes 356/607 and 018 were resistant to moxifloxacin, rifampicin, erythromycin and clindamycin. The results obtained highlight the need to raise awareness to the microbiological diagnosis of CDI among clinicians and to implement and harmonize diagnostic methods for CDI in Italian laboratories in the perspective of a future national
Vindigni, Stephen M; Broussard, Elizabeth K; Surawicz, Christina M
Clostridium difficile infection is increasingly common with a high risk of recurrence despite antibiotic treatment. In cases of recurrent C. difficile infection, fecal microbiota transplant (FMT) is a highly effective treatment option promoting the restoration of normal gut microbiota. Furthermore, preliminary uncontrolled evidence demonstrates possible benefit of FMT in the management of some cases of inflammatory bowel disease and chronic constipation. In addition to presenting an overview of FMT, we discuss the role of probiotics, a more common approach to modifying the intestinal microbiome. Probiotics have been utilized broadly for many disease processes, including gastrointestinal, cardiovascular and allergic disease settings, although with limited and inconsistent results. Multiple potential areas for research are also identified.
Imlay, Hannah; Kaul, Daniel; Rao, Krishna
Background: Clostridium difficile infection is a healthcare-associated infection resulting in significant morbidity. Although immunosuppression is associated with Clostridium difficile infection acquisition and adverse outcomes, the epidemiology of Clostridium difficile infection in HIV-infected patients has been little studied in the era of antiretroviral therapy. This study identifies the risk factors for acquisition of Clostridium difficile infection in HIV-infected patients. Methods: A retrospective, propensity score–matched case–control study design was employed, with patients selected from our institution’s outpatient HIV clinic. Clostridium difficile infection cases were defined as having positive stool testing plus an appropriate clinical presentation. The propensity score was generated via multiple logistic regression from year of HIV diagnosis, age at first contact, duration of follow-up, gender, and initial CD4 count. Results: The 46 cases included were matched to a total of 180 controls. Prior antibiotic treatment was a significant predictor of Clostridium difficile infection (odds ratio: 13, 95% confidence interval: 3.49–48.8, p < .001) as was number of hospital admissions in the preceding year (odds ratio: 4.02, confidence interval: 1.81–8.94, p < .001). Having both proton pump inhibitor use and CD4 count <200 cells/µL significantly increased odds of Clostridium difficile infection in the multivariable model (odds ratio: 15.17, confidence interval: 1.31–175.9, p = .021). Conclusion: As in the general population, frequent hospitalizations and exposure to antimicrobials are independent predictors of Clostridium difficile infection acquisition in patients with HIV. Additionally, low CD4 count and proton pump inhibitor use are new potentially modifiable variables that can be targeted for prevention of Clostridium difficile infection in future interventional studies. PMID:28348742
A recent report by the Department of Health, Clostridium Difficile Infection: How to deal with the problem - a board to ward approach, is a revised set of guidelines based on best practice and key recommendations for the NHS to ensure the control of Clostridium difficile infection (CDI). It takes into account a national framework for clinical governance which did not previously exist, a framework that gives significant weight to infection control as a matter of patient safety, and highlights that all clinicians have a personal responsibility for infection prevention and control. It puts the onus on Trust management and PCTs to ensure that measures are in place to prevent and manage CDI according to best evidence. However, the report fails to explain how these measures will have an impact on finance and resources on an already burdened system. The author explains how much of the report is comparable with the one published in 1994, and highlights many of its limitations within the busy hospital setting. Reducing CDI is achievable, as many hospitals are showing large reductions in their CDI rates. Healthcare workers must be applauded for their success in reducing CDI, but there is more to be done.
Essenmacher, Alex C.; Khurram, Nazish; Bismack, Gregory T.
Reactive arthritis is an acute, aseptic, inflammatory arthropathy following an infectious process but removed from the site of primary infection. It is often attributed to genitourinary and enteric pathogens, such as Chlamydia, Salmonella, Shigella, Campylobacter, and Yersinia, in susceptible individuals. An uncommon and less recognized cause of this disease is preceding colonic infection with Clostridium difficile, an organism associated with pseudomembranous colitis and diarrhea in hospitalized patients and those recently exposed to antibiotics. Recognition of this association may be complicated by non-specific presentation of diarrhea, the interval between gastrointestinal and arthritic symptoms, and the wide differential in mono- and oligoarthritis. We present the case of a 61-year-old, hospitalized patient recently treated for C. difficile colitis who developed sudden, non-traumatic, right knee pain and swelling. Physical examination and radiographs disclosed joint effusion, and sterile aspiration produced cloudy fluid with predominant neutrophils and no growth on cultures. Diagnostic accuracy is enhanced by contemporaneous laboratory investigations excluding other entities such as gout and rheumatoid arthritis and other infections that typically precede reactive arthritis. Contribution of Clostridium infection to reactive arthritis is an obscure association frequently difficult to prove, but this organism is warranted inclusion in the differential of reactive arthritis. PMID:26908381
Clostridium difficile (C. difficile) infection (CDI) is the most common cause of healthcare-associated infections in US hospitals. The epidemic strain NAP1/BI/ribotype 027 accounts for outbreaks worldwide, with increasing mortality and severity. CDI is acquired from an endogenous source or from spores in the environment, most easily acquired during the hospital stay. The use of antimicrobials disrupts the intestinal microflora enabling C. difficile to proliferate in the colon and produce toxins. Clinical diagnosis in symptomatic patients requires toxin detection from stool specimens and rarely in combination with stool culture to increase sensitivity. However, stool culture is essential for epidemiological studies. Oral metronidazole is the recommended therapy for milder cases of CDI and oral vancomycin or fidaxomicin for more severe cases. Treatment of first recurrence involves the use of the same therapy used in the initial CDI. In the event of a second recurrence oral vancomycin often given in a tapered dose or intermittently, or fidaxomicin may be used. Fecal transplantation is playing an immense role in therapy of recurrent CDI with remarkable results. Fulminant colitis and toxic megacolon warrant surgical intervention. Novel approaches including new antibiotics and immunotherapy against CDI or its toxins appear to be of potential value. PMID:27065726
Gweon, Tae-Geun; Choi, Myung-Gyu; Baeg, Myong Ki; Lim, Chul-Hyun; Park, Jae Myung; Lee, In Seok; Kim, Sang Woo; Lee, Dong-Gun; Park, Yeon Joon; Lee, Jong Wook
AIM: To investigate the incidence and clinical outcome of Clostridium difficile (C. difficile) associated diarrhea (CDAD) in patients with hematologic disease. METHODS: We retrospectively reviewed the medical records of patients who underwent C. difficile testing in a tertiary hospital in 2011. The incidence and risk factors for CDAD and its clinical course including recurrence and mortality were assessed in patients with hematologic disease and compared with those in patients with nonhematologic disease. RESULTS: About 320 patients were diagnosed with CDAD (144 patients with hematologic disease; 176 with nonhematologic disease). The incidence of CDAD in patients with hematologic disease was estimated to be 36.7 cases/10000 patient hospital days, which was higher than the 5.4 cases/10000 patient hospital days in patients with nonhematologic disease. Recurrence of CDAD was more frequent in patients with hematologic disease compared to those with nonhematologic disease (18.8% vs 8.5%, P < 0.01), which was associated with higher re-use of causative antibiotics for CDAD. Mortality due to CDAD did not differ between the two groups. Multivariate analysis showed that intravenous immunoglobulin was the only significant factor associated with a lower rate of recurrence of CDAD in patients with hematologic disease. CONCLUSION: The incidence and recurrence of CDAD was higher in patients with hematologic disease than in those with nonhematologic disease. PMID:24914383
Usacheva, Elena A; Jin, Jian-P; Peterson, Lance R
Clostridium difficile infection (CDI) is a significant healthcare concern worldwide, and C. difficile is recognised as the most frequent aetiological agent of infectious healthcare-associated diarrhoea in hospitalised adult patients. The clinical manifestation of CDI varies from self-limited diarrhoea to life-threatening colitis. Such a broad disease spectrum can be explained by the impact of host factors. Currently, a complex CDI aetiology is widely accepted, acknowledging the interaction between bacteria and the host. C. difficile strains producing clostridial toxins A and B are considered toxigenic and can cause disease; those not producing the toxins are non-pathogenic. A person colonised with a toxigenic strain will not necessarily develop CDI. It is imperative to recognise patients with active disease from those only colonised with this pathogen and to implement appropriate treatment. This can be achieved by diagnostics that rely on host factors specific to CDI. This review will focus on major aspects of CDI pathogenesis and molecular mechanisms, describing host factors in disease progression and assessment of the host response in order to facilitate the development of CDI-specific diagnostics.
The widespread use of antibiotics has led Clostridium difficile infection (CDI) to become a common problem with pronounced medical and economic effects. The recurrence of CDI after treatment with standard antibiotics is becoming more common with the emergence of more resistant strains of C. difficile. As CDI is an antibiotic-associated disease, further treatment with antibiotic is best avoided. As the gut flora is severely disturbed in CDI, approaches that restore the gut microbiota may become good alternative modes of CDI therapies. Fecal microbiota transplantation (FMT) is the procedure of transplantation of fecal bacteria from a healthy donor individual into a patient for restoration of the normal colonic flora. Thus, FMT helps in the eradication of C. difficile and resolution of clinical symptoms such as diarrhea, cramping, and urgency. Though this approach to treatment is not new, presently, it has become an alternative and promising way of combating infections. The procedure is not in regular use because of the time required to identify a suitable donor, the risk of introducing opportunistic pathogens, and a general patient aversion to the transplant. However, FMT is gaining popularity because of its success rate as a panacea for recurrent attacks of CDI and is being increasingly used in clinical practice. This review describes the rationale, the indications, the results, the techniques, the potential donors, the benefits as well as the complications of fecal microbiota instillation to CDI patients in order to restore the normal gut flora.
Clostridium difficile is one of the many aetiological agents of antibiotic associated diarrhoea and is implicated in 15-25 per cent of the cases. The organism is also involved in the exacearbation of inflammatory bowel disease and extracolonic manifestations. Due to increase in the incidence of C. difficile infection (CDI), emergence of hypervirulent strains, and increased frequency of recurrence, the clinical management of the disease has become important. The management of CDI is based on disease severity, and current antibiotic treatment options are limited to vancomycin or metronidazole in the developing countries. this review article briefly describes important aspects of CDI, and the new drug, fidaxomicin, for its treatment. Fidaxomicin is particularly active against C.difficile and acts by inhibition of RNA synthesis. Clinical trials done to compare the efficacy and safety of fidaxomicin with that of vancomycin in treating CDI concluded that fidaxomicin was non-inferior to vancomycin for treatment of CDI and that there was a significant reduction in recurrences. The bactericidal properties of fidaxomicin make it an ideal alternative for CDI treatment. However, fidaxomicin use should be considered taking into account the potential benefits of the drug, along with the medical requirements of the patient, the risks of treatment and the high cost of fidaxomicin compared to other treatment regimens.
Virk, Hafeez Ul Hassan; Inayat, Faisal
Context: Takotsubo cardiomyopathy (TCM) mimics acute coronary syndrome and is accompanied by reversible left ventricular apical ballooning in the absence of angiographically significant coronary artery stenosis. It is a transient condition that typically precedes physical or emotional triggers. Case Report: We describe the case of a 65-year-old woman who presented to our institution with symptomatic Clostridium difficile infection. 24 hours after admission, the patient complained of severe, retrosternal chest pain. Electrocardiogram showed diffuse elevation of ST-segment in the chest leads; however, coronary angiography demonstrated normal coronary arteries. Therein, an echocardiography was performed, which revealed apical ballooning and hypercontractile base with global left ventricular hypokinesis. These features were consistent with TCM. The patient was managed conservatively. Repeat echocardiogram 2 weeks later showed resolution of heart failure. Conclusion: To our research, this is the first report of TCM caused by C. difficile infection. Clinicians involved in the care of patients with C. difficile infection must be aware of this complication and should consider TCM in those who develop atypical chest pain. PMID:27583241
Knight, Daniel R.; Elliott, Briony; Chang, Barbara J.; Perkins, Timothy T.
SUMMARY Clostridium difficile infection (CDI) is the leading cause of antimicrobial and health care-associated diarrhea in humans, presenting a significant burden to global health care systems. In the last 2 decades, PCR- and sequence-based techniques, particularly whole-genome sequencing (WGS), have significantly furthered our knowledge of the genetic diversity, evolution, epidemiology, and pathogenicity of this once enigmatic pathogen. C. difficile is taxonomically distinct from many other well-known clostridia, with a diverse population structure comprising hundreds of strain types spread across at least 6 phylogenetic clades. The C. difficile species is defined by a large diverse pangenome with extreme levels of evolutionary plasticity that has been shaped over long time periods by gene flux and recombination, often between divergent lineages. These evolutionary events are in response to environmental and anthropogenic activities and have led to the rapid emergence and worldwide dissemination of virulent clonal lineages. Moreover, genome analysis of large clinically relevant data sets has improved our understanding of CDI outbreaks, transmission, and recurrence. The epidemiology of CDI has changed dramatically over the last 15 years, and CDI may have a foodborne or zoonotic etiology. The WGS era promises to continue to redefine our view of this significant pathogen. PMID:26085550
Durham, David P; Olsen, Margaret A; Dubberke, Erik R; Galvani, Alison P; Townsend, Jeffrey P
To quantify the effect of hospital and community-based transmission and control measures on Clostridium difficile infection (CDI), we constructed a transmission model within and between hospital, community, and long-term care-facility settings. By parameterizing the model from national databases and calibrating it to C. difficile prevalence and CDI incidence, we found that hospitalized patients with CDI transmit C. difficile at a rate 15 (95% CI 7.2-32) times that of asymptomatic patients. Long-term care facility residents transmit at a rate of 27% (95% CI 13%-51%) that of hospitalized patients, and persons in the community at a rate of 0.1% (95% CI 0.062%-0.2%) that of hospitalized patients. Despite lower transmission rates for asymptomatic carriers and community sources, these transmission routes have a substantial effect on hospital-onset CDI because of the larger reservoir of hospitalized carriers and persons in the community. Asymptomatic carriers and community sources should be accounted for when designing and evaluating control interventions.
Culligan, Eamonn P.; Sleator, Roy D.
Clostridium difficile is a major cause of morbidity and mortality worldwide, causing over 400,000 infections and approximately 29,000 deaths in the United States alone each year. C. difficile is the most common cause of nosocomial diarrhoea in the developed world, and, in recent years, the emergence of hyper-virulent (mainly ribotypes 027 and 078, sometimes characterised by increased toxin production), epidemic strains and an increase in the number of community-acquired infections has caused further concern. Antibiotic therapy with metronidazole, vancomycin or fidaxomicin is the primary treatment for C. difficile infection (CDI). However, CDI is unique, in that, antibiotic use is also a major risk factor for acquiring CDI or recurrent CDI due to disruption of the normal gut microbiota. Therefore, there is an urgent need for alternative, non-antibiotic therapeutics to treat or prevent CDI. Here, we review a number of such potential treatments which have emerged from advances in the field of microbiome research. PMID:27657145
Zhang, Wen; Cheng, Ying; Du, Pengcheng; Zhang, Yuanyuan; Jia, Hongbing; Li, Xianping; Wang, Jing; Han, Na; Qiang, Yujun; Chen, Chen; Lu, Jinxing
Clostridium difficile, the etiological agent of Clostridium difficile infection (CDI), is a gram-positive, spore-forming bacillus that is responsible for ∼20% of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. Previous data have shown that a substantial proportion (11%) of the C. difficile genome consists of mobile genetic elements, including seven conjugative transposons. However, the mechanism underlying the formation of a mosaic genome in C. difficile is unknown. The type-IV secretion system (T4SS) is the only secretion system known to transfer DNA segments among bacteria. We searched genome databases to identify a candidate T4SS in C. difficile that could transfer DNA among different C. difficile strains. All T4SS gene clusters in C. difficile are located within genomic islands (GIs), which have variable lengths and structures and are all conjugative transposons. During the horizontal-transfer process of T4SS GIs within the C. difficile population, the excision sites were altered, resulting in different short-tandem repeat sequences among the T4SS GIs, as well as different chromosomal insertion sites and additional regions in the GIs.
Gweon, Tae Geun; Lee, Kyung Jin; Kang, Dong Hoon; Park, Sung Soo; Kim, Kyung Hoon; Seong, Hyeon Jin; Ban, Tae Hyun; Moon, Sung Jin; Kim, Jin Su; Kim, Sang Woo
Clostridium difficile infection. The mortality rate of fulminant C. difficile infection is reported to be as high as 50%. Fecal microbiota transplantation is a highly effective treatment in patients with recurrent or refractory C. difficile infection. However, there are few published articles on the use of such transplantation for fulminant C. difficile infection. Here, we report on a patient with toxic megacolon complicated by C. difficile infection who was treated successfully with fecal mi-crobiota transplantation. (Gut Liver, 2015;9:247-250).
Lee, Bruce Y; Popovich, Michael J; Tian, Ye; Bailey, Rachel R; Ufberg, Paul J; Wiringa, Ann E; Muder, Robert R
Efforts are currently underway to develop a vaccine against Clostridium difficile infection (CDI). We developed two decision analytic Monte Carlo computer simulation models: (1) an Initial Prevention Model depicting the decision whether to administer C. difficile vaccine to patients at-risk for CDI and (2) a Recurrence Prevention Model depicting the decision whether to administer C. difficile vaccine to prevent CDI recurrence. Our results suggest that a C. difficile vaccine could be cost-effective over a wide range of C. difficile risk, vaccine costs, and vaccine efficacies especially, when being used post-CDI treatment to prevent recurrent disease.
Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian
This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. PMID:25411174
Schneeberg, Alexander; Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian
This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray.
Girinathan, Brintha Parasumanna; Braun, Sterling; Sirigireddy, Apoorva Reddy; Lopez, Jose Espinola; Govind, Revathi
Clostridium difficile is the principal cause of antibiotic-associated diarrhea. Major metabolic requirements for colonization and expansion of C. difficile after microbiota disturbance have not been fully determined. In this study, we show that glutamate utilization is important for C. difficile to establish itself in the animal gut. When the gluD gene, which codes for glutamate dehydrogenase (GDH), was disrupted, the mutant C. difficile was unable to colonize and cause disease in a hamster model. Further, from the complementation experiment it appears that extracellular GDH may be playing a role in promoting C. difficile colonization and disease progression. Quantification of free amino acids in the hamster gut during C. difficile infection showed that glutamate is among preferred amino acids utilized by C. difficile during its expansion. This study provides evidence of the importance of glutamate metabolism for C. difficile pathogenesis. PMID:27467167
Gorbach, Sherwood L
In 1975 John Bartlett began trials investigating the problem of antibiotic-associated diarrhea and pseudomembranous colitis. His work led the discovery of Clostridium difficile and he identified it as the leading cause of hospital-associated infections.
This document provides an update to the Agency’s interim guidance for the efficacy evaluation of antimicrobial pesticides that are labeled for treating hard non-porous surfaces in healthcare settings contaminated with spores of Clostridium difficile.
Chassaing, Benoit; Adekunle, Oluwaseyi; Mattei, Lisa M.; Edwards, Adrianne N.; McBride, Shonna M.; Bushman, Frederic D.; Gewirtz, Andrew T.
ABSTRACT Herein, we report the genome sequence of a Clostridium difficile strain isolated from the feces of antibiotic-treated C57BL/6 mice. We have named this strain, which differs considerably from those of the previously sequenced C. difficile strains, LEM1. PMID:28385835
Deshpande, Abhishek; Pant, Chaitanya; Anderson, Michael P; Donskey, Curtis J; Sferra, Thomas J
To determine whether the incidence of Clostridium difficile infection continues to increase in hospitalized pediatric patients, we evaluated data from a United States national inpatient database. For the period of 2003 to 2009, we found an increasing trend in the incidence of C. difficile infection. These data suggest greater effort be given to prevent and treat this infection in children.
The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America with the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer t...
Wilcox, Mark H; Gerding, Dale N; Poxton, Ian R; Kelly, Ciaran; Nathan, Richard; Birch, Thomas; Cornely, Oliver A; Rahav, Galia; Bouza, Emilio; Lee, Christine; Jenkin, Grant; Jensen, Werner; Kim, You-Sun; Yoshida, Junichi; Gabryelski, Lori; Pedley, Alison; Eves, Karen; Tipping, Robert; Guris, Dalya; Kartsonis, Nicholas; Dorr, Mary-Beth
Background Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. Methods We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. Results In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, -10.1 percentage points; 95% confidence interval [CI], -15.9 to -4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, -9.9 percentage points; 95% CI, -15.5 to -4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, -11.6 percentage points; 95% CI, -17.4 to -5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, -10.7 percentage points; 95% CI, -16.4 to -5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab
Pruitt, Rory N.; Chumbler, Nicole M.; Rutherford, Stacey A.; Farrow, Melissa A.; Friedman, David B.; Spiller, Ben; Lacy, D. Borden
The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.
Shields, Kelsey; Araujo-Castillo, Roger V.; Theethira, Thimmaiah G.; Alonso, Carolyn D.; Kelly, Ciaran
Clostridium difficile infection (CDI) is increasingly prevalent, dangerous and challenging to prevent and manage. Despite intense national and international attention the incidence of primary and of recurrent CDI (PCDI and RCDI, respectively) have risen rapidly throughout the past decade. Of major concern is the increase in cases of RCDI resulting in substantial morbidity, morality and economic burden. RCDI management remains challenging as there is no uniformly effective therapy, no firm consensus on optimal treatment, and reliable data regarding RCDI-specific treatment options is scant. Novel therapeutic strategies are critically needed to rapidly, accurately, and effectively identify and treat patients with, or at-risk for, RCDI. In this review we consider the factors implicated in the epidemiology, pathogenesis and clinical presentation of RCDI, evaluate current management options for RCDI and explore novel and emerging therapies. PMID:25930686
Infection due to Clostridium difficile is currently the main cause of hospital acquired gastrointestinal disease. Its prevalence in the elderly population is higher due to there being many associated risk factors in this age group, such as comorbidity, frequent exposure to the healthcare or residential home setting, immunosenescence, greater consumption of antibiotics, and antiacids. The diagnostic techniques have notably improved in the last few years, which could also account for an increase in its diagnosis. The new expert consensus recommendations propose stratifying the clinical situation of the patient in order to choose the treatment option. Therapeutic options have recently been included in the new Clinical Guidelines, such as flidaxomicin or fecal transplants, with encouraging results, particularly for the control of frequent recurrences.
Brown, William R
Clostridium difficile infection (CDI) is an increasingly common and severe international health problem. Customary treatment of this infection, usually with antibiotics, is often ineffective and its recurrence is common. In recent years the treatment of recurrent or refractory CDI by the transfer of stool from an uninfected person, so called fecal "microbiota transplantation" has become recognized as effective and generally safe. The effectiveness of this novel treatment is incompletely defined but is likely to be due to its correction of the intestinal dysbiosis that characterizes the disease. Practical methods for the administration of the transplantation have been described. This review summarizes the current reported experiences with fecal microbiota transplantation in the treatment for CDI.
Kerstens, Jeroen; de Gheldere, Charles; Vanclooster, Patrick
We present the case of a 58-year-old man who underwent urgent blowhole colostomy for toxic megacolon (TM) secondary to Clostridium difficile infection (CDI). This infection occurred under antibiotic coverage with amoxicillin-clavulanic acid, four days after laparoscopic sigmoidectomy in our hospital. Although prospective clinical research regarding the surgical management of TM is lacking, decompressive procedures like blowhole colostomy are reported to carry a high risk of postoperative morbidity and mortality and are widely regarded as obsolete. Subtotal or total colectomy with end ileostomy is currently considered the procedure of choice. After presenting our case, we discuss the literature available on the subject to argue that the scarce evidence on the optimal surgical treatment for TM is primarily based on TM associated with inflammatory bowel diseases (IBD) and that there might be a rationale for considering minimally invasive procedures like blowhole colostomy for CDI-associated TM. PMID:28097034
Clostridium difficile (CD)-associated diarrhoea is currently the most prevalent nosocomial diarrhoea worldwide. Many characteristics of CD pathogenicity remain poorly understood. Recent data strongly indicate the importance of an RNA network for the control of gene expression in CD. More than 200 regulatory RNAs have been identified by deep sequencing and targeted approaches, including Hfq-dependent trans riboregulators, cis-antisense RNAs, CRISPR RNAs, and c-di-GMP-responsive riboswitches. These regulatory RNAs are involved in the control of major processes in the CD infection cycle, for example motility, biofilm formation, adhesion, sporulation, stress response, and defence against bacteriophages. We will discuss recent advances in elucidation of the original features of RNA-based mechanisms in this important enteropathogen. This knowledge may pave the way for further discoveries in this emergent field.
Tamma, Pranita D.; Sandora, Thomas J.
The incidence of Clostridium difficile infection (CDI) in children has increased over the past decade. In recent years, new and intriguing data on pediatric CDI have emerged. Community-onset infections are increasingly recognized, even in children who have not previously received antibiotics. A hypervirulent strain is responsible for up to 20% of pediatric CDI cases. Unique risk factors for CDI in children have been identified. Advances in diagnostic testing strategies, including the use of nucleic acid amplification tests, have raised new questions about the optimal approach to diagnosing CDI in children. Novel therapeutic options are available for adult patients with CDI, raising questions about the use of these agents in children. Updated recommendations about infection prevention and control measures are now available. We summarize these recent developments in pediatric CDI in this review and also highlight remaining knowledge gaps that should be addressed in future research efforts. PMID:23687578
RICCIARDI, R.; HARRIMAN, K.; BAXTER, N. N.; HARTMAN, L. K.; TOWN, R. J.; VIRNIG, B. A.
SUMMARY Hospital-level predictors of high rates of ‘Clostridium difficile-associated disease’ (CDAD) were evaluated in over 2300 hospitals across California, Arizona, and Minnesota. American Hospital Association data were used to determine hospital characteristics associated with high rates of CDAD. Significant correlations were found between hospital rates of CDAD, common infections and other identified pathogens. Hospitals in urban areas had higher average rates of CDAD; yet, irrespective of geographic location, hospital rates of CDAD were associated with other infections. In addition, hospitals with ‘high CDAD’ rates had slower turnover of beds and were more likely to offer transplant services. These results reveal large differences in rates of CDAD across regions. Hospitals with high rates of CDAD have high rates of other common infections, suggesting a need for broad infection control policies. PMID:17686193
Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Azarnia Tehran, Domenico; Montecucco, Cesare; Barth, Holger
The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.
Wilcox, Mark H.; Chalmers, James D.; Nord, Carl E.; Freeman, Jane; Bouza, Emilio
The incidence of Clostridium difficile infection (CDI) in Europe has increased markedly since 2000. Previous meta-analyses have suggested a strong association between cephalosporin use and CDI, and many national programmes on CDI control have focused on reducing cephalosporin usage. Despite reductions in cephalosporin use, however, rates of CDI have continued to rise. This review examines the potential association of CDI with cephalosporins, and considers other factors that influence CDI risk. EUCLID (the EUropean, multicentre, prospective biannual point prevalence study of CLostridium difficile Infection in hospitalized patients with Diarrhoea) reported an increase in the annual incidence of CDI from 6.6 to 7.3 cases per 10 000 patient bed-days from 2011–12 to 2012–13, respectively. While CDI incidence and cephalosporin usage varied widely across countries studied, there was no clear association between overall cephalosporin prescribing (or the use of any particular cephalosporin) and CDI incidence. Moreover, variations in the pharmacokinetic and pharmacodynamic properties of cephalosporins of the same generation make categorization by generation insufficient for predicting impact on gut microbiota. A multitude of additional factors can affect the risk of CDI. Antibiotic choice is an important consideration; however, CDI risk is associated with a range of antibiotic classes. Prescription of multiple antibiotics and a long duration of treatment are key risk factors for CDI, and risk also differs across patient populations. We propose that all of these are factors that should be taken into account when selecting an antibiotic, rather than focusing on the exclusion of individual drug classes. PMID:27659735
Wilcox, Mark H; Chalmers, James D; Nord, Carl E; Freeman, Jane; Bouza, Emilio
The incidence of Clostridium difficile infection (CDI) in Europe has increased markedly since 2000. Previous meta-analyses have suggested a strong association between cephalosporin use and CDI, and many national programmes on CDI control have focused on reducing cephalosporin usage. Despite reductions in cephalosporin use, however, rates of CDI have continued to rise. This review examines the potential association of CDI with cephalosporins, and considers other factors that influence CDI risk. EUCLID (the EUropean, multicentre, prospective biannual point prevalence study of CLostridium difficile Infection in hospitalized patients with Diarrhoea) reported an increase in the annual incidence of CDI from 6.6 to 7.3 cases per 10 000 patient bed-days from 2011-12 to 2012-13, respectively. While CDI incidence and cephalosporin usage varied widely across countries studied, there was no clear association between overall cephalosporin prescribing (or the use of any particular cephalosporin) and CDI incidence. Moreover, variations in the pharmacokinetic and pharmacodynamic properties of cephalosporins of the same generation make categorization by generation insufficient for predicting impact on gut microbiota. A multitude of additional factors can affect the risk of CDI. Antibiotic choice is an important consideration; however, CDI risk is associated with a range of antibiotic classes. Prescription of multiple antibiotics and a long duration of treatment are key risk factors for CDI, and risk also differs across patient populations. We propose that all of these are factors that should be taken into account when selecting an antibiotic, rather than focusing on the exclusion of individual drug classes.
Deng, Kai; Plaza-Garrido, Angela; Torres, J Antonio; Paredes-Sabja, Daniel
Clostridium difficile's presence has been reported in meat products stored typically at low temperatures. This study evaluated the viability in phosphate buffer saline (PBS) of spores from epidemic C. difficile strain R20291 (4.6 log CFU/ml) and M120 (7.8 log CFU/ml). Viability was assessed during 4 months at -80 °C, -20 °C, 4 °C (refrigeration), and 23 °C (room temperature), and after 10 freeze (-20 °C)/thaw (+23 °C) cycles. Although spore viability decreased, significant viability was still observed after 4 months at -20 °C, i.e., 3.5 and 3.9 log CFU/ml and -80 °C, i.e., 6.0 and 6.1 log CFU/ml for strains R20291 and M120, respectively. The same trend was observed for M120 at 4 °C and 23 °C, while for R20291 the viability change was non-significant at 4 °C but increased significantly at 23 °C (p > 0.05). After 10 freeze-thaw cycles, viability of both strains decreased but a significant fraction remained viable (4.3 and 6.3 log CFU/ml for strain R20291 and M120, respectively). Strikingly, both strains showed higher viability in a meat model than in PBS. A small but significant decrease (p < 0.05) from 6.7 to 6.3 log CFU/ml in M120 viability was observed after 2-month storage in the meat model while the decrease from an initial 3.4 log CFU/ml observed for R20291 was non-significant (p = 0.12). In summary, C. difficile spores can survive low-temperature conditions for up to 4 months.
Dubois, Thomas; Dancer-Thibonnier, Marie; Monot, Marc; Hamiot, Audrey; Bouillaut, Laurent; Soutourina, Olga; Martin-Verstraete, Isabelle
The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601. PMID:27297391
Vickers, Richard J; Tillotson, Glenn; Goldstein, Ellie J C; Citron, Diane M; Garey, Kevin W; Wilcox, Mark H
Clostridium difficile infection (CDI) is the leading cause of infectious healthcare-associated diarrhoea. Recurrent CDI increases disease morbidity and mortality, posing a high burden to patients and a growing economic burden to the healthcare system. Thus, there exists a significant unmet and increasing medical need for new therapies for CDI. This review aims to provide a concise summary of CDI in general and a specific update on ridinilazole (formerly SMT19969), a novel antibacterial currently under development for the treatment of CDI. Owing to its highly targeted spectrum of activity and ability to spare the normal gut microbiota, ridinilazole provides significant advantages over metronidazole and vancomycin, the mainstay antibiotics for CDI. Ridinilazole is bactericidal against C. difficile and exhibits a prolonged post-antibiotic effect. Furthermore, treatment with ridinilazole results in decreased toxin production. A phase 1 trial demonstrated that oral ridinilazole is well tolerated and specifically targets clostridia whilst sparing other faecal bacteria. Phase 2 and 3 trials will hopefully further our understanding of the clinical utility of ridinilazole for the treatment of CDI.
Båverud, V; Gunnarsson, A; Karlsson, M; Franklin, A
The antimicrobial susceptibility of 50 Clostridium difficile isolates, 36 of them from horse feces and 14 from environmental sites, was determined by broth microdilution. The antimicrobial agents tested were avilamycin, cephalothin, chloramphenicol, clindamycin, erythromycin, gentamicin, neomycin, oxacillin, oxytetracycline, penicillin, spiramycin, streptomycin, trimethoprim/sulfamethoxazole, vancomycin, and virginiamycin. All isolates were susceptible to vancomycin (MIC =1 microg/ml). The MICs of erythromycin, oxytetracycline, spiramycin, and virginiamycin showed a bimodal distribution. Compared with the majority of isolates, the MICs of erythromycin (MIC > 16 microg/ml), oxytetracycline (MIC >/=32 microg/ml), spiramycin (MIC > 16 microg/ml), and virginiamycin (MIC 8-16 microg/ml) were higher for 18 isolates. Those were mainly isolated from horses at animal hospitals and further from environmental sites at a stud farm. In contrast, all isolates, except one, from healthy foals had low MICs of erythromycin, spiramycin, virginiamycin, and oxytetracycline. The isolates from soil in public parks had also low MICs of these antimicrobial agents. Broth microdilution appeared both reliable and reproducible for susceptibility testing of C. difficile. The method was also readily performed and the MIC endpoints were easily read.
Wright, Anne; Wait, Robin; Begum, Shajna; Crossett, Ben; Nagy, Judit; Brown, Katherine; Fairweather, Neil
Clostridium difficile is a bacterium that causes disease of the large intestine, particularly after treatment with antibiotics. The bacterium produces two toxins (A and B) that are responsible for the pathology of the disease. In addition, a number of bacterial virulence factors associated with adhesion to the gut have previously been identified, including the cell wall protein Cwp66, the high-molecular weight surface layer protein (HMW-SLP) and the flagella. As the genome sequence predicts many other cell wall associated proteins, we have investigated the diversity of proteins in cell wall extracts, with the aim of identifying further virulence factors. We have used a number of methods to remove the proteins associated with the cell wall of C. difficile. Two of the resulting extracts, obtained using low pH glycine treatment and lysozyme digestion of the cell wall, have been analysed in detail by two-dimensional electrophoresis and mass spectrometry. One hundred and nineteen spots, comprising 49 different proteins, have been identified. The two surface layer proteins (SLPs) are the most abundant proteins, and we have also found components of the flagellum. Interestingly, we have also determined that a number of paralogs of the HMW-SLP are expressed, and these could represent targets for further investigation as virulence factors.
Wilcox, Mark H; Planche, Tim; Fang, Ferric C; Gilligan, Peter
With the recognition of several serious outbreaks of Clostridium difficile infection in the industrialized world coupled with the development of new testing technologies for detection of this organism, there has been renewed interest in the laboratory diagnosis of C. difficile infection. Two factors seem to have driven much of this interest. First, the recognition that immunoassays for detection of C. difficile toxins A and B, for many years the most widely used tests for C. difficile infection diagnosis, were perhaps not as sensitive as previously believed at a time when attributed deaths to C. difficile infections were showing a remarkable rise. Second, the availability of FDA-approved commercial and laboratory-developed PCR assays which could detect toxigenic strains of C. difficile provided a novel and promising testing approach for diagnosing this infection. In this point-counterpoint on the laboratory diagnosis of C. difficile infection, we have asked two experts in C. difficile infection diagnosis, Ferric Fang, who has recently published two articles in the Journal of Clinical Microbiology advocating the use of PCR as a standalone test (see this author's references 12 and 28), and Mark Wilcox, who played a key role in developing the IDSA/SHEA guidelines on Clostridium difficile infection (see Wilcox and Planche's reference 1), along with his colleague, Tim Planche, to address the following question: what is the current role of algorithmic approaches to the diagnosis of C. difficile infection?
Denys, Gerald A
The Portrait Toxigenic Clostridium difficile assay is a rapid, qualitative assay for the detection of the tcdB gene of C. difficile in stool specimens from patients suspected of C. difficile infections, and received 510(k) clearance by the US FDA in March 2012. The Portrait Toxigenic C. difficile assay combines novel blocked-primer-mediated helicase-dependent multiplex amplification (bpHDA) technology and chip-based detection in an automated sample-to-result format. The assay requires minimal sample preparation and results are available within 90 min. In a multicenter evaluation, the Portrait Toxigenic C. difficile assay had a sensitivity of 98.2% and specificity of 92.8% compared with toxigenic culture. A comparative study between the Portrait Toxigenic C. difficile assay and three FDA-cleared molecular assays for the detection of toxigenic C. difficile exhibited a high degree of agreement (93.8-97.5%). The Portrait Toxigenic C. difficile assay provides a simple, cost-effective method with broad applicability to panel-based approaches, potentially simplifying workflow.
Alcalá, Luis; Reigadas, Elena; Marín, Mercedes; Fernández-Chico, Antonia; Catalán, Pilar; Bouza, Emilio
We compared two multistep diagnostic algorithms based on C. Diff Quik Chek Complete and, as confirmatory tests, GenomEra C. difficile and Xpert C. difficile. The sensitivity, specificity, positive predictive value, and negative predictive value were 87.2%, 99.7%, 97.1%, and 98.3%, respectively, for the GenomEra-based algorithm and 89.7%, 99.4%, 95.5%, and 98.6%, respectively, for the Xpert-based algorithm. GenomEra represents an alternative to Xpert as a confirmatory test of a multistep algorithm for Clostridium difficile infection (CDI) diagnosis.
Dannheim, Henning; Riedel, Thomas; Neumann-Schaal, Meina; Bunk, Boyke; Schober, Isabel; Spröer, Cathrin; Chibani, Cynthia Maria; Gronow, Sabine; Liesegang, Heiko; Overmann, Jörg; Schomburg, Dietmar
We resequenced the genome of Clostridium difficile 630Δerm (DSM 28645), a model strain commonly used for the generation of insertion mutants. The genome sequence was obtained by a combination of single-molecule real-time (SMRT) and Illumina sequencing technology. Detailed manual curation and comparison to the previously published genomic sequence revealed sequence differences including inverted regions and the presence of plasmid pCD630. Manual curation of our previously deposited genome sequence of the parental strain 630 (DSM 27543) led to an improved genome sequence. In addition, the sequence of the transposon Tn5397 was completely identified. We manually revised the current manual annotation of the initial sequence of strain 630 and modified either gene names, gene product names or assigned EC numbers of 57 % of genes. The number of hypothetical and conserved hypothetical proteins was reduced by 152. This annotation was used as a template to annotate the most recent genome sequences of the strains 630Δerm and 630. Based on the genomic analysis, several new metabolic features of C. difficile are proposed and could be supported by literature and subsequent experiments.
Trubiano, Jason A; Gardiner, Bradley; Kwong, Jason C; Ward, Peter; Testro, Adam G; Charles, Patrick G P
We describe a case of faecal microbiota transplantation (FMT) used for severe binary toxin-positive Clostridium difficile infection in an intensive care setting. The patient was admitted to the ICU of a tertiary hospital and failed traditional maximal pharmacological management. Adjunctive therapy with FMT given through gastroscopy resulted in resolution of the C. difficile-related symptoms. Although there is a growing experience with FMT for recurrent C. difficile infection, published evidence in severe disease is very limited. In a landscape of increasingly severe C. difficile infection, adjunctive FMT may be considered a useful early treatment option.
Kronman, Matthew P; Nielson, Heather J; Adler, Amanda L; Giefer, Matthew J; Wahbeh, Ghassan; Singh, Namita; Zerr, Danielle M; Suskind, David L
Fecal microbiota transplantation (FMT) is a safe and effective therapy for adults with recurrent Clostridium difficile colitis, but data regarding FMT in children are limited and focus on colonoscopic administration of FMT. We present 10 consecutive children who received FMT via nasogastric tube for treatment of recurrent C difficile infection. Median age was 5.4 years, and 30% were receiving simultaneous immunosuppression. Median follow-up was 44 days, and 90% of patients resolved their C difficile infection; one patient relapsed 2 months later after receiving antibiotics. FMT via nasogastric tube appears safe, well tolerated, and effective in treating pediatric recurrent C difficile colitis.
Gardiner, David F; Rosenberg, Talia; Zaharatos, Jerry; Franco, David; Ho, David D
Clostridium difficile is a pathogen with increasing severity for which host antibody responses provide protection from disease. DNA vaccination has several advantages compared to traditional vaccine methods, however no study has examined this platform against C. difficile toxins. A synthetic gene was created encoding the receptor-binding domain (RBD) of C. difficile toxin A, optimized for expression in human cells. Gene expression was examined in vitro. Mice were inoculated and then challenged with parenteral toxin A. Vaccination provided high titer antibodies and protected mice from death. This represents the first report of DNA vaccine inducing neutralizing antibodies to C. difficile toxin A.
Carroll, Karen C.
SUMMARY Clostridium difficile is a formidable nosocomial and community-acquired pathogen, causing clinical presentations ranging from asymptomatic colonization to self-limiting diarrhea to toxic megacolon and fulminant colitis. Since the early 2000s, the incidence of C. difficile disease has increased dramatically, and this is thought to be due to the emergence of new strain types. For many years, the mainstay of C. difficile disease diagnosis was enzyme immunoassays for detection of the C. difficile toxin(s), although it is now generally accepted that these assays lack sensitivity. A number of molecular assays are commercially available for the detection of C. difficile. This review covers the history and biology of C. difficile and provides an in-depth discussion of the laboratory methods used for the diagnosis of C. difficile infection (CDI). In addition, strain typing methods for C. difficile and the evolving epidemiology of colonization and infection with this organism are discussed. Finally, considerations for diagnosing C. difficile disease in special patient populations, such as children, oncology patients, transplant patients, and patients with inflammatory bowel disease, are described. As detection of C. difficile in clinical specimens does not always equate with disease, the diagnosis of C. difficile infection continues to be a challenge for both laboratories and clinicians. PMID:23824374
Han, Sang Bong; Chang, Jiyoung; Shin, Sang Hyun; Park, Kang Gyun; Lee, Gun Dong; Park, Yong Gyu; Park, Yeon-Joon
We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMérieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80℃, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.
Pituch, Hanna; Kreft, Deborah; Obuch-Woszczatyński, Piotr; Wultańska, Dorota; Meisel-Mikołajczyk, Felicja; Łuczak, Mirosław; van Belkum, Alex
Clinically relevant Clostridium difficile strains usually produce toxins A and B. Some C. difficile strains can produce an additional binary toxin. We report clonality among five strains carrying all toxin genes from Polish patients with C. difficile-associated diarrhea. In another strain, possible recombination between binary toxin genes is documented. PMID:15635019
Marsh, Jane W.; Schlackman, Jessica L.; Harrison, Lee H.
The prevalence of Clostridium difficile in retail meat samples has varied widely. The food supply may be a source for C. difficile infections. A total of 102 ground meat and sausage samples from 3 grocers in Pittsburgh, PA, were cultured for C. difficile. Brand A pork sausages were resampled between May 2011 and January 2012. Two out of 102 (2.0%) meat products initially sampled were positive for C. difficile; both were pork sausage from brand A from the same processing facility (facility A). On subsequent sampling of brand A products, 10/19 samples from processing facility A and 1/10 samples from 3 other facilities were positive for C. difficile. The isolates recovered were inferred ribotype 078, comprising 6 genotypes. The prevalence of C. difficile in retail meat may not be as high as previously reported in North America. When contamination occurs, it may be related to events at processing facilities. PMID:22504814
In the past decade Clostridium difficile has become a bacterial pathogen of global significance. Epidemic strains have spread throughout hospitals, while community acquired infections and other sources ensure a constant inoculation of spores into hospitals. In response to the increasing medical burden, a new C. difficile antibiotic, fidaxomicin, was approved in 2011 for the treatment of C. difficile-associated diarrhea. Rudimentary fecal transplants are also being trialed as effective treatments. Despite these advances, therapies that are more effective against C. difficile spores and less damaging to the resident gastrointestinal microbiome and that reduce recurrent disease are still desperately needed. However, bringing a new treatment for C. difficile infection to market involves particular challenges. This review covers the current drug discovery pipeline, including both small molecule and biologic therapies, and highlights the challenges associated with in vitro and in vivo models of C. difficile infection for drug screening and lead optimization. PMID:25760275
Diniz, Amanda Nádia; Silva, Rodrigo Otávio Silveira; Oliveira Junior, Carlos Augusto; Pierezan, Felipe; Lobato, Francisco Carlos Faria
The aim of this study was to report two cases of Clostridium perfringens type A and Clostridium difficile co-infection in adult dogs. Both animals were positive for A/B toxin. Toxigenic C. difficile and C. perfringens type A positive for NetE and NetF-encoding genes were isolated. This report reinforces the necessity of studying a possible synergism of C. difficile and C. perfringens in enteric disorders.
Semenyuk, Ekaterina G; Laning, Michelle L; Foley, Jennifer; Johnston, Pehga F; Knight, Katherine L; Gerding, Dale N; Driks, Adam
The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.
Péchiné, Séverine; Denève, Cécile; Le Monnier, Alban; Hoys, Sandra; Janoir, Claire; Collignon, Anne
Clostridium difficile is a pathogen responsible for diarrhoea and colitis, particularly after antibiotic treatment. We evaluated the C. difficile protease Cwp84, found to be associated with the S-layer proteins, as a vaccine antigen to limit the C. difficile intestinal colonization and therefore the development of the infection in a clindamycin-treated hamster model. First, we evaluated the immune response and the animal protection against death induced by several immunization routes: rectal, intragastric and subcutaneous. Antibody production was variable according to the immunization routes. In addition, serum Cwp84 antibody titres did not always correlate with animal protection after challenge with a toxigenic C. difficile strain. The best survival rate was observed with the rectal route of immunization. Then, in a second assay, we selected this immunization route to perform a larger immunization assay including a Cwp84 immunized group and a control group. Clostridium difficile intestinal colonization and survival rate, as well as the immune response were examined. Clostridium difficile hamster challenge resulted in a 26% weaker and slower C. difficile intestinal colonization in the immunized group. Furthermore, hamster survival in the Cwp84 immunized group was 33% greater than that of the control group, with a significant statistical difference.
Bibbò, Stefano; Lopetuso, Loris Riccardo; Ianiro, Gianluca; Di Rienzo, Teresa; Gasbarrini, Antonio
Recurrent Clostridium difficile infection represents a burdensome clinical issue whose epidemiology is increasing worldwide. The pathogenesis is not yet completely known. Recent observations suggest that the alteration of the intestinal microbiota and impaired innate immunity may play a leading role in the development of recurrent infection. Various factors can cause dysbiosis. The causes most involved in the process are antibiotics, NSAIDs, acid suppressing therapies, and age. Gut microbiota impairment can favor Clostridium difficile infection through several mechanisms, such as the alteration of fermentative metabolism (especially SCFAs), the alteration of bile acid metabolism, and the imbalance of antimicrobial substances production. These factors alter the intestinal homeostasis promoting the development of an ecological niche for Clostridium difficile and of the modulation of immune response. Moreover, the intestinal dysbiosis can promote a proinflammatory environment, whereas Clostridium difficile itself modulates the innate immunity through both toxin-dependent and toxin-independent mechanisms. In this narrative review, we discuss how the intestinal microbiota modifications and the modulation of innate immune response can lead to and exacerbate Clostridium difficile infection. PMID:24995345
Rodriguez, C; Korsak, N; Taminiau, B; Avesani, V; Van Broeck, J; Brach, P; Delmée, M; Daube, G
This study investigates the contamination of foods and surfaces with Clostridium difficile in a single nursing home. C. difficile PCR-ribotype 078 was found in one food sample and in none of the tested surfaces. These results indicate that food and surfaces are an unlikely source of C. difficile infection in this setting.
Fountain, Eric M; Moses, Maggie C; Park, Lawrence P; Woods, Christopher W; Arepally, Gowthami M
Clostridium difficile infection (CDI) is a common cause of nosocomial diarrhea and colitis. The incidence and prognostic significance of thrombocytopenia as related to mode of acquisition (hospital vs. community), NAP1/027 strain, and disease severity has not been examined. We performed a single-institution retrospective analysis of all adult inpatients from 2013 to 2014 diagnosed with CDI during their hospitalization to document the incidence/prevalence of thrombocytopenia and associated outcomes. Severe disease was defined by a composite endpoint of inpatient death, death within 30 days of discharge, presence of septic shock, or need for colectomy during hospitalization. Of the 533 patients diagnosed with CDI, moderate thrombocytopenia (platelet count <100 × 10(9)/L at time of CDI diagnosis) was present in 15 % of the total cohort and incident thrombocytopenia developed in 3 % of patients after admission. Thrombocytopenia was more common in hospital-acquired disease and associated with increased length of stay, but was not associated with treatment failure. Those with moderate thrombocytopenia were more likely to have severe disease, after controlling for white blood cell count, albumin, and creatinine. Moderate thrombocytopenia is associated with poor prognosis and is a potential risk stratification tool for severe CDI.
Yin, Jui-Chieh; Fei, Chun-Hsien; Lo, Yen-Chen; Hsiao, Yu-Yuan; Chang, Jyun-Cyuan; Nix, Jay C.; Chang, Yuan-Yu; Yang, Lee-Wei; Huang, I-Hsiu; Wang, Shuying
Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26–PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB. PMID:27921010
Drekonja, Dimitri M
Clostridium difficile infection (CDI) has increased in incidence and severity, and is now among the most common nosocomial infections. Several agents are available for the initial treatment of CDI, some of which are rarely used, and none of which is clearly superior for initial clinical cure. Fidaxomicin appears to offer a benefit in terms of preventing recurrent disease, although the cost-benefit ratio is debated. Recurrent CDI is a major challenge, occurring after 15-30% of initial episodes. The treatment of recurrent CDI is difficult, with sparse evidence available to support any particular agent. Fecal microbiota therapy, also known as 'stool transplantation', appears to be highly effective, although availability is currently limited, and the regulatory environment is in flux. Synthetic stool products and an orally available fecal microbiota therapy product are both under investigation, which may address the problem of availability. As with most infectious diseases, an effective vaccine would be a welcome addition to our armamentarium, but none is currently available.
Chan, Khee-Siang; Lee, Wen-Ying; Yu, Wen-Liang
Cytomegalovirus (CMV) colitis usually occurs in immunocompromised patients with human immunodeficiency virus infection, organ transplantation, and malignancy receiving chemotherapy or ulcerative colitis receiving immunosuppressive agents. However, CMV colitis is increasingly recognized in immunocompetent hosts. Notably, CMV colitis coexisting with Clostridium difficile infection (CDI) in apparently healthy individuals has been published in recent years, which could result in high morbidity and mortality. CMV colitis is a rare but possible differential diagnosis in immunocompetent patients with abdominal pain, watery, or especially bloody diarrhea, which could be refractory to standard treatment for CDI. As a characteristic of CDI, however, pseudomembranous colitis may be only caused by CMV infection. Real-time CMV-polymerase chain reaction (PCR) for blood and stool samples may be a useful and noninvasive diagnostic strategy to identify CMV infection when treatment of CDI eventually fails to show significant benefits. Quantitative CMV-PCR in mucosal biopsies may increase the diagnostic yield of traditional histopathology. CMV colitis is potentially life-threatening if severe complications occur, such as sepsis secondary to colitis, massive colorectal bleeding, toxic megacolon, and colonic perforation, so that may necessitate pre-emptive antiviral treatment for those who are positive for CMV-PCR in blood and/or stool samples while pending histological diagnosis.
Tauxe, William M.; Haydek, John P.; Rebolledo, Paulina A.; Neish, Emma; Newman, Kira L.; Ward, Angela; Dhere, Tanvi; Kraft, Colleen S.
Background: The objective of this study was to describe the safety of fecal microbiota transplant (FMT) for Clostridium difficile infection (CDI) among older adults. Methods: We performed a case review of all FMT recipients aged 65 or older treated at Emory University Hospital, a tertiary care and referral center for Georgia and surrounding states. Results: CDI resolved in 27 (87%) of 31 respondents, including three individuals who received multiple FMTs. Among four whose CDI was not resolved at follow up, three respondents did well initially before CDI recurred, and one individual never eradicated his CDI despite repeating FMT. During the study, five deaths and eight serious adverse events requiring hospitalization were reported within the study group during the follow-up period. Fecal transplant was not a causative factor in these events. The most common adverse event reported in 4 (13%) of 31 respondents was subjective worsening of arthritis. Conclusion: FMT is a generally safe and effective treatment option for older adults with CDI. PMID:27134658
Khanafer, Nagham; Oltra, Luc; Hulin, Monique; Dauwalder, Olivier; Vandenesch, Francois; Vanhems, Philippe
Abstract The epidemiology of Clostridium difficile infection (CDI) has changed with an increase in incidence and severity. Prospective surveillance was therefore implemented in a French university hospital to monitor the characteristics of patients at risk and to recognize local trends. Between 2007 and 2014, all hospitalized patients (≥18 years) with CDI were included. During the survey, the mean incidence rate of CDI was 2.9 per 10,000 hospital-days. In all, 590 patients were included. Most of the episodes were healthcare-associated (76.1%). The remaining cases were community-acquired (18.1%) and unknown (5.9%). The comparison with healthcare-associated cases showed that the community-acquired group had a lower rate of antimicrobial exposure (P < 0.001), proton pump inhibitor (P < 0.001), and immunosuppressive drugs (P = 0.02). Over the study period, death occurred in 61 patients (10.3%), with 18 (29.5%) being related to CDI according to the physician in charge of the patient. Active surveillance of CDI is required to obtain an accurate picture of the real dimensions of CDI. PMID:27281101
Guerrero-Araya, Enzo; Plaza-Garrido, Angela; Díaz-Yañez, Fernando; Pizaro-Guajardo, Marjorie; Valenzuela, Sandro L.; Meneses, Claudio; Gil, Fernando
Clostridium paraputrificum is a gut microbiota member reported in several cases of bacteremia and coinfections. So far, only one genome sequence of a C. paraputrificum (AGR2156) isolate is available. Here, we present the draft genome of C. paraputrificum strain 373-A1, isolated from stools from a patient with C. difficile infection. PMID:27811092
Guerrero-Araya, Enzo; Plaza-Garrido, Angela; Díaz-Yañez, Fernando; Pizaro-Guajardo, Marjorie; Valenzuela, Sandro L; Meneses, Claudio; Gil, Fernando; Castro-Nallar, Eduardo; Paredes-Sabja, Daniel
Clostridium paraputrificum is a gut microbiota member reported in several cases of bacteremia and coinfections. So far, only one genome sequence of a C. paraputrificum (AGR2156) isolate is available. Here, we present the draft genome of C. paraputrificum strain 373-A1, isolated from stools from a patient with C. difficile infection.
Sartelli, Massimo; Malangoni, Mark A; Abu-Zidan, Fikri M; Griffiths, Ewen A; Di Bella, Stefano; McFarland, Lynne V; Eltringham, Ian; Shelat, Vishal G; Velmahos, George C; Kelly, Ciarán P; Khanna, Sahil; Abdelsattar, Zaid M; Alrahmani, Layan; Ansaloni, Luca; Augustin, Goran; Bala, Miklosh; Barbut, Frédéric; Ben-Ishay, Offir; Bhangu, Aneel; Biffl, Walter L; Brecher, Stephen M; Camacho-Ortiz, Adrián; Caínzos, Miguel A; Canterbury, Laura A; Catena, Fausto; Chan, Shirley; Cherry-Bukowiec, Jill R; Clanton, Jesse; Coccolini, Federico; Cocuz, Maria Elena; Coimbra, Raul; Cook, Charles H; Cui, Yunfeng; Czepiel, Jacek; Das, Koray; Demetrashvili, Zaza; Di Carlo, Isidoro; Di Saverio, Salomone; Dumitru, Irina Magdalena; Eckert, Catherine; Eckmann, Christian; Eiland, Edward H; Enani, Mushira Abdulaziz; Faro, Mario; Ferrada, Paula; Forrester, Joseph Derek; Fraga, Gustavo P; Frossard, Jean Louis; Galeiras, Rita; Ghnnam, Wagih; Gomes, Carlos Augusto; Gorrepati, Venkata; Ahmed, Mohamed Hassan; Herzog, Torsten; Humphrey, Felicia; Kim, Jae Il; Isik, Arda; Ivatury, Rao; Lee, Yeong Yeh; Juang, Paul; Furuya-Kanamori, Luis; Karamarkovic, Aleksandar; Kim, Peter K; Kluger, Yoram; Ko, Wen Chien; LaBarbera, Francis D; Lee, Jae Gil; Leppaniemi, Ari; Lohsiriwat, Varut; Marwah, Sanjay; Mazuski, John E; Metan, Gokhan; Moore, Ernest E; Moore, Frederick Alan; Nord, Carl Erik; Ordoñez, Carlos A; Júnior, Gerson Alves Pereira; Petrosillo, Nicola; Portela, Francisco; Puri, Basant K; Ray, Arnab; Raza, Mansoor; Rems, Miran; Sakakushev, Boris E; Sganga, Gabriele; Spigaglia, Patrizia; Stewart, David B; Tattevin, Pierre; Timsit, Jean Francois; To, Kathleen B; Tranà, Cristian; Uhl, Waldemar; Urbánek, Libor; van Goor, Harry; Vassallo, Angela; Zahar, Jean Ralph; Caproli, Emanuele; Viale, Pierluigi
In the last two decades there have been dramatic changes in the epidemiology of Clostridium difficile infection (CDI), with increases in incidence and severity of disease in many countries worldwide. The incidence of CDI has also increased in surgical patients. Optimization of management of C difficile, has therefore become increasingly urgent. An international multidisciplinary panel of experts prepared evidenced-based World Society of Emergency Surgery (WSES) guidelines for management of CDI in surgical patients.
Arvand, Mardjan; Bettge-Weller, Gudrun
Clostridium difficile-isolates associated with CDI in different healthcare facilities in Hesse were analysed. The most common ribotypes were 001 (31.1%) and 027 (27.0%). The proportion of ribotype 027 among regional C. difficile-isolates was 10.8% in North Hesse, 17.2% in Middle Hesse, and 33.5% in the Rhine-Main Metropolitan Area. In the latter region, ribotype 027 was the most prevalent ribotype.
Varshney, Jyotika B; Very, Katherine J; Williams, Jen L; Hegarty, John P; Stewart, David B; Lumadue, Jeanne; Venkitanarayanan, Kumar; Jayarao, Bhushan M
A study was conducted to determine the prevalence of Clostridium difficile and characterize C. difficile isolates from human stool and retail grocery meat samples. Human stool samples (n=317) were obtained from a clinical laboratory and meat samples (n=303) were collected from 8 retail grocery stores from October 2011 through September 2012 from Centre County of Pennsylvania and were examined for C. difficile. C. difficile was isolated from 16.7% of stool samples (n=317) and 6.9%, 11.5%, 14.5%, and 7.8% of beef (n=72), pork (n=78), turkey (n=76), and chicken (n=77) samples, respectively. Six different toxin gene profiles were detected in all human and meat isolates of C. difficile based on the presence or absence of toxin genes tcdA, tcdB, and cdtA and cdtB. Interestingly, 75.6% of the human C. difficile isolates lacked any deletion in the tcdC gene (139-bp), whereas a 39-bp deletion was observed in 61.3% of the C. difficile strains isolated from meat samples. C. difficile from meat samples were more susceptible to clindamycin, moxifloxacin, vancomycin, and metronidazole than C. difficile isolates from human samples. Twenty-five different ribotypes were identified in human and meat C. difficile isolates. In conclusion, significant genotypic and phenotypic differences were observed between human and meat isolates of C. difficile; however, a few C. difficile isolates from meat-in particular ribotypes 078, PA01, PA05, PA16, and PA22 with unique profiles (toxin gene, tcdC gene size and antimicrobial resistance profiles)-were similar to human C. difficile isolates.
Shan, Jinyu; Patel, Krusha V; Hickenbotham, Peter T; Nale, Janet Y; Hargreaves, Katherine R; Clokie, Martha R J
Prophages are encoded in most genomes of sequenced Clostridium difficile strains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity of C. difficile prophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific to C. difficile myoviruses and siphoviruses. Subsequent PCR analysis of 16 clinical C. difficile ribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups of C. difficile phages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, including Clostridium perfringens, Clostridium botulinum, and Bacillus spp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains of C. difficile and correlates the molecular data to the morphological observation.
Shan, Jinyu; Patel, Krusha V.; Hickenbotham, Peter T.; Nale, Janet Y.; Hargreaves, Katherine R.
Prophages are encoded in most genomes of sequenced Clostridium difficile strains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity of C. difficile prophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific to C. difficile myoviruses and siphoviruses. Subsequent PCR analysis of 16 clinical C. difficile ribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups of C. difficile phages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, including Clostridium perfringens, Clostridium botulinum, and Bacillus spp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains of C. difficile and correlates the molecular data to the morphological observation. PMID:22706062
El Feghaly, Rana E; Stauber, Jennifer L; Tarr, Phillip I; Haslam, David B
Clostridium difficile infections in children are increasing. In this cohort study, we enrolled 62 children with diarrhea and C difficile. We performed polymerase chain reaction assays to detect viral agents of gastroenteritis and quantify C difficile burden. Fifteen (24%) children diagnosed as having C difficile infection had a concomitant viral co-infection. These patients tended to be younger and had a higher C difficile bacterial burden than children with no viral co-infections (median difference = 565,957 cfu/mL; P = 0.011), but were clinically indistinguishable. The contribution of viral co-infection to C difficile disease in children warrants future investigation.
Polage, Christopher R; Solnick, Jay V; Cohen, Stuart H
Diarrhea is common among hospitalized patients but the causes are distinct from those of diarrhea in the community. We review existing data about the epidemiology of nosocomial diarrhea and summarize recent progress in understanding the mechanisms of diarrhea. Clinicians should recognize that most cases of nosocomial diarrhea have a noninfectious etiology, including medications, underlying illness, and enteral feeding. Apart from Clostridium difficile, the frequency of infectious causes such as norovirus and toxigenic strains of Clostridium perfringens, Klebsiella oxytoca, Staphylococcus aureus, and Bacteroides fragilis remains largely undefined and test availability is limited. Here we provide a practical approach to the evaluation and management of nosocomial diarrhea when tests for C. difficile are negative.
Khoshdel, Abolfazl; Habibian, Roya; Parvin, Neda; Doosti, Abbas; Famouri, Fatemeh; Eshraghi, Ali; Hafizi, Massoud
Clostridium difficile is recognized as a major cause of nosocomial acquired antibiotic-associated diarrhea and pseudomembranous colitis. It is a significant financial burden on modern healthcare resources. This study aimed to assess the molecular characterization of C. difficile strains isolated from children under 5 years old suffered from nosocomial diarrhea. One hundred diarrheic and 130 non-diarrheic fecal samples were collected from pediatrics less than 5 years old. Samples were cultured and C. difficile isolates were subjected to the PCR technique to study the distribution of ribotypes of C. difficile using P3 and P5 primers. Fifty-two out of 100 samples (52 %) were positive for C. difficile. The prevalence of bacterium in healthy children was 4.61 %. Total prevalence of C. difficile in diarrheic girls and boys were 48.9 and 54.7 %, respectively. Thirteen to twenty-four month age children had the highest prevalence of C. difficile. The most commonly detected ribotypes in the C. difficile isolates of Iranian pediatrics were RT027 (11.52 %), R1 (9.61 %) and R13 (7.68 %). The ribotypes of all of the six bacterial isolates of healthy children was not diagnosed. According to the presence of C. difficile and R27 ribotype, a continued genotype surveillance of this bacterium is necessary to monitor changes in the prevalence of certain strains and to identify the emergence of new strains that could affect future vaccine strategies.
Stone, Nathan E.; Sidak-Loftis, Lindsay C.; Sahl, Jason W.; Vazquez, Adam J.; Wiggins, Kristin B.; Gillece, John D.; Hicks, Nathan D.; Schupp, James M.; Busch, Joseph D.; Keim, Paul; Wagner, David M.
Nosocomial acquisition of Clostridium difficile is well documented, yet recent studies have highlighted the importance of community acquired infections and identified community associated reservoirs for this pathogen. Multiple studies have implicated companion pets and farm animals as possible sources of community acquired C. difficile infections in humans. To explore the potential role of pet dogs in human C. difficile infections we systematically collected canine fecal samples (n = 197) in Flagstaff, AZ. Additionally, nineteen fecal samples were collected at a local veterinary clinic from diarrheic dogs. We used these combined samples to investigate important questions regarding C. difficile colonization in pet canines: 1) What is the prevalence and diversity of C. difficile in this companion pet population, and 2) Do C. difficile isolates collected from canines genetically overlap with isolates that cause disease in humans? We used a two-step sequence typing approach, including multilocus sequence typing to determine the overall genetic diversity of C. difficile present in Flagstaff canines, and whole-genome sequencing to assess the fine-scale diversity patterns within identical multilocus sequence types from isolates obtained within and among multiple canine hosts. We detected C. difficile in 17% of the canine fecal samples with 10% containing toxigenic strains that are known to cause human disease. Sequencing analyses revealed similar genotypes in dogs and humans. These findings suggest that companion pets are a potential source of community acquired C. difficile infections in humans. PMID:27723795
Vyas, Dinesh; L'esperance, Heidi E; Vyas, Arpita
Fecal enemas were first reported to successfully treat life threatening enterocolitis in 1958, but fecal therapy to treat Clostridium difficile (C. difficile) infection has remained esoteric and not well investigated until recently. In the past few years, systematic reviews of case series and case reports of fecal microbiota transplant for recurrent C. difficile infection have become available and validate use of fecal transplant for C. difficile enterocolitis. Methods of fecal transplant reported in the literature include: nasogastric tube, gastroscope, duodenal tube, colonoscopy, rectal tube, and fecal enemas administered at home; no method has been shown to be superior. A recent randomized study published in New England Journal of Medicine found fecal transplant to be superior to oral vancomycin alone in treatment of recurrent C. difficile enterocolitis. The significance of this trial cannot be underestimated as it lends credibility to the idea of intentionally using microbes to combat disease, providing an alternative to the older paradigm of disease eradication through use of antimicrobials.
Arruda, Paulo H. E.; Madson, Darin M.; Ramirez, Alejandro; Rowe, Eric W.; Songer, J. Glenn
Although Clostridium difficile infection (CDI) is a common disease in swine, there is a lack of prevention strategies. The objectives of this study were to evaluate: i) the effectiveness of Lactobacillus spp. and ii) non-toxigenic C. difficile (NTCD) as prevention for the development of CDI in piglets. Cesarean-derived piglets (N = 150) were randomly assigned to 6 groups: GROUP 1 — negative control (n = 10); GROUP 2 — NTCD only (n = 13); GROUP 3 — Lactobacillus spp. only (n = 14); GROUP 4 — positive control (challenged with toxigenic C. difficile strain) (n = 35); GROUP 5 — NTCD and challenged with the toxigenic C. difficile strain (n = 34); and GROUP 6 — Lactobacillus spp. and challenged with the toxigenic C. difficile strain (n = 44). Piglets which received NTCD showed lower prevalence of toxin-positive feces, mesocolonic edema, and microscopic lesions compared with positive control piglets. Administration of Lactobacillus spp. did not reveal clear benefits. PMID:26834271
Broecker, Felix; Martin, Christopher E; Wegner, Erik; Mattner, Jochen; Baek, Ju Yuel; Pereira, Claney L; Anish, Chakkumkal; Seeberger, Peter H
Infections with Clostridium difficile increasingly cause morbidity and mortality worldwide. Bacterial surface glycans including lipoteichoic acid (LTA) were identified as auspicious vaccine antigens to prevent colonization. Here, we report on the potential of synthetic LTA glycans as vaccine candidates. We identified LTA-specific antibodies in the blood of C. difficile patients. Therefore, we evaluated the immunogenicity of a semi-synthetic LTA-CRM197 glycoconjugate. The conjugate elicited LTA-specific antibodies in mice that recognized natural LTA epitopes on the surface of C. difficile bacteria and inhibited intestinal colonization of C. difficile in mice in vivo. Our findings underscore the promise of synthetic LTA glycans as C. difficile vaccine candidates.
Arruda, Paulo H E; Madson, Darin M; Ramirez, Alejandro; Rowe, Eric W; Songer, J Glenn
Although Clostridium difficile infection (CDI) is a common disease in swine, there is a lack of prevention strategies. The objectives of this study were to evaluate: i) the effectiveness of Lactobacillus spp. and ii) non-toxigenic C. difficile (NTCD) as prevention for the development of CDI in piglets. Cesarean-derived piglets (N = 150) were randomly assigned to 6 groups: GROUP 1 - negative control (n = 10); GROUP 2 - NTCD only (n = 13); GROUP 3 - Lactobacillus spp. only (n = 14); GROUP 4 - positive control (challenged with toxigenic C. difficile strain) (n = 35); GROUP 5 - NTCD and challenged with the toxigenic C. difficile strain (n = 34); and GROUP 6 - Lactobacillus spp. and challenged with the toxigenic C. difficile strain (n = 44). Piglets which received NTCD showed lower prevalence of toxin-positive feces, mesocolonic edema, and microscopic lesions compared with positive control piglets. Administration of Lactobacillus spp. did not reveal clear benefits.
Rodriguez, Cristina; Warszawski, Nathalie; Korsak, Nicolas; Taminiau, Bernard; Van Broeck, Johan; Delmée, Michel; Daube, Georges
Despite increasing interest in the bacterium, the methodology for Clostridium difficile recovery has not yet been standardized. Cycloserine-cefoxitin fructose taurocholate (CCFT) has historically been the most used medium for C. difficile isolation from human, animal, environmental, and food samples, and presumptive identification is usually based on colony morphologies. However, CCFT is not totally selective. This study describes the recovery of 24 bacteria species belonging to 10 different genera other than C. difficile, present in the environment and foods of a retirement establishment that were not inhibited in the C. difficile selective medium. These findings provide insight for further environmental and food studies as well as for the isolation of C. difficile on supplemented CCFT.
Spivack, Jordan G; Eppes, Stephen C; Klein, Joel D
This retrospective cohort analysis examined the risk factors, symptoms, and severity of disease associated with C. difficile in pediatric inpatients. Risk factors for a C. difficile-positive test were an oncologic diagnosis, diarrhea of more than 2 days' duration, and gastrointestinal symptoms, especially abdominal pain. Over a 3.5-year period, there was a total of 22 C. difficile-positive patients, and most had mild, self-limiting diarrheal illness. No cases of C. difficile diarrhea were identified. Seventy-eight percent of the C. difficile-positive patients were found to have alternate risk factors for diarrhea. Our data indicate that C. difficile rarely causes severe diarrhea in pediatric inpatients and that C. difficile testing should be limited to patients with severe prolonged diarrhea and abdominal pain.
Moono, Peter; Putsathit, Papanin; Knight, Daniel R; Squire, Michele M; Hampson, David J; Foster, Niki F; Riley, Thomas V
Clostridium difficile is commonly associated with healthcare-related infections in humans, and is an emerging pathogen in food animal species. There is potential for transmission of C. difficile from animals or animal products to humans. This study aimed to determine if C. difficile RT 237 had persisted in a Western Australian piggery or if there had been a temporal change in C. difficile diversity. C. difficile carriage in litters with and without diarrhea was investigated, as was the acquisition of C. difficile over time using cohort surveys. Rectal swabs were obtained from piglets aged 1-10 days to determine prevalence of C. difficile carriage and samples were obtained from 20 piglets on days 1, 7, 13, 20, and 42 of life to determine duration of shedding. Isolation of C. difficile from feces was achieved by selective enrichment culture. All isolates were characterized by standard molecular typing. Antimicrobial susceptibility testing was performed on selected isolates (n = 29). Diarrheic piglets were more likely to shed C. difficile than the non-diseased (p = 0.0124, χ2). In the cohort study, C. difficile was isolated from 40% samples on day 1, 50% on day 7, 20% on day 13, and 0% on days 20 and 42. All isolates were RT 237 and no antimicrobial resistance was detected. The decline of shedding of C. difficile to zero has public health implications because slaughter age pigs have a low likelihood of spreading C. difficile to consumers via pig meat.
Ray, Arnab; Smith, Robert; Breaux, Jacob
Background Clostridium difficile infection (CDI) accounts for 20%-30% of cases of antibiotic-associated diarrhea and is the most commonly recognized cause of infectious diarrhea in healthcare settings. The incidence of CDI is rising, while the effectiveness of antibiotics for treatment decreases with recurrent episodes. The use of fecal microbiota transplantation (FMT) for cure of CDI has been reported since 1958, and the worldwide cure rate is reported to be 93%. We report our experience with FMT for the treatment of CDI. Methods We performed a retrospective chart review of patients undergoing FMT for CDI at Ochsner Clinic Foundation from August 2012 to November 2013. FMT was administered via colonoscopy for patients with recurrent or severe CDI. Stool donors were screened for infections in the majority of cases. Results FMT was performed in 20 CDI patients. The 16 female and 4 male patients ranged in age from 27 to 89 years (mean 62 years). The average duration of illness from diagnosis to treatment was 49.6 weeks, based on available data. Only 3 donors were unscreened for infectious pathogens. Nine donors were related to the recipients by blood; most of the other donors were spouses. The average length of follow-up after FMT was 3 months. No recurrences of CDI after treatment have been documented. Adverse events reported after treatment included abdominal cramping, bloating, flatulence, and nausea that resolved. Conclusion Although the US Food and Drug Administration currently considers FMT an experimental therapy, we demonstrate that FMT is safe, well tolerated, and effective for recurrent and severe CDI. PMID:25598718
Norman, K N; Harvey, R B; Scott, H M; Hume, M E; Andrews, K; Brawley, A D
The objectives of this study were to compare the prevalence of Clostridium difficile (Cd) among different age and production groups of swine in a vertically integrated swine operation in Texas in 2006 and to compare our isolates to other animal and human isolates. Results are based on 131 Cd isolates from 1008 swine fecal samples and pork trim samples (overall prevalence of 13%). The prevalence (number positive/number tested in production type) of Cd was different between the groups (P
Permpoonpattana, Patima; Phetcharaburanin, Jutarop; Mikelsone, Anna; Dembek, Marcin; Tan, Sisareuth; Brisson, Marie-Clémence; La Ragione, Roberto; Brisson, Alain R; Fairweather, Neil; Hong, Huynh A; Cutting, Simon M
Spores of Clostridium difficile play a key role in the dissemination of this important human pathogen, and until recently little has been known of their functional characteristics. Genes encoding six spore coat proteins (cotA, cotB, cotCB, cotD, cotE, and sodA) were disrupted by ClosTron insertional mutagenesis. Mutation of one gene, cotA, presented a major structural defect in spore assembly, with a clear misassembly of the outermost layers of the spore coat. The CotA protein is most probably subject to posttranslational modification and could play a key role in stabilizing the spore coat. Surprisingly, mutation of the other spore coat genes did not affect the integrity of the spore, although for the cotD, cotE, and sodA mutants, enzyme activity was reduced or abolished. This could imply that these enzymatic proteins are located in the exosporium or alternatively that they are structurally redundant. Of the spore coat proteins predicted to carry enzymatic activity, three were confirmed to be enzymes using both in vivo and in vitro methods, the latter using recombinant expressed proteins. These were a manganese catalase, encoded by cotD, a superoxide dismutase (SOD), encoded by sodA, and a bifunctional enzyme with peroxiredoxin and chitinase activity, encoded by cotE. These enzymes being exposed on the spore surface would play a role in coat polymerization and detoxification of H2O2. Two additional proteins, CotF (a tyrosine-rich protein and potential substrate for SodA) and CotG (a putative manganese catalase) were shown to be located at the spore surface.
Hübner, Claudia; Hübner, Nils-Olaf; Muhr, Michaela; Claus, Franziska; Leesch, Henning; Kramer, Axel; Flessa, Steffen
Zielsetzung: Die Clostridium-difficile-assoziierte Diarrhö (CDAD) bedingt hohe finanzielle Belastungen für Gesundheitssysteme weltweit. Wie bei allen nosokomial erworbenen Infektionen ist ein verlängerter Krankenhausaufenthalt der wesentliche Kostentreiber. Bisherige Kostenstudien beziehen sich nur auf Krankenhausabrechnungsdaten vor Einführung des DRG-Entgeltsystems und den Vergleich von Verweildauer zu nicht infizierten Patienten. Eine Erhebung tatsächlicher Kosten steht bislang aus. Methode: Anhand einer retrospektiven Analyse wurden Daten der Universitätsmedizin Greifswald von Patienten mit einer stationär behandelten CDAD über einen 1-Jahres-Zeitraum ausgewertet. Über eine Identifizierung von CDAD-relevanten Behandlungsprozessen wurden die Kosten von Hygienemaßnahmen, Arzneimittel und Labor sowie Erlösausfälle bedingt durch Bettensperrungen und Verweildauerverlängerungen berechnet. Ergebnisse: 19 Patienten wurden in die Analyse eingeschlossen. Im Durchschnitt fallen pro CDADPatient zusätzliche Gesamtkosten in Höhe von 5.262,96 € an. Erlösausfälle aufgrund der verlängerten Verweildauer stellen mit 2.555,59 € pro Fall den höchsten Anteil dar, gefolgt von den Erlösausfällen aufgrund von Bettensperrungen während der Isolierung mit 2.413,08 € pro Fall. Insgesamt ergeben diese Opportunitätskosten einen Anteil von 94,41% an den Gesamtkosten. Die Kosten für Hygienemaßnahmen (253,98 €), Arzneimittel (22,88 €) und Labor (17,44 €) sind dem gegenüber gering.Schlussfolgerung: Die CDAD führt zu deutlichen Mehrkosten für das Krankenhaus. Unsere Erhebung der tatsächlichen Kosten bestätigt bisherige Studienergebnisse.
Zilberberg, Marya D.; Shorr, Andrew F.; Jesdale, William M.; Tjia, Jennifer; Lapane, Kate
Abstract We explored the epidemiology and outcomes of Clostridium difficile infection (CDI) recurrence among Medicare patients in a nursing home (NH) whose CDI originated in acute care hospitals. We conducted a retrospective, population-based matched cohort combining Medicare claims with Minimum Data Set 3.0, including all hospitalized patients age ≥65 years transferred to an NH after hospitalization with CDI 1/2011-11/2012. Incident CDI was defined as ICD-9-CM code 008.45 with no others in prior 60 days. CDI recurrence was defined as (within 60 days of last day of CDI treatment): oral metronidazole, oral vancomycin, or fidaxomicin for ≥3 days in part D file; or an ICD-9-CM code for CDI (008.45) during a rehospitalization. Cox proportional hazards and linear models, adjusted for age, gender, race, and comorbidities, examined mortality within 60 days and excess hospital days and costs, in patients with recurrent CDI compared to those without. Among 14,472 survivors of index CDI hospitalization discharged to an NH, 4775 suffered a recurrence. Demographics and clinical characteristics at baseline were similar, as was the risk of death (24.2% with vs 24.4% without). Median number of hospitalizations was 2 (IQR 1–3) among those with and 0 (IQR 0–1) among those without recurrence. Adjusted excess hospital days per patient were 20.3 (95% CI 19.1–21.4) and Medicare reimbursements $12,043 (95% CI $11,469–$12,617) in the group with a recurrence. Although recurrent CDI did not increase the risk of death, it was associated with a far higher risk of rehospitalization, excess hospital days, and costs to Medicare. PMID:28272217
Gerding, D. N.; Hecht, D. W.; Louie, T.; Nord, C. E.; Talbot, G. H.; Cornely, O. A.; Buitrago, M.; Best, E.; Sambol, S.; Osmolski, J. R.; Kracker, H.; Locher, H. H.; Charef, P.; Wilcox, M.
Objectives The aim of this study was to evaluate the susceptibilities of Clostridium difficile isolates to cadazolid, a novel antibiotic for the treatment of C. difficile infection. Methods Ribotyping and susceptibilities were determined for C. difficile isolates from a multicentre, double-blind, Phase 2 study of oral cadazolid in patients with C. difficile infection (NCT01222702, ClinicalTrials.gov; EudraCT 2010-020941-29, European Clinical Trials Database). Patients were randomized to receive 250, 500 or 1000 mg of cadazolid twice daily or 125 mg of vancomycin four times daily, for 10 days. MICs of cadazolid, vancomycin, fidaxomicin, linezolid and moxifloxacin were determined at baseline for all patients and post-baseline for patients with clinical failure or recurrence, using the agar dilution method. Results Seventy-eight of 84 patients had an evaluable toxigenic C. difficile isolate at baseline. The most frequent PCR ribotype was 027 (15.4%). Cadazolid MICs for baseline isolates (including epidemic strain 027) ranged from 0.06 to 0.25 mg/L. Baseline cadazolid MICs were similar to those of fidaxomicin and lower than those of vancomycin, linezolid and moxifloxacin. For each clinical outcome group (clinical cure, clinical failure, sustained clinical response and clinical failure or recurrence), the baseline cadazolid MIC range was 0.06–0.25 mg/L. Mean (min–max) cadazolid faecal concentration (μg/g) on day 5 was 884 (101–2710), 1706 (204–4230) and 3226 (1481–12 600) for the doses 250, 500 and 1000 mg, respectively. Conclusions For all cadazolid doses, the faecal concentration was in excess of several thousand-fold the MIC90 for C. difficile. The MIC of cadazolid for all C. difficile isolates, including epidemic strains, was low and in the same narrow range regardless of treatment outcome. PMID:26433782
Roberts, April; McGlashan, Joanna; Al-Abdulla, Ibrahim; Ling, Roger; Denton, Harriet; Green, Steve; Coxon, Ruth; Landon, John
Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed. PMID:22144483
O'Horo, John; Safdar, Nasia
Clostridium difficile is the most common infectious cause of nosocomial healthcare-associated diarrhea. The increasing prevalence of C difficile, spread in the community, virulence and frequent relapse has created an urgent need to identify new effective treatments for C. difficile infection. Among these, intravenous immunoglobulin (IVIG) is used for cases of severe C. difficile infection. We undertook a systematic review to examine the published literature pertaining to the use of immunoglobulin for C. difficile infection. Four retrospective studies and five case reports that addressed the use of IVIG for the treatment of C. difficile infection were identified. One study on the use of oral immunoglobulin was identified. Although overall there appear to be benefits to using IVIG in recurrent severe disease, the small sample sizes and lack of control groups in three of the four studies do not allow recommendations to be made regarding the use of immunoglobulin in C. difficile infection. Further research is urgently needed to clarify the role of immunoglobulin--intravenous or oral--for the treatment of C. difficile infection.
Mehta, Krunal K.; Paskaleva, Elena E.; Wu, Xia; Grover, Navdeep; Mundra, Ruchir V.; Chen, Kevin; Zhang, Yongrong; Yang, Zhiyong; Feng, Hanping; Dordick, Jonathan S.; Kane, Ravi S.
Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. PMID:27260850
Mooyottu, Shankumar; Kollanoor-Johny, Anup; Flock, Genevieve; Bouillaut, Laurent; Upadhyay, Abhinav; Sonenshein, Abraham L.; Venkitanarayanan, Kumar
Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo. PMID:24625665
Mooyottu, Shankumar; Kollanoor-Johny, Anup; Flock, Genevieve; Bouillaut, Laurent; Upadhyay, Abhinav; Sonenshein, Abraham L; Venkitanarayanan, Kumar
Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo.
Moono, Peter; Lim, Su Chen; Riley, Thomas V.
Clostridium difficile is a well-established hospital pathogen. Recently, it has been detected increasingly in patients without hospital contact. Given this rise in community associated infections with C. difficile, we hypothesized that the environment could play an important role in transmission of spores outside the hospital. Lawn samples (311) collected in public spaces in the metropolitan area of Perth, Western Australia, from February to June 2016 were cultured for C. difficile. C. difficile was isolated from the samples by direct and enrichment culture, and characterized by standard molecular methods using toxin gene PCR and ribotyping. The overall prevalence of C. difficile was 59%, new lawn (≤4 months old) was twice as likely as old lawn (>4 months old) to test positive (OR = 2.3; 95%CI 1.16–4.57, p = 0.015) and 35 C. difficile ribotypes were identified with toxigenic ribotype 014/020 (39%) predominating. The highest viable count from lawn soil samples was 1200 CFU/g. These results show that lawns in Perth, Western Australia, harbor toxigenic C. difficile, an important finding. The source of lawn contamination is likely related to modern practice of producing “roll-out” lawn. Further work should focus on identifying specific management practices that lead to C. difficile contamination of lawn to inform prevention and control measures. PMID:28145453
Bandelj, Petra; Blagus, Rok; Briski, France; Frlic, Olga; Vergles Rataj, Aleksandra; Rupnik, Maja; Ocepek, Matjaz; Vengust, Modest
Farm animals have been suggested to play an important role in the epidemiology of Clostridium difficile infection (CDI) in the community. The purpose of this study was to evaluate risk factors associated with C. difficile dissemination in family dairy farms, which are the most common farming model in the European Union. Environmental samples and fecal samples from cows and calves were collected repeatedly over a 1 year period on 20 mid-size family dairy farms. Clostridium difficile was detected in cattle feces on all farms using qPCR. The average prevalence between farms was 10% (0-44.4%) and 35.7% (3.7-66.7%) in cows and calves, respectively. Bacterial culture yielded 103 C. difficile isolates from cattle and 61 from the environment. Most C. difficile isolates were PCR-ribotype 033. A univariate mixed effect model analysis of risk factors associated dietary changes with increasing C. difficile prevalence in cows (P = 0.0004); and dietary changes (P = 0.004), breeding Simmental cattle (P = 0.001), mastitis (P = 0.003) and antibiotic treatment (P = 0.003) in calves. Multivariate analysis of risk factors found that dietary changes in cows (P = 0.0001) and calves (P = 0.002) increase C. difficile prevalence; mastitis was identified as a risk factor in calves (P = 0.001). This study shows that C. difficile is common on dairy farms and that shedding is more influenced by farm management than environmental factors. Based on molecular typing of C. difficile isolates, it could also be concluded that family dairy farms are currently not contributing to increased CDI incidence.
Reske, Kimberly A.; Seiler, Sondra; Hink, Tiffany; Kwon, Jennie H.
Asymptomatic colonization may contribute to Clostridium difficile transmission. Few data identify which patients are at risk for colonization. We performed a prospective cohort study of C. difficile colonization and risk factors for C. difficile acquisition and loss in hospitalized patients. Patients admitted to medical or surgical wards at a tertiary care hospital were enrolled; interviews and chart review were performed to determine patient demographics, C. difficile infection (CDI) history, medications, and health care exposures. Stool samples/rectal swabs were collected at enrollment and discharge; stool samples from clinical laboratory tests were also included. Samples were cultured for C. difficile, and the isolates were tested for toxins A and B and ribotyped. Chi-square tests and univariate logistic regression were used for the analyses. Two hundred thirty-five patients were enrolled. Of the patients, 21% were colonized with C. difficile (toxigenic and nontoxigenic) at admission and 24% at discharge. Ribotype 027 accounted for 6% of the strains at admission and 12% at discharge. Of the patients colonized at admission, 78% were also colonized at discharge. Cephalosporin use was associated with C. difficile acquisition (47% of patients who acquired C. difficile versus 25% of patients who did not; P = 0.03). β-lactam–β-lactamase inhibitor combinations were associated with a loss of C. difficile colonization (36% of patients who lost C. difficile colonization versus 8% of patients colonized at both admission and discharge; P = 0.04), as was metronidazole (27% versus 3%; P = 0.03). Antibiotic use affects the epidemiology of asymptomatic C. difficile colonization, including acquisition and loss, and it requires additional study. PMID:25987626
Waslawski, Sheila; Lo, Eugene S; Ewing, Sarah A; Young, Vincent B; Aronoff, David M; Sharp, Susan E; Novak-Weekley, Susan M; Crist, Arthur E; Dunne, W Michael; Hoppe-Bauer, Joan; Johnson, Michelle; Brecher, Stephen M; Newton, Duane W; Walk, Seth T
Capillary-based PCR ribotyping was used to quantify the presence/absence and relative abundance of 98 Clostridium difficile ribotypes from clinical cases of disease at health care institutions in six states of the United States. Regionally important ribotypes were identified, and institutions in close proximity did not necessarily share more ribotype diversity than institutions that were farther apart.
Banach, David B; Francois, Jeannette; Blash, Stephanie; Patel, Gopi; Jenkins, Stephen G; LaBombardi, Vincent; Kreiswirth, Barry N; Srinivasan, Arjun; Calfee, David P
Active surveillance to identify asymptomatic carriers of carbapenem-resistant Enterobacteriaceae (CRE) is a recommended strategy for CRE control in healthcare facilities. Active surveillance using stool specimens tested for Clostridium difficile is a relatively low-cost strategy to detect CRE carriers. Further evaluation of this and other risk factor-based active surveillance strategies is warranted.
Floods can contaminate potable water and other resources, thus increasing the potential for fecal-oral transmission of pathogens. Clostridium difficile is a bacterium that can spread by water and cause acute gastrointestinal illness. It often affects older adults who are hospital...
Martin, Christopher E; Broecker, Felix; Eller, Steffen; Oberli, Matthias A; Anish, Chakkumkal; Pereira, Claney L; Seeberger, Peter H
Clostridium difficile is a leading cause of severe nosocomial infections. Cell-surface carbohydrate antigens are promising vaccine candidates. Here we report the first total synthesis of oligomers of the lipoteichoic acid antigen repeating unit. Synthetic glycan microarrays revealed anti-glycan antibodies in the blood of patients that help to define epitopes for vaccine development.
Guandalini, S; Fasano, A; Migliavacca, M; Verga, M C; Mastrantonio Gianfrilli, P; Ferrara, A; Alessio, M; Malamisura, B; Galati, P; Pantosti, A
To elucidate the pathophysiological changes leading to postantibiotic diarrhoea caused by Clostridium difficile and its cytotoxin, oral ampicillin was given to rabbits, and jejunal, ileal, and caecal segments of those that developed diarrhoea were investigated in vitro. The rabbits that, in response to treatment, harboured Clostridium difficile in their colonic lumen were studied, and the results expressed according to the presence or absence of Clostridium difficile and/or its cytotoxin. Thus, we refer to either CD+ or CD- segments. The influx of glucose, phenylalanine, glycylphenylalanine, and lysine across the brush border of jejunum and ileum of CD+ segments was severely impaired, while only slightly blunted in CD-. No significant change was detected in the influx of glutamic acid in the jejunum of all treated animals and in the CD- ilea. Morphologic damage in ileum and caecum of CD+ was also more evident than in CD-. Transepithelial ion transport across short circuited ileal mucosa (CD+ and CD-) revealed secretory changes in Cl net transport that were more marked in CD-. We conclude that: (1) Clostridium difficile may also colonise the upper intestinal tract, where it induces morphological and functional damage, severely impairing nutrient absorption; and (2) the ileum contributes to the diarrhoea caused by CD even when the micro-organism is confined to the more distal gut by showing moderate impairment of nutrient absorption and marked electrolyte secretion. PMID:3396947
Dubberke, Erik R; Nyazee, Humaa A; Yokoe, Deborah S; Mayer, Jeanmarie; Stevenson, Kurt B; Mangino, Julie E; Khan, Yosef M; Fraser, Victoria J
Automated surveillance using electronically available data has been found to be accurate and save time. An automated Clostridium difficile infection (CDI) surveillance algorithm was validated at 4 Centers for Disease Control and Prevention Epicenter hospitals. Electronic surveillance was highly sensitive, specific, and showed good to excellent agreement for hospital-onset; community-onset, study facility-associated; indeterminate; and recurrent CDI.
The incidence and severity of disease associated with toxigenic Clostridium difficile (Cd) have increased in hospitals in North America from the emergence of newer, more virulent strains of Cd. Toxigenic Cd has been isolated from food animals and retail meat with potential implications of transfer ...
Bunk, Boyke; Thürmer, Andrea; Spröer, Cathrin; Brzuszkiewicz, Elzbieta; Abt, Birte; Gronow, Sabine; Liesegang, Heiko; Daniel, Rolf; Overmann, Jörg
We resequenced the complete genome of the virulent and multidrug-resistant pathogen Clostridium difficile strain 630. A combination of single-molecule real-time and Illumina sequencing technology revealed the presence of an additional rRNA gene cluster, additional tRNAs, and the absence of a transposon in comparison to the published and reannotated genome sequence. PMID:25858846
John, Santhosh Gheevarghese; Dominguez, Cristian; Chandiramani, Vijay; Vemulappalli, Tejo
Patient: Male, 63 Final Diagnosis: Cytomegalo virus (CMV) infection Symptoms: Diarrhea Medication:— Clinical Procedure:— Specialty: Infectious Diseases Objective: Unusual clinical course Background: Coinfection with cytomegalovirus in a patient with Clostridium difficile persistent diarrhea and colitis can lead to a delay in diagnosis and treatment. Case Report: A 63-year-old man with squamous cell carcinoma of the lower lip, status post surgical resection and currently on chemoradiation presented with intractable diarrhea and abdominal pain. Initial workup showed Clostridium difficile diarrhea with pancolitis. Diarrhea persisted despite being on antibiotics and bacteriological cure for C. difficile. Further noninvasive work up revealed associated cytomegalovirus infection, and patient had a dramatic response to ganciclovir without any relapse. Conclusions: Physicians should be cognizant about other causes of diarrhea and colitis in immunocompromised patient when treatment for primary diagnosis fails to resolve their symptoms. PMID:24298304
Ghantoji, Shashank S; Stibich, Mark; Stachowiak, Julie; Cantu, Sherry; Adachi, Javier A; Raad, Issam I; Chemaly, Roy F
The standard for Clostridium difficile surface decontamination is bleach solution at a concentration of 10 % of sodium hypochlorite. Pulsed xenon UV light (PX-UV) is a means of quickly producing germicidal UV that has been shown to be effective in reducing environmental contamination by C. difficile spores. The purpose of this study was to investigate whether PX-UV was equivalent to bleach for decontamination of surfaces in C. difficile infection isolation rooms. High-touch surfaces in rooms previously occupied by C. difficile infected patients were sampled after discharge but before and after cleaning using either bleach or non-bleach cleaning followed by 15 min of PX-UV treatment. A total of 298 samples were collected by using a moistened wipe specifically designed for the removal of spores. Prior to disinfection, the mean contamination level was 2.39 c.f.u. for bleach rooms and 22.97 for UV rooms. After disinfection, the mean level of contamination for bleach was 0.71 c.f.u. (P = 0.1380), and 1.19 c.f.u. (P = 0.0017) for PX-UV disinfected rooms. The difference in final contamination levels between the two cleaning protocols was not significantly different (P = 0.9838). PX-UV disinfection appears to be at least equivalent to bleach in the ability to decrease environmental contamination with C. difficile spores. Larger studies are needed to validate this conclusion.
Sarker, Mahfuzur R; Paredes-Sabja, Daniel
Clostridium difficile infections (CDIs) occur when antibiotic therapy disrupts the gastrointestinal flora, favoring infected C. difficile spores to germinate, outgrow, colonize and produce toxins. During CDI, C. difficile vegetative cells initiate the process of sporulation allowing a fraction of the spores to remain adhered to the intestinal surfaces. These spores, which are unaffected by antibiotic therapy commonly used for CDIs, then germinate, outgrow and recolonize the host's GI tract causing relapse of CDI. Consequently, the germination and colonization processes can be considered as the earliest and most essential steps for the development as well as relapse of CDI. The aim of this review is to provide an overview on the molecular basis involved in C. difficile spore germination and colonization.
Vincent, Caroline; Manges, Amee R.
Clostridium difficile infection (CDI) is the most important cause of nosocomial diarrhea. Broad-spectrum antimicrobials have profound detrimental effects on the structure and diversity of the indigenous intestinal microbiota. These alterations often impair colonization resistance, allowing the establishment and proliferation of C. difficile in the gut. Studies involving animal models have begun to decipher the precise mechanisms by which the intestinal microbiota mediates colonization resistance against C. difficile and numerous investigations have described gut microbiota alterations associated with C. difficile colonization or infection in human subjects. Fecal microbiota transplantation (FMT) is a highly effective approach for the treatment of recurrent CDI that allows the restoration of a healthy intestinal ecosystem via infusion of fecal material from a healthy donor. The recovery of the intestinal microbiota after FMT has been examined in a few reports and work is being done to develop custom bacterial community preparations that could be used as a replacement for fecal material. PMID:27025623
Theriot, Casey M; Young, Vincent B
Antibiotics disturb the gastrointestinal tract microbiota and in turn reduce colonization resistance against Clostridium difficile. The mechanism for this loss of colonization resistance is still unknown but likely reflects structural (microbial) and functional (metabolic) changes to the gastrointestinal tract. Members of the gut microbial community shape intestinal metabolism that provides nutrients and ultimately supports host immunity. This review will discuss how antibiotics alter the structure of the gut microbiota and how this impacts bacterial metabolism in the gut. It will also explore the chemical requirements for C. difficile germination, growth, toxin production and sporulation. Many of the metabolites that influence C. difficile physiology are products of gut microbial metabolism including bile acids, carbohydrates and amino acids. To restore colonization resistance against C. difficile after antibiotics a targeted approach restoring both the structure and function of the gastrointestinal tract is needed. PMID:24335555
Friedman-Moraco, R J; Mehta, A K; Lyon, G M; Kraft, C S
Fecal microbiota transplantation (FMT) has been shown to be safe and efficacious in individuals with refractory Clostridium difficile. It has not been widely studied in individuals with immunosuppression due to concerns about infectious complications. We describe two solid organ transplant recipients, one lung and one renal, in this case report that both had resolution of their diarrhea caused by C. difficile after FMT. Both recipients required two FMTs to achieve resolution of their symptoms and neither had infectious complications. Immunosuppressed individuals are at high risk for acquisition of C. difficile and close monitoring for infectious complications after FMT is necessary, but should not preclude its use in patients with refractory disease due to C. difficile. Sequential FMT may be used to achieve cure in these patients with damaged microbiota from antibiotic use and immunosuppression.
Bien, Justyna; Palagani, Vindhya
Gut microbiota is a compilation of microorganisms dwelling in the entire mammalian gastrointestinal tract. They display a symbiotic relationship with the host contributing to its intestinal health and disease. Even a slight fluctuation in this equipoise may be deleterious to the host, leading to many pathological conditions like Clostridium difficile infection or inflammatory bowel disease (IBD). In this review, we focus on the role of microbial dysbiosis in initiation of C. difficile infection and IBD, and we also touch upon the role of specific pathogens, particularly C. difficile, as causative agents of IBD. We also discuss the molecular mechanisms activated by C. difficile that contribute to the development and exacerbation of gastrointestinal disorders. PMID:23320050
Wang, Su; Rustandi, Richard R; Lancaster, Catherine; Hong, Laura G; Thiriot, David S; Xie, Jinfu; Secore, Susan; Kristopeit, Adam; Wang, Sheng-Ching; Heinrichs, Jon H
Clostridium difficile is the leading cause of hospital-acquired diarrhea, also known as C. difficile associated diarrhea. The two major toxins, toxin A and toxin B are produced by most C. difficile bacteria, but some strains, such as BI/NAP1/027 isolates, produce a third toxin called binary toxin. The precise biological role of binary toxin is not clear but it has been shown to be a cytotoxin for Vero cells. We evaluated the toxicity of these toxins in mice and hamsters and found that binary toxin causes death in both animals similar to toxins A and B. Furthermore, immunization of mice with mutant toxoids of all three toxins provided protection upon challenge with native toxins. These results support the concept that binary toxin contributes to the pathogenicity of C. difficile and provide a method for monitoring the toxicity of binary toxin components in vaccines.
Hussack, Greg; Tanha, Jamshid
Clostridium difficile continues to be one of the most prevalent hospital-acquired bacterial infections in the developed world, despite the recent introduction of a novel and effective antibiotic agent (fidaxomicin). Alternative approaches under investigation to combat the anaerobic Gram-positive bacteria include fecal transplantation therapy, vaccines, and antibody-based immunotherapies. In this review, we catalog the recent advances in antibody-based approaches under development and in the clinic for the treatment of C. difficile infection. By and large, inhibitory antibodies that recognize the primary C. difficile virulence factors, toxin A and toxin B, are the most popular passive immunotherapies under investigation. We provide a detailed summary of the toxin epitopes recognized by various antitoxin antibodies and discuss general trends on toxin inhibition efficacy. In addition, antibodies to other C. difficile targets, such as surface-layer proteins, binary toxin, motility factors, and adherence and colonization factors, are introduced in this review. PMID:27536153
Engevik, Melinda A.; Engevik, Kristen A.; Yacyshyn, Mary Beth; Wang, Jiang; Hassett, Daniel J.; Darien, Benjamin; Yacyshyn, Bruce R.
Clostridium difficile infection (CDI) is principally responsible for hospital acquired, antibiotic-induced diarrhea and colitis and represents a significant financial burden on our healthcare system. Little is known about C. difficile proliferation requirements, and a better understanding of these parameters is critical for development of new therapeutic targets. In cell lines, C. difficile toxin B has been shown to inhibit Na+/H+ exchanger 3 (NHE3) and loss of NHE3 in mice results in an altered intestinal environment coupled with a transformed gut microbiota composition. However, this has yet to be established in vivo in humans. We hypothesize that C. difficile toxin inhibits NHE3, resulting in alteration of the intestinal environment and gut microbiota. Our results demonstrate that CDI patient biopsy specimens have decreased NHE3 expression and CDI stool has elevated Na+ and is more alkaline compared with stool from healthy individuals. CDI stool microbiota have increased Bacteroidetes and Proteobacteria and decreased Firmicutes phyla compared with healthy subjects. In vitro, C. difficile grows optimally in the presence of elevated Na+ and alkaline pH, conditions that correlate to changes observed in CDI patients. To confirm that inhibition of NHE3 was specific to C. difficile, human intestinal organoids (HIOs) were injected with C. difficile or healthy and CDI stool supernatant. Injection of C. difficile and CDI stool decreased NHE3 mRNA and protein expression compared with healthy stool and control HIOs. Together these data demonstrate that C. difficile inhibits NHE3 in vivo, which creates an altered environment favored by C. difficile. PMID:25552580
Engevik, Melinda A; Engevik, Kristen A; Yacyshyn, Mary Beth; Wang, Jiang; Hassett, Daniel J; Darien, Benjamin; Yacyshyn, Bruce R; Worrell, Roger T
Clostridium difficile infection (CDI) is principally responsible for hospital acquired, antibiotic-induced diarrhea and colitis and represents a significant financial burden on our healthcare system. Little is known about C. difficile proliferation requirements, and a better understanding of these parameters is critical for development of new therapeutic targets. In cell lines, C. difficile toxin B has been shown to inhibit Na(+)/H(+) exchanger 3 (NHE3) and loss of NHE3 in mice results in an altered intestinal environment coupled with a transformed gut microbiota composition. However, this has yet to be established in vivo in humans. We hypothesize that C. difficile toxin inhibits NHE3, resulting in alteration of the intestinal environment and gut microbiota. Our results demonstrate that CDI patient biopsy specimens have decreased NHE3 expression and CDI stool has elevated Na(+) and is more alkaline compared with stool from healthy individuals. CDI stool microbiota have increased Bacteroidetes and Proteobacteria and decreased Firmicutes phyla compared with healthy subjects. In vitro, C. difficile grows optimally in the presence of elevated Na(+) and alkaline pH, conditions that correlate to changes observed in CDI patients. To confirm that inhibition of NHE3 was specific to C. difficile, human intestinal organoids (HIOs) were injected with C. difficile or healthy and CDI stool supernatant. Injection of C. difficile and CDI stool decreased NHE3 mRNA and protein expression compared with healthy stool and control HIOs. Together these data demonstrate that C. difficile inhibits NHE3 in vivo, which creates an altered environment favored by C. difficile.
Fairley, Derek J; McKenna, James P; Stevenson, Mike; Weaver, Jeremy; Gilliland, Carol; Watt, Alison; Coyle, Peter V
Using a Clostridium difficile glutamate dehydrogenase (GDH) immunoassay and a sensitive C. difficile toxin A/B immunoassay, human stool specimens from patients with diarrhoea (n = 1085) were classified as either GDH positive/toxin negative, or GDH positive/toxin positive. Overall, 528/725 (73%) of the GDH-positive/toxin-negative specimens contained viable C. difficile, and 433/528 (82%) of these C. difficile isolates were PCR positive for the toxin gene pathogenicity locus. Overall, 867/1078 (80%) of the GDH-positive specimens contained viable C. difficile, and 433/725 (60%) of the GDH-positive/toxin-negative specimens contained a toxigenic C. difficile strain. The diversity of toxigenic C. difficile ribotypes isolated from toxin-negative specimens (n = 433) and toxin-positive specimens (n = 339) was significantly different (P < 0.0001). Specifically, the presence of ribotype 078 strains was very strongly associated (P < 0.0001) with detection of toxin in clinical specimens using a sensitive toxin immunoassay. Specimens positive for ribotype 078 were almost twice as likely to be toxin positive as opposed to toxin negative (risk ratio = 1.90, 95% confidence interval 1.64-2.19). In contrast, other circulating ribotypes were seen with similar frequency in specimens with and without detectable toxin. This supports the view that ribotype 078 strains may be more virulent than other common ribotypes in terms of toxin production.
Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of Clostridium difficile (C. difficile) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic C. difficile (NTCD) strains in preventing CDI due to highly toxigenic C. difficile (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to C. difficile cells. Herein, by monitoring the field screening behavior of the C. difficile cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest C. difficile colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope. PMID:27547818
Tsai, Bo-Yang; Ko, Wen-Chien; Chen, Ter-Hsin; Wu, Ying-Chen; Lan, Po-Han; Chen, Yi-Hsuan; Hung, Yuan-Pin; Tsai, Pei-Jane
Clostridium difficile is the major cause of nosocomial diarrhea. We have previously demonstrated that in southern Taiwan, severe C. difficile-associated diarrhea (CDAD) cases were due to the C. difficile RT 126 strain infection, indicating the arrival of an epidemic C. difficile clone in southern Taiwan. RT126 has a close genetic relationship with RT078. However, the RT078 family is the predominant strain of C. difficile in animals worldwide, particularly in swine. In this study, we surveyed C. difficile strains isolated from swine at several farms in Taiwan from August 2011 to March 2015. We found that all swine strains, namely RT078 (32.5%, 37 of 114), RT126 (28.9%, 33 of 114) and RT127 (37.7%, 43 of 114), belonged to the toxigenic RT078 family. All strains had high gyrA mutation rate (57.9%, 66/114), which was linked to quinolone resistance. Notably, Rep-PCR revealed that 3 RT078 animal strains had the same fingerprint as human RT078 clinical isolates; their phylogenic relationship was closely related to the whole gene sequences of tcdB, thus suggesting zoonotic potential for C. difficile infection in Taiwan.
Ling, Zongxin; Liu, Xia; Jia, Xiaoyun; Cheng, Yiwen; Luo, Yueqiu; Yuan, Li; Wang, Yuezhu; Zhao, Chunna; Guo, Shu; Li, Lanjuan; Xu, Xiwei; Xiang, Charlie
Increasing evidence suggests that altered intestinal microbial composition and function result in an increased risk of Clostridium difficile-associated diarrhoea (CDAD); however, the specific changes of intestinal microbiota in children suffering from CDAD and their associations with C. difficile strain toxigenicity are poorly understood. High-throughput pyrosequencing showed that reduced faecal bacterial diversity and dramatic shifts of microbial composition were found in children with CDAD. The Firmicutes/Bacteroidetes ratio was increased significantly in patients with CDAD, which indicated that dysbiosis of faecal microbiota was closely associated with CDAD. C. difficile infection resulted in an increase in lactate-producing phylotypes, with a corresponding decrease in butyrate-producing bacteria. The decrease in butyrate and lactate buildup impaired intestinal colonisation resistance, which increased the susceptibility to C. difficile colonisation. Strains of C. difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive, and were associated with unusually abundant Enterococcus, which implies that the C. difficile toxins have different impacts on the faecal microbiota of children. Greater understanding of the relationships between disruption of the normal faecal microbiota and colonisation with C. difficile that produces different toxins might lead to improved treatment.
Lee, Won-Kil; Selasi, Gati Noble; Na, Seok Hyeon; Kwon, Hyo Il; Kim, Yoo Jeong; Lee, Hae Sook; Song, Kyung Eun; Shin, Jeong Hwan; Lee, Je Chul
Clostridium difficile is one of the main etiological agents causing antibiotic-associated diarrhea. This study investigated the genetic diversity of 70 toxigenic C. difficile isolates from two Korean hospitals by employing toxinotyping, ribotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Toxin gene amplification resulted in 68 A⁺B⁺ and two A-B+ isolates. Most isolates (95.7–100%) were susceptible to daptomycin, metronidazole, and vancomycin. Seventy C. difficile isolates were classified into five toxinotypes, 19 ribotypes, 16 sequence types (STs), and 33 arbitrary pulsotypes. All C. difficile isolates of ribotype 018 (n = 38) were classified into ST17, which was the most prevalent ST in both hospitals. However, C. difficile isolates of ST17 (ribotype 018) exhibited pulsotypes that differed by hospital. ST2 (ribotype 014/020), 8 (ribotypes 002), 17 (ribotype 018), and 35 (ribotypes 015) were detected in both hospitals, whereas other STs were unique to each hospital. Statistical comparison of the different typing methods revealed that ribotyping and PFGE were highly predictive of STs. In conclusion, our epidemiological study indicates that C. difficile infections in both hospitals are associated with the persistence of endemic clones coupled with the emergence of many unique clones. A combination of MLST with PFGE or ribotyping could be useful for monitoring epidemic C. difficile strains and the emergence of new clones in hospitals. PMID:28355266
Stokely, Janelle N; Niendorf, Sandra; Taube, Stefan; Hoehne, Marina; Young, Vincent B; Rogers, Mary AM; Wobus, Christiane E
Objective Human norovirus (HuNoV) and Clostridium difficile are common causes of infectious gastroenteritis in adults in the US. However, limited information is available regarding HuNoV and C. difficile coinfections. Our study was designed to evaluate the prevalence of HuNoV and C. difficile coinfections among adult patients in a hospital setting and disease symptomatology. Study design and setting For a cross-sectional analysis, 384 fecal samples were tested for the presence of C. difficile toxins from patients (n=290), whom the provider suspected of C. difficile infections. Subsequent testing was then performed for HuNoV genogroups I and II. Multinomial logistic regression was performed to determine symptoms more frequently associated with coinfections. Results The final cohort consisted of the following outcome groups: C. difficile (n=196), C. difficile + HuNoV coinfection (n=40), HuNoV only (n=12), and neither (n=136). Coinfected patients were more likely to develop nausea, gas, and abdominal pain and were more likely to seek treatment in the winter season compared with individuals not infected or infected with either pathogen alone. Conclusion Our study revealed that patients with coinfection are more likely to experience certain gastrointestinal symptoms, in particular abdominal pain, suggesting an increased severity of disease symptomatology in coinfected patients. PMID:27418856
Ling, Zongxin; Liu, Xia; Jia, Xiaoyun; Cheng, Yiwen; Luo, Yueqiu; Yuan, Li; Wang, Yuezhu; Zhao, Chunna; Guo, Shu; Li, Lanjuan; Xu, Xiwei; Xiang, Charlie
Increasing evidence suggests that altered intestinal microbial composition and function result in an increased risk of Clostridium difficile-associated diarrhoea (CDAD); however, the specific changes of intestinal microbiota in children suffering from CDAD and their associations with C. difficile strain toxigenicity are poorly understood. High-throughput pyrosequencing showed that reduced faecal bacterial diversity and dramatic shifts of microbial composition were found in children with CDAD. The Firmicutes/Bacteroidetes ratio was increased significantly in patients with CDAD, which indicated that dysbiosis of faecal microbiota was closely associated with CDAD. C. difficile infection resulted in an increase in lactate-producing phylotypes, with a corresponding decrease in butyrate-producing bacteria. The decrease in butyrate and lactate buildup impaired intestinal colonisation resistance, which increased the susceptibility to C. difficile colonisation. Strains of C. difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive, and were associated with unusually abundant Enterococcus, which implies that the C. difficile toxins have different impacts on the faecal microbiota of children. Greater understanding of the relationships between disruption of the normal faecal microbiota and colonisation with C. difficile that produces different toxins might lead to improved treatment. PMID:25501371
Zackular, Joseph P.; Moore, Jessica L.; Jordan, Ashley T.; Juttukonda, Lillian J.; Noto, Michael J.; Nicholson, Maribeth R.; Crews, Jonathan D.; Semler, Matthew W.; Zhang, Yaofang; Ware, Lorraine B.; Washington, M. Kay; Chazin, Walter J.; Caprioli, Richard M.; Skaar, Eric P.
Clostridium difficile is the most commonly reported nosocomial pathogen in the United States and is an urgent public health concern worldwide1. Over the past decade, incidence, severity, and costs associated with C. difficile infection (CDI) have increased dramatically2. CDI is most commonly initiated by antibiotic-mediated disruption of the gut microbiota; however, non-antibiotic associated CDI cases are well documented and on the rise3,4. This suggests that unexplored environmental, nutrient, and host factors likely influence CDI. Here we show that excess dietary zinc (Zn) significantly alters the gut microbiota and in turn reduces the threshold of antibiotics needed to confer susceptibility to C. difficile infection. In mice colonized with C. difficile, excess dietary Zn severely exacerbates C. difficile-associated disease by increasing toxin activity and altering the host immune response. In addition, we show that the Zn binding S100 protein calprotectin is antimicrobial against C. difficile and an essential component of the innate immune response to CDI. Together, these data suggest that nutrient Zn levels play a key role in determining susceptibility to CDI and severity of disease, and that calprotectin-mediated metal limitation is an important factor in the host immune response to C. difficile. PMID:27668938
Seekatz, Anna M; Theriot, Casey M; Molloy, Caitlyn T; Wozniak, Katherine L; Bergin, Ingrid L; Young, Vincent B
Recurrent Clostridium difficile infection (CDI) is of particular concern among health care-associated infections. The role of the microbiota in disease recovery is apparent given the success of fecal microbiota transplantation (FMT) for recurrent CDI. Here, we present a murine model of CDI relapse to further define the microbiota recovery following FMT. Cefoperazone-treated mice were infected with C. difficile 630 spores and treated with vancomycin after development of clinical disease. Vancomycin treatment suppressed both C. difficile colonization and cytotoxin titers. However, C. difficile counts increased within 7 days of completing treatment, accompanied by relapse of clinical signs. The administration of FMT immediately after vancomycin cleared C. difficile and decreased cytotoxicity within 1 week. The effects of FMT on the gut microbiota community were detectable in recipients 1-day posttransplant. Conversely, mice not treated with FMT remained persistently colonized with high levels of C. difficile, and the gut microbiota in these mice persisted at low diversity. These results suggest that full recovery of colonization resistance against C. difficile requires the restoration of a specific community structure.
Norman, Keri N; Scott, H Morgan; Harvey, Roger B; Norby, Bo; Hume, Michael E; Andrews, Kathleen
Recently, an apparent rise in the number of cases attributed to community-acquired Clostridium difficile infection has led researchers to explore additional sources of infection. The finding of C. difficile in food animals and retail meat has raised concern about potential food-borne and occupational exposures. The objective of this study was to compare C. difficile isolated from a closed population of healthy individuals consisting of both humans and swine in order to investigate possible food safety and occupational risks for exposure. Using a multistep enrichment isolation technique, we identified 11.8% of the human wastewater samples and 8.6% of the swine samples that were positive for C. difficile. The prevalences of C. difficile in swine production groups differed significantly (P < 0.05); however, the prevalences in the two human occupational group cohorts did not differ significantly (P = 0.81). The majority of the human and swine isolates were similar based on multiple typing methods. The similarity in C. difficile prevalence in the human group cohorts suggests a low occupational hazard, while a greatly decreased prevalence of C. difficile in later-stage swine production groups suggests a diminished risk for food-borne exposure. The similarity of strains in the two host species suggests the possibility of a common environmental source for healthy individuals in a community setting.
Ghose, Chandrabali; Eugenis, Ioannis; Sun, Xingmin; Edwards, Adrianne N; McBride, Shonna M; Pride, David T; Kelly, Ciarán P; Ho, David D
Clostridium difficile is a Gram-positive bacillus and is the leading cause of toxin-mediated nosocomial diarrhea following antibiotic use. C. difficile flagella play a role in colonization, adherence, biofilm formation, and toxin production, which might contribute to the overall virulence of certain strains. Human and animal studies indicate that anti-flagella immune responses may play a role in protection against colonization by C. difficile and subsequent disease outcome. Here we report that recombinant C. difficile flagellin (FliC) is immunogenic and protective in a murine model of C. difficile infection (CDI) against a clinical C. difficile strain, UK1. Passive protection experiments using anti-FliC polyclonal serum in mice suggest this protection to be antibody-mediated. FliC immunization also was able to afford partial protection against CDI and death in hamsters following challenge with C. difficile 630Δerm. Additionally, immunization against FliC does not have an adverse effect on the normal gut flora of vaccinated hamsters as evidenced by comparing the fecal microbiome of vaccinated and control hamsters. Therefore, the use of FliC as a vaccine candidate against CDI warrants further testing.
Boudry, Pierre; Semenova, Ekaterina; Monot, Marc; Datsenko, Kirill A.; Lopatina, Anna; Sekulovic, Ognjen; Ospina-Bedoya, Maicol; Fortier, Louis-Charles; Severinov, Konstantin; Dupuy, Bruno
ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. PMID:26330515
Jackson-Rosario, Sarah; Cowart, Darin; Myers, Andrew; Tarrien, Rebecca; Levine, Rodney L; Scott, Robert A; Self, William Thomas
Clostridium difficile is a nosocomial pathogen whose incidence and importance are on the rise. Previous work in our laboratory characterized the central role of selenoenzyme-dependent Stickland reactions in C. difficile metabolism. In this work we have identified, using mass spectrometry, a stable complex formed upon reaction of auranofin (a gold-containing drug) with selenide in vitro. X-ray absorption spectroscopy supports the structure that we proposed on the basis of mass-spectrometric data. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium ((75)Se) in selenoproteins in both Escherichia coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth medium significantly reduces the inhibitory action of auranofin on the growth of C. difficile. On the basis of these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. This study demonstrates that targeting selenium metabolism provides a new avenue for antimicrobial development against C. difficile and other selenium-dependent pathogens.
Dubberke, Erik R.; Haslam, David B.; Lanzas, Cristina; Bobo, Linda D.; Burnham, Carey-Ann D.; Gröhn, Yrjö T.; Tarr, Phillip I.
Summary Clostridium difficile is a well recognized pathogen of humans and animals. Although C. difficile was first identified over 70 years ago, much remains unknown in regards to the primary source of human acquisition and its pathobiology. These deficits in our knowledge have been intensified by dramatic increases in both the frequency and severity of disease in humans over the last decade. The changes in C. difficile epidemiology might be due to the emergence of a hypervirulent stain of C. difficile, aging of the population, altered risk of developing infection with newer medications, and/or increased exposure to C. difficile outside of hospitals. In recent years there have been numerous reports documenting C. difficile contamination of various foods, and reports of similarities between strains that infect animals and strains that infect humans as well. The purposes of this review are to highlight the many challenges to diagnosing, treating, and preventing C. difficile infection in humans, and to stress that collaboration between human and veterinary researchers is needed to control this pathogen. PMID:21223531
Baines, Simon D.; Wilcox, Mark H.
Clostridium difficile infection (CDI) remains a substantial burden on healthcare systems and is likely to remain so given our reliance on antimicrobial therapies to treat bacterial infections, especially in an aging population in whom multiple co-morbidities are common. Antimicrobial agents are a key component in the aetiology of CDI, both in the establishment of the infection and also in its treatment. The purpose of this review is to summarise the role of antimicrobial agents in primary and recurrent CDI; assessing why certain antimicrobial classes may predispose to the induction of CDI according to a balance between antimicrobial activity against the gut microflora and C. difficile. Considering these aspects of CDI is important in both the prevention of the infection and in the development of new antimicrobial treatments. PMID:27025625
Genth, Harald; Schelle, Ilona; Just, Ingo
Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the “Large clostridial glycosylating toxins.” These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB—together with Toxin A (TcdA)—is causative for the pseudomembranous colitis (PMC). Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn2+ > Co2+ > Mg2+ >> Ca2+, Cu2+, Zn2+. TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn2+ and 180 µM for Mg2+. TcsL and TcdB further require co-stimulation by monovalent K+ (not by Na+). Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K+ and Mg2+ (rather than Mn2+) in mammalian target cells. PMID:27089365
Bermejo, C; Maseda, E; Salgado, P; Gabilondo, G; Gilsanz, F
The epidemiology of Clostridium difficile infection has changed in the past decade. The incidence rate of community acquired cases has increased in patients with no typical risk factors. We present a patient who was diagnosed with community-acquired Clostridium difficile infection who presented with acute abdominal pain, and subsequently developed acute renal failure and septic shock. We describe the diagnosis, treatment and outcome and brief review of the literature.
Johnson, Steven W; Kanatani, Meganne; Humphries, Romney M; Uslan, Daniel Z
The impact of a new Clostridium difficile nucleic acid amplification test (NAAT) on antibiotic utilization in patients with suspected C difficile infection was assessed. This single-center, cross-sectional study of 270 patients demonstrated that the use of NAAT decreased antibiotic expenditure by reducing prolonged empiric days of therapy in these patients.
Gheorghe, Romina; Eastmond, Jennifer; Miller, Mark A.
In 54/64 subjects with nosocomial diarrhea, fecal calprotectin levels correlated with the results of stool samples tested for Clostridium difficile toxin gene by PCR. Fecal calprotectin levels can be used as an adjunctive measure to PCR to support the diagnosis of C. difficile infection. PMID:26354814
Anosova, Natalie G; Brown, Anna M; Li, Lu; Liu, Nana; Cole, Leah E; Zhang, Jinrong; Mehta, Hersh; Kleanthous, Harry
Clostridium difficile infection (CDI) has been identified as the leading cause of nosocomial diarrhoea and pseudomembranous colitis associated with antibiotic therapy. Recent epidemiological changes as well as increases in the number of outbreaks of strains associated with increased virulence and higher mortality rates underscore the importance of identifying alternatives to antibiotics to manage this important disease. Animal studies have clearly demonstrated the roles that toxins A and B play in gut inflammation as well as diarrhoea; therefore it is not surprising that serum anti-toxin A and B IgG are associated with protection against recurrent CDI. In humans, strong humoral toxin-specific immune responses elicited by natural C. difficile infection is associated with recovery and lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. The first generation of C. difficile vaccine that contained inactivated toxin A and B was found to be completely protective against death and diarrhoea in the hamster C. difficile challenge model. When tested in young healthy volunteers in Phase I clinical trials, this investigational vaccine was shown to be safe and immunogenic. Moreover, in a separate study this vaccine was able to prevent further relapses in three out of three patients who had previously suffered from chronic relapsing C. difficile-associated diarrhoea. Herein we examined the immunogenicity and protective activity of a next-generation Sanofi Pasteur two-component highly purified toxoid vaccine in a C. difficile hamster model. This model is widely recognized as a stringent and relevant choice for the evaluation of novel treatment strategies against C. difficile and was used in preclinical testing of the first-generation vaccine candidate. Intramuscular (i.m.) immunizations with increasing doses of this adjuvanted toxoid vaccine protected hamsters from mortality and disease symptoms in a dose-dependent manner. ELISA
Di Bella, Stefano; Ascenzi, Paolo; Siarakas, Steven; Petrosillo, Nicola; di Masi, Alessandra
Clostridium difficile infection (CDI) has significant clinical impact especially on the elderly and/or immunocompromised patients. The pathogenicity of Clostridium difficile is mainly mediated by two exotoxins: toxin A (TcdA) and toxin B (TcdB). These toxins primarily disrupt the cytoskeletal structure and the tight junctions of target cells causing cell rounding and ultimately cell death. Detectable C. difficile toxemia is strongly associated with fulminant disease. However, besides the well-known intestinal damage, recent animal and in vitro studies have suggested a more far-reaching role for these toxins activity including cardiac, renal, and neurologic impairment. The creation of C. difficile strains with mutations in the genes encoding toxin A and B indicate that toxin B plays a major role in overall CDI pathogenesis. Novel insights, such as the role of a regulator protein (TcdE) on toxin production and binding interactions between albumin and C. difficile toxins, have recently been discovered and will be described. Our review focuses on the toxin-mediated pathogenic processes of CDI with an emphasis on recent studies. PMID:27153087
Buffie, Charlie G.; Bucci, Vanni; Stein, Richard R.; McKenney, Peter T.; Ling, Lilan; Gobourne, Asia; No, Daniel; Liu, Hui; Kinnebrew, Melissa; Viale, Agnes; Littmann, Eric; van den Brink, Marcel R. M.; Jenq, Robert R.; Taur, Ying; Sander, Chris; Cross, Justin R.; Toussaint, Nora C.; Xavier, Joao B.; Pamer, Eric G.
The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens. Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens. Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients. Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear. Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile. Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans. Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion. Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency. These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.
Bakker, Dennis; Smits, Wiep Klaas; Kuijper, Ed J.; Corver, Jeroen
In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis, sometimes resulting in colectomy or death. The main virulence factors of C. difficile are toxin A and toxin B. Besides the genes encoding these toxins (tcdA and tcdB), the pathogenicity locus (PaLoc) also contains genes encoding a sigma factor (tcdR) and a putative anti-sigma factor (tcdC). The important role of TcdR as a sigma factor for toxin expression is undisputed, whereas the role of TcdC as an anti-sigma factor, inhibiting toxin expression, is currently the subject of debate. To clarify the role of TcdC in toxin expression, we generated an isogenic ClosTron-based mutant of tcdC in Clostridium difficile strain 630Δ Erm (CT::tcdC) and determined the transcription levels of the PaLoc genes and the expression levels of the toxins in the wild type strain and the tcdC mutant strain. We found only minor differences in transcription levels of the PaLoc genes between the wild type and CT::tcdC strains and total toxin levels did not significantly differ either. These results suggest that in C. difficile 630Δerm TcdC is not a major regulator of toxin expression under the conditions tested. PMID:22912837
Mayer, Melinda J.; Narbad, Arjan; Gasson, Michael J.
Clostridium difficile infection is increasing in both frequency and severity, with the emergence of new highly virulent strains highlighting the need for more rapid and effective methods of control. Here, we show that bacteriophage endolysin can be used to inhibit and kill C. difficile. The genome sequence of a novel bacteriophage that is active against C. difficile was determined, and the bacteriophage endolysin gene was subcloned and expressed in Escherichia coli. The partially purified endolysin was active against 30 diverse strains of C. difficile, and importantly, this group included strains of the major epidemic ribotype 027 (B1/NAP1). In contrast, a range of commensal species that inhabit the gastrointestinal tract, including several representatives of the clostridium-like Firmicutes, were insensitive to the endolysin. This endolysin provides a platform for the generation of both therapeutic and detection systems to combat the C. difficile problem. To investigate a method for the protected delivery and production of the lysin in the gastrointestinal tract, we demonstrated the expression of active CD27L endolysin in the lactic acid bacterium Lactococcus lactis MG1363. PMID:18708505
Background This study was conducted to evaluate the faecal occurrence and characterization of Clostridium difficile in clinically healthy dogs (N = 50) and in dogs with diarrhea (N = 20) in the Stockholm-Uppsala region of Sweden. Findings Clostridium difficile was isolated from 2/50 healthy dogs and from 2/20 diarrheic dogs. Isolates from healthy dogs were negative for toxin A and B and for the tcdA and tcdB genes. Both isolates from diarrheic dogs were positive for toxin B and for the tcdA and tcdB genes. The C. difficile isolates from healthy dogs had PCR ribotype 009 (SE-type 6) and 010 (SE-type 3) whereas both isolates from dogs with diarrhoea had the toxigenic ribotype 014 (SE-type 21). One of the isolates from healthy dogs was initially resistant to metronidazole. Conclusions This study revealed presence of toxigenic C. difficile in faecal samples of diarrheic dogs and low number of non- toxigenic isolates in healthy dogs from Uppsala-Stockholm region in Sweden. However, more comprehensive studies are warranted to investigate the role of C. difficile in gastrointestinal disease in dogs. PMID:23497714
Russello, Giuseppe; Russo, Antonio; Sisto, Francesca; Scaltrito, Maria Maddalena; Farina, Claudio
We evaluated a three-step algorithm for laboratory diagnosis of Clostridium difficile-associated diarrhoea (CDAD). First, stool specimens were screened using an EIA test for glutamate dehydrogenase detection. Screen-positive specimens were tested by a rapid cytotoxintoxin A/B assay and subjected to stool culture. All cultures positive for C. difficile underwent toxigenic culture. The results showed that toxigenic culture allowed us to recover 37/156 (24.4%) stool samples harbouring toxigenic C. difficile that would have been missed by using faecal cytotoxin assay alone. This determined an increase in infection prevalence of 4.2% (from 11.4% to 15.6 %). Furthermore, to characterize the clinical Clostridium difficile isolates and the distribution of PCR ribotypes circulating in the San Carlo Borromeo hospital, molecular typing using semi-automated repetitive-sequence-based PCR (rep- PCR) and PCR ribotyping, and an evaluation of the antibiotic resistance were also performed. Among them, 71 indistinguishable strains were detected by rep-PCR and 83 by PCR-ribotyping revealing C. difficile outbreaks in our hospital. A total of 6 different ribotypes were obtained by PCR ribotyping. The most frequent ribotype was 018 (88.2%) that also showed resistance to moxifloxacin. In one case, uncommon PCR ribotype 186 was also identified.
Donnelly, M. Lauren; Fimlaid, Kelly A.
ABSTRACT The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes. IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized
Francis, Michael B.
ABSTRACT Classically, dormant endospores are defined by their resistance properties, particularly their resistance to heat. Much of the heat resistance is due to the large amount of dipicolinic acid (DPA) stored within the spore core. During spore germination, DPA is released and allows for rehydration of the otherwise-dehydrated core. In Bacillus subtilis, 7 proteins are encoded by the spoVA operon and are important for DPA release. These proteins receive a signal from the activated germinant receptor and release DPA. This DPA activates the cortex lytic enzyme CwlJ, and cortex degradation begins. In Clostridium difficile, spore germination is initiated in response to certain bile acids and amino acids. These bile acids interact with the CspC germinant receptor, which then transfers the signal to the CspB protease. Activated CspB cleaves the cortex lytic enzyme, pro-SleC, to its active form. Subsequently, DPA is released from the core. C. difficile encodes orthologues of spoVAC, spoVAD, and spoVAE. Of these, the B. subtilis SpoVAC protein was shown to be capable of mechanosensing. Because cortex degradation precedes DPA release during C. difficile spore germination (opposite of what occurs in B. subtilis), we hypothesized that cortex degradation would relieve the osmotic constraints placed on the inner spore membrane and permit DPA release. Here, we assayed germination in the presence of osmolytes, and we found that they can delay DPA release from germinating C. difficile spores while still permitting cortex degradation. Together, our results suggest that DPA release during C. difficile spore germination occurs though a mechanosensing mechanism. IMPORTANCE Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway
Chambers, Christopher J.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi
Sortase enzymes are responsible for covalent anchoring of specific proteins to the peptidoglycan of the cell wall of gram-positive bacteria. In some gram-positive bacteria (e.g. Staphylococcus aureus), sortases have been found to be essential for pathogenesis and their inhibitors are under development as potential novel therapeutics. Here we provide the first report on the structural characterisation of the C. difficile sortase. An active site mutant was crystallised and its structure determined to 2.55 Å by X-ray diffraction to provide structural insight into its catalytic mechanism. In order to elucidate the role of the sortase in the cell wall biogenesis, a C. difficile sortase knockout strain was constructed by intron mutagenesis. Characterisation of this mutant led to the discovery that the putative adhesin CD0386 is anchored to the peptidoglycan of C. difficile by the sortase SrtB and that an SPKTG peptide motif is involved in the transpeptidation reaction with the C. difficile peptidoglycan. In an animal model for C. difficile infection, the SrtB mutant caused disease at a similar rate of onset as the wild type strain. In conclusion, our detailed study shows that the SrtB enzyme from C. difficile does not play an essential role in pathogenesis. PMID:25801974
Dubberke, Erik R.; Han, Zhuolin; Bobo, Linda; Hink, Tiffany; Lawrence, Brenda; Copper, Susan; Hoppe-Bauer, Joan; Burnham, Carey-Ann D.; Dunne, William Michael
Asymptomatic Clostridium difficile colonization is common in hospitalized patients. Existing C. difficile assay comparisons lack data on severity of diarrhea or patient outcomes, limiting the ability to interpret their results in regard to the diagnosis of C. difficile infection (CDI). The objective of this study was to measure how including patient presentation with the C. difficile assay result impacted assay performance to diagnose CDI. Stool specimens from 150 patients that met inclusion and exclusion criteria were selected. Nine methods to detect C. difficile in stool were evaluated. All patients were interviewed prospectively to assess diarrhea severity. We then assessed how different reference standards, with and without the inclusion of patient presentation, impact the sensitivity, specificity, and positive and negative predictive values of the assays to diagnose CDI. There were minimal changes in sensitivity; however, specificity was significantly lower for the assays Tox A/B II, C. diff Chek-60, BD GeneOhm Cdiff, Xpert C. difficile, and Illumigene C. difficile and for toxigenic culture (P was <0.01 for all except Tox A/B II from fresh stool, for which the P value was 0.016) when the reference standard was recovery of toxigenic C. difficile from stool plus the presence of clinically significant diarrhea compared to when the reference standard was having at least four assays positive while ignoring diarrhea severity. There were 15 patients whose assay result was reported as negative but subsequently found to be positive by at least four assays in the comparison. None suffered from any CDI-related adverse events. In conclusion, clinical presentation is important when interpreting C. difficile diagnostic assays. PMID:21697328
Bobr, Aleh; Kuskowski, Michael A.; Johnston, Brian D.; Sadowsky, Michael J.; Khoruts, Alexander
Recurrent Clostridium difficile infection (R-CDI) is common and difficult to treat, potentially necessitating fecal microbiota transplantation (FMT). Although C. difficile spores persist in the hospital environment and cause infection, little is known about their potential presence or importance in the household environment. Households of R-CDI subjects in the peri-FMT period and of geographically matched and age-matched controls were analyzed for the presence of C. difficile. Household environmental surfaces and fecal samples from humans and pets in the household were examined. Households of post-FMT subjects were also examined (environmental surfaces only). Participants were surveyed regarding their personal history and household cleaning habits. Species identity and molecular characteristics of presumptive C. difficile isolates from environmental and fecal samples were determined by using the Pro kit (Remel, USA), Gram staining, PCR, toxinotyping, tcdC gene sequencing, and pulsed-field gel electrophoresis (PFGE). Environmental cultures detected C. difficile on ≥1 surface in 8/8 (100%) peri-FMT households, versus 3/8 (38%) post-FMT households and 3/8 (38%) control households (P = 0.025). The most common C. difficile-positive sites were the vacuum (11/27; 41%), toilet (8/30; 27%), and bathroom sink (5/29; 17%). C. difficile was detected in 3/36 (8%) fecal samples (two R-CDI subjects and one household member). Nine (90%) of 10 households with multiple C. difficile-positive samples had a single genotype present each. In conclusion, C. difficile was found in the household environment of R-CDI patients, but whether it was found as a cause or consequence of R-CDI is unknown. If household contamination leads to R-CDI, effective decontamination may be protective. PMID:26921425
Orden, Cristina; Blanco, Jose L; Álvarez-Pérez, Sergio; Garcia-Sancho, Mercedes; Rodriguez-Franco, Fernando; Sainz, Angel; Villaescusa, Alejandra; Harmanus, Celine; Kuijper, Ed; Garcia, Marta E
The prevalence of Clostridium difficile in 107 dogs with diverse digestive disorders attended in a Spanish veterinary teaching hospital was assessed. The microorganism was isolated from 13 dogs (12.1%) of different disease groups. Isolates belonged to PCR ribotypes 078, 106, 154 and 430 (all of them toxigenic) and 110 (non-toxigenic), and were resistant to several antimicrobial drugs. Notably, seven isolates obtained from different dogs displayed stable resistance to metronidazole. The results of this study provide further evidence that dogs can act as a reservoir of C. difficile strains of epidemic ribotypes with resistance to multiple antibiotics.
Wullt, M; Norén, T; Ljungh, A; Åkerlund, T
IgG antibodies against Clostridium difficile toxins A and B were followed in controls and in patients with an initial C. difficile infection (CDI). Of the 50 CDI patients, 38 were cured and 12 developed recurrence. Compared to controls, patients had significantly lower anti-toxin A and B IgGs at inclusion, but the subsequent levels rose slightly regardless of clinical outcome. The results imply that the general serum reactivity against toxins A and B in the population reduces the risk of CDI, which suggests implications for vaccine strategies.
Kaslow, David C; Shiver, John W
Both Clostridium difficile and Staphylococcus aureus asymptomatically colonize a significant percentage of humans, particularly during the first year of life. The epidemiology of both has been and continues to be quite dynamic; presently, we are in the midst of epidemics of infections by C. difficile and S. aureus. These ancient microbes are now armed with more potent virulence factors, which have extended their reach from the hospital into community settings, and from the elderly and debilitated hosts into the younger and otherwise healthy population. This review presents some emerging concepts that will likely shape efforts to develop active and passive immunization interventions in response to the reemergence of these bacterial pathogens.
Roberts, Adam P; Allan, Elaine; Mullany, Peter
Clostridium difficile infection (CDI) is now recognised as the main cause of healthcare associated diarrhoea. Over the recent years there has been a change in the epidemiology of CDI with certain related strains dominating infection. These strains have been termed hyper-virulent and have successfully spread across the globe. Many C. difficile strains have had their genomes completely sequenced allowing researchers to build up a very detailed picture of the contribution of horizontal gene transfer to the adaptive potential, through the acquisition of mobile DNA, of this organism. Here, we review and discuss the contribution of mobile genetic elements to the biology of this clinically important pathogen.
Indra, Alexander; Schmid, Daniela; Huhulescu, Steliana; Simons, Erica; Hell, Markus; Stickler, Karl; Allerberger, Franz
A prospective, noninterventional survey was conducted among Clostridium difficile positive patients identified in the time period of July until October 2012 in 18 hospitals distributed across all nine Austrian provinces. Participating hospitals were asked to send stool samples or isolates from ten successive patients with C.difficile infection to the National Clostridium difficile Reference Laboratory at the Austrian Agency for Health and Food Safety for PCR-ribotyping and in vitro susceptibility testing. A total of 171 eligible patients were identified, including 73 patients with toxin-positive stool specimens and 98 patients from which C. difficile isolates were provided. Of the 159 patients with known age, 127 (74.3%) were 65 years or older, the median age was 76 years (range: 9-97 years), and the male to female ratio 2.2. Among these patients, 73% had health care-associated and 20% community-acquired C. difficile infection (indeterminable 7%). The all-cause, 30-day mortality was 8.8% (15/171). Stool samples yielded 46 different PCR-ribotypes, of which ribotypes 027 (20%), 014 (15.8%), 053 (10.5%), 078 (5.3%), and 002 (4.7%) were the five most prevalent. Ribotype 027 was found only in the provinces Vienna, Burgenland, and Lower Austria. Severe outcome of C. difficile infection was found to be associated with ribotype 053 (prevalence ratio: 3.04; 95% CI: 1.24, 7.44), not with the so-called hypervirulent ribotypes 027 and 078. All 027 and 053 isolates exhibited in vitro resistance against moxifloxacin. Fluoroquinolone use in the health care setting must be considered as a factor favoring the spread of these fluoroquinolone resistant C. difficile clones.
Collins, Laura E; Lynch, Mark; Marszalowska, Izabela; Kristek, Maja; Rochfort, Keith; O'Connell, Mary; Windle, Henry; Kelleher, Dermot; Loscher, Christine E
Clostridium difficile is the leading cause of hospital-acquired diarrhoea worldwide, and if the bacterium is not cleared effectively it can pose a risk of recurrent infections and complications such as colitis, sepsis and death. In this study we demonstrate that surface layer proteins from the one of the most frequently acquired strains of C. difficile, activate mechanisms in murine macrophage in vitro that are associated with clearance of bacterial infection. Surface layer proteins (SLPs) isolated from C. difficile induced the production of pro-inflammatory cytokines and chemokines and increased macrophage migration and phagocytotic activity in vitro. Furthermore, we also observed up-regulation of a number of cell surface markers on the macrophage, which are important in pathogen recognition and antigen presentation. The effects of SLPs on macrophages were reversed in the presence of a p38 inhibitor, indicating the potential importance of this signalling protein in how SLP activates the immune system. In conclusion this study shows that surface layer proteins from a common strain of C. difficile can activate a clearance response in macrophage and suggests that these proteins are important in clearance of C. difficile infection. Understanding how the immune system clears C. difficile infection could offer important insights for new treatment strategies.
Shilling, Michael; Matt, Laurie; Rubin, Evelyn; Visitacion, Mark Paul; Haller, Nairmeen A; Grey, Scott F; Woolverton, Christopher J
Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide; in addition, the proliferation of antibiotic-resistant C. difficile is becoming a significant problem. Virgin coconut oil (VCO) has been shown previously to have the antimicrobial activity. This study evaluates the lipid components of VCO for the control of C. difficile. VCO and its most active individual fatty acids were tested to evaluate their antimicrobial effect on C. difficile in vitro. The data indicate that exposure to lauric acid (C12) was the most inhibitory to growth (P<.001), as determined by a reduction in colony-forming units per milliliter. Capric acid (C10) and caprylic acid (C8) were inhibitory to growth, but to a lesser degree. VCO did not inhibit the growth of C. difficile; however, growth was inhibited when bacterial cells were exposed to 0.15-1.2% lipolyzed coconut oil. Transmission electron microscopy (TEM) showed the disruption of both the cell membrane and the cytoplasm of cells exposed to 2 mg/mL of lauric acid. Changes in bacterial cell membrane integrity were additionally confirmed for VCO and select fatty acids using Live/Dead staining. This study demonstrates the growth inhibition of C. difficile mediated by medium-chain fatty acids derived from VCO.
Romano, Maria R; Leuzzi, Rosanna; Cappelletti, Emilia; Tontini, Marta; Nilo, Alberto; Proietti, Daniela; Berti, Francesco; Costantino, Paolo; Adamo, Roberto; Scarselli, Maria
Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen is not currently available. C. difficile strains produce two main toxins (TcdA and TcdB) and express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most C. difficile ribotypes offering the opportunity of the development of a carbohydrate-based vaccine. In this paper, we evaluate the efficacy, in naive mice model, of PSII glycoconjugates where recombinant toxins A and B fragments (TcdA_B2 and TcdB_GT respectively) have been used as carriers. Both glycoconjugates elicited IgG titers anti-PSII although only the TcdB_GT conjugate induced a response comparable to that obtained with CRM197. Moreover, TcdA_B2 and TcdB_GT conjugated to PSII retained the ability to elicit IgG with neutralizing activity against the respective toxins. These results are a crucial proof of concept for the development of glycoconjugate vaccines against C. difficile infection (CDI) that combine different C. difficile antigens to potentially prevent bacterial colonization of the gut and neutralize toxin activity.
Hunt, Jonathan J.
SUMMARY Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile, now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade. PMID:24296572
Pilling, Sally; Vernon, Jonathan; Wilcox, Mark H.
ABSTRACT Treatments for Clostridium difficile infection remain limited, despite the introduction of fidaxomicin, and development of new agents is necessary. We determined the in vitro susceptibilities of 199 prevalent or emerging Clostridium difficile PCR ribotypes to MCB3681, a novel investigational quinolonyl-oxazolidinone, and 8 comparators (metronidazole, vancomycin, fidaxomicin, moxifloxacin, ciprofloxacin, clindamycin, tigecycline, and linezolid). MCB3681 showed good activity against C. difficile with no evidence of MCB3681 resistance in isolates showing either moxifloxacin or linezolid resistance or both moxifloxacin and linezolid resistance. PMID:27993853
Kaur, Sukhminderjit; Vaishnavi, Chetana; Prasad, Kaushal Kishor; Ray, Pallab; Kochhar, Rakesh
Background & objectives: Clostridium difficile-associated disease (CDAD) remains an important nosocomial ailment. Antimicrobial therapy used for CDAD gives inconsistent results. This experimental study was planned to investigate the beneficial effects of Lactobacillus acidophilus and epidermal growth factor (EGF) for CDAD management. Methods: Among 10 groups of BALB/c mice (6 in each), group 1 served as controls receiving no inoculum. Animals in groups 2-10 received C. difficile, those in groups 3, 6 and 9 received L. acidophilus and those in groups 4, 7 and 10 received EGF after C. difficile inoculation. Animals in groups 5-7 were pre-treated with ampicillin and those in groups 8-10 with lansoprazole prior to C. difficile. The animals were killed and investigated for colonisation by C. difficile and toxin production, myeloperoxidase (MPO) activity and histopathology. Results: Colonisation by C. difficile was found to be significantly different (P<0.001) in the various groups. C. difficile toxin titres and MPO activity were significantly lower in animals given L. acidophilus and EGF after ampicillin (groups 6 and 7) and lansoprazole (groups 9 and 10). The severity of acute inflammation was also significantly less (P<0.05) in caecal and colonic segments of animals in groups 6 and 7 compared to those in group 5. Although the severity of acute inflammation was less in the caecal and colonic segment of animals in groups 9 and 10, the reduction was not significant compared to group 8. Interpretation & conclusions: Our findings showed that the administration of L. acidophilus and EGF reduced the severity of C. difficile infection in the experimental animals. PMID:21537099
Legaria, M C; Lumelsky, G; Rosetti, S
Clostridium difficile is responsible for 15-25% of all cases of antibiotic associated diarrhea. The incidence of infection with this organism is increasing in hospitals worldwide, consequent to the widespread use of broad-spectrum antibiotics. Although the clinical and financial impact of nosocomial C. difficile infection is believed to be significant, only limited information is available on the importance of C. difficile as a cause of diarrhea in Argentina. The aim of the study was to evaluate the impact and diagnosis methods of CDAD from symptomatic patients in a general hospital from Argentina. Consecutive diarrheal stool samples from symptomatic patients from a General Hospital in Argentina were screened for toxigenic C. difficile between April 2000 and April 2001. Toxins were detected in stools by the Premier Cytoclone A+B EIA. Each specimen was examined for toxigenic C. difficile strains by culture. From 104 specimens, 40 (38.5%) [32 of 87 patients (36.8%)] were positive and 64 (61.5%) [55 of 87 patients (63.2%)] were negative by stool toxin assay and/or toxigenic culture. In 11 of 40 positives samples C. difficile toxins were detected only by toxigenic culture. Five (15.6%) patients presented with symptomatic recurrences. Toxin-negative strains were not isolated. This data indicates that the high prevalence of toxigenic strains of C. difficile is of concern in routine diagnostic testing for C. difficile toxins in our study population. Detection of toxins in stools by EIA, coupled with testing strains for toxigenicity only in those cases in which direct toxin assay produces negative results, may be a satisfactory strategy. CDAD is an emerging nosocomial problem in our hospital. It will be necessary to evaluate the epidemiology and measures to control nosocomial spread.
Kim, Eun Jin; Lee, Sung Hyun; Tchah, Hann
Purpose Clinical symptoms associated with Clostridium difficile infection (CDI) can vary widely. Carrier state without apparent symptoms is relatively common during infancy. The objective of this study was to determine the association of C. difficile colonization with bowel habit change and the effect of C. difficile colonization treatment on restoration of normal bowel habit. Methods Between 2006 and 2014, infants at 1 to 12 months of age with diarrhea for more than 2 weeks who did not improve with conservative care were recruited from Gachon University Gil Medical Center. Infants who were followed up for at least 7 days were included. The presence or absence of C. difficile colonization, effect of metronidazole, and other medical records were reviewed. To determine the association between CDI and bowel habit change, logistic regression analysis was used. Results Of a total of 126 infants, 74 (58.7%) were male patients. Of the 126 patients, 27 (21.4%) had C. difficile colonization. Significant (p<0.05) risk factors for C. difficile colonization included artificial milk feeding (odds ratio [OR], 4.310; 95% confidence interval [CI], 1.564-11.878), prior rotavirus vaccination (OR, 4.322; 95% CI, 1.018-18.349), and antibiotic use (OR, 4.798; 95% CI, 1.430-16.101). There was improvement in bowel habit after metronidazole therapy (OR, 0.34; 95% CI, 0.15-0.79; p<0.05), regardless of the presence or absence of C. difficile colonization. Conclusion There was no significant correlation between bowel habit change and C. difficile colonization during infancy. However, metronidazole can be used as an optional method to manage functional gastrointestinal disorders.
Baldan, Rossella; Bianchini, Valentina; Ossi, Cristina; Cichero, Paola; Cirillo, Daniela M.
Clostridium difficile infection (CDI), one of the most common hospital-acquired infections, is increasing in incidence and severity with the emergence and diffusion of hypervirulent strains. CDI is precipitated by antibiotic treatment that destroys the equilibrium of the gut microbiota. Human α-defensin 5 (HD5), the most abundant enteric antimicrobial peptide, is a key regulator of gut microbiota homeostasis, yet it is still unknown if C. difficile, which successfully evades killing by other host microbicidal peptides, is susceptible to HD5. We evaluated, by means of viability assay, fluorescence-activated cell sorter (FACS) analysis, and electron microscopy, the antimicrobial activities of α-defensins 1 and 5 against a panel of C. difficile strains encompassing the most prevalent epidemic and hypervirulent PCR ribotypes in Europe (012, 014/020, 106, 018, 027, and 078). Here we show that (i) concentrations of HD5 within the intestinal physiological range produced massive C. difficile cell killing; (ii) HD5 bactericidal activity was mediated by membrane depolarization and bacterial fragmentation with a pattern of damage peculiar to C. difficile bacilli, compared to commensals like Escherichia coli and Enterococcus faecalis; and (iii) unexpectedly, hypervirulent ribotypes were among the most susceptible to both defensins. These results support the notion that HD5, naturally present at very high concentrations in the mucosa of the small intestine, could indeed control the very early steps of CDI by killing C. difficile bacilli at their germination site. As a consequence, HD5 can be regarded as a good candidate for the containment of hypervirulent C. difficile strains, and it could be exploited in the therapy of CDI and relapsing C. difficile-associated disease. PMID:25547793
Mehta, Krunal K; Paskaleva, Elena E; Wu, Xia; Grover, Navdeep; Mundra, Ruchir V; Chen, Kevin; Zhang, Yongrong; Yang, Zhiyong; Feng, Hanping; Dordick, Jonathan S; Kane, Ravi S
Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc.
Shaughnessy, Megan K; Bobr, Aleh; Kuskowski, Michael A; Johnston, Brian D; Sadowsky, Michael J; Khoruts, Alexander; Johnson, James R
Recurrent Clostridium difficile infection (R-CDI) is common and difficult to treat, potentially necessitating fecal microbiota transplantation (FMT). Although C. difficilespores persist in the hospital environment and cause infection, little is known about their potential presence or importance in the household environment. Households of R-CDI subjects in the peri-FMT period and of geographically matched and age-matched controls were analyzed for the presence ofC. difficile Household environmental surfaces and fecal samples from humans and pets in the household were examined. Households of post-FMT subjects were also examined (environmental surfaces only). Participants were surveyed regarding their personal history and household cleaning habits. Species identity and molecular characteristics of presumptive C. difficile isolates from environmental and fecal samples were determined by using the Pro kit (Remel, USA), Gram staining, PCR, toxinotyping, tcdC gene sequencing, and pulsed-field gel electrophoresis (PFGE). Environmental cultures detected C. difficile on ≥1 surface in 8/8 (100%) peri-FMT households, versus 3/8 (38%) post-FMT households and 3/8 (38%) control households (P= 0.025). The most common C. difficile-positive sites were the vacuum (11/27; 41%), toilet (8/30; 27%), and bathroom sink (5/29; 17%).C. difficile was detected in 3/36 (8%) fecal samples (two R-CDI subjects and one household member). Nine (90%) of 10 households with multiple C. difficile-positive samples had a single genotype present each. In conclusion,C. difficile was found in the household environment of R-CDI patients, but whether it was found as a cause or consequence of R-CDI is unknown. If household contamination leads to R-CDI, effective decontamination may be protective.
Peltier, Johann; Courtin, Pascal; El Meouche, Imane; Catel-Ferreira, Manuella; Chapot-Chartier, Marie-Pierre; Lemée, Ludovic; Pons, Jean-Louis
Primary antibiotic treatment of Clostridium difficile intestinal diseases requires metronidazole or vancomycin therapy. A cluster of genes homologous to enterococcal glycopeptides resistance vanG genes was found in the genome of C. difficile 630, although this strain remains sensitive to vancomycin. This vanG-like gene cluster was found to consist of five ORFs: the regulatory region consisting of vanR and vanS and the effector region consisting of vanG, vanXY and vanT. We found that 57 out of 83 C. difficile strains, representative of the main lineages of the species, harbour this vanG-like cluster. The cluster is expressed as an operon and, when present, is found at the same genomic location in all strains. The vanG, vanXY and vanT homologues in C. difficile 630 are co-transcribed and expressed to a low level throughout the growth phases in the absence of vancomycin. Conversely, the expression of these genes is strongly induced in the presence of subinhibitory concentrations of vancomycin, indicating that the vanG-like operon is functional at the transcriptional level in C. difficile. Hydrophilic interaction liquid chromatography (HILIC-HPLC) and MS analysis of cytoplasmic peptidoglycan precursors of C. difficile 630 grown without vancomycin revealed the exclusive presence of a UDP-MurNAc-pentapeptide with an alanine at the C terminus. UDP-MurNAc-pentapeptide [d-Ala] was also the only peptidoglycan precursor detected in C. difficile grown in the presence of vancomycin, corroborating the lack of vancomycin resistance. Peptidoglycan structures of a vanG-like mutant strain and of a strain lacking the vanG-like cluster did not differ from the C. difficile 630 strain, indicating that the vanG-like cluster also has no impact on cell-wall composition.
Drigo, Ilenia; Mazzolini, Elena; Bacchin, Cosetta; Tonon, Elena; Puiatti, Cinzia; Bano, Luca; Spigaglia, Patrizia; Barbanti, Fabrizio; Agnoletti, Fabrizio
Clostridium difficile is an important cause of enteric disease in humans and animals. Recent studies demonstrated a genetic overlap between C. difficile isolated from animals and humans suggesting animals as possible reservoir for human pathogenic strains. This study was a preliminary investigation on the occurrence of C. difficile in rabbits raised in industrial holdings for food production and aimed to characterise isolates and estimate their antimicrobial susceptibility. C. difficile isolates were characterized by toxin profiles, toxinotyping and PCR-ribotyping. The MICs of six antibiotics were determined using E-test. Between 2007 and 2013, 285 industrial holdings (representing 40% of the national census) submitted rabbits to our laboratory for diagnostic purposes, among these holdings, groups of three to five post-weaned rabbits were sampled once by convenience. 1279 samples of caecal content were collected. The overall isolation rate of C. difficile from the enteric specimen was 3% (38/1279), with no difference among animals affected or not by enteric disorders. Among isolates 66% (25/38) were toxigenic. Sixteen different PCR-ribotypes (RTs) were identified. Among the toxigenic strains RT-014/020, RT-078 and RT-012 were found in at least three rabbit holdings. According to the ECOFF threshold, 82% (31/38) C. difficile isolates displayed a reduced susceptibility to at least one and 18% (7/38) to three tested antimicrobials. Rabbits are colonized by heterogeneous C. difficile ribotypes many of which are commonly isolated in humans. One third of isolates displayed a reduced susceptibility to MTZ, the first choice antimicrobial for human CDI treatment. According to our findings rabbits are a potential source of C. difficile for humans.
Krutova, Marcela; Matejkova, Jana; Kuijper, Ed J; Drevinek, Pavel; Nyc, Otakar
In 2014, 18 hospitals in the Czech Republic participated in a survey of the incidence of Clostridium difficile infections (CDI) in the country. The mean CDI incidence was 6.1 (standard deviation (SD):7.2) cases per 10,000 patient bed-days and 37.8 cases (SD: 41.4) per 10,000 admissions. The mean CDI testing frequency was 39.5 tests (SD: 25.4) per 10,000 patient bed-days and 255.8 tests (SD: 164.0) per 10,000 admissions. A total of 774 C. difficile isolates were investigated, of which 225 (29%) belonged to PCR ribotype 176, and 184 isolates (24%) belonged to PCR ribotype 001. Multilocus variable-number tandem repeat analysis (MLVA) revealed 27 clonal complexes formed by 84% (190/225) of PCR ribotype 176 isolates, and 14 clonal complexes formed by 77% (141/184) of PCR ribotype 001 isolates. Clonal clusters of PCR ribotypes 176 and 001 were observed in 11 and 7 hospitals, respectively. Our data demonstrate the spread of two C. difficile PCR ribotypes within 18 hospitals in the Czech Republic, stressing the importance of standardising CDI testing protocols and implementing mandatory CDI surveillance in the country.
Ganc, Arnaldo José; Ganc, Ricardo Leite; Reimão, Sílvia Mansur; Frisoli Junior, Alberto; Pasternak, Jacyr
Clostridium difficile is the major etiological agent of pseudomembranous colitis and is found in up to 20% of adult inpatients. The recommended treatment is antibiotic therapy with metronidazole and/or vancomycin. However, the recurrence rate may reach up to 25% and it increases in each episode. The newest alternative to treat diarrhea due to recurrent Clostridium difficile is fecal microbiota transplantation. The procedure was performed in 12 patients, with a 6-month follow-up on 10 of them. Of the ten cases, bacterial recurrence was diagnosed in only one patient, after a course of antibiotic to treat urinary tract infection, without presenting with diarrhea. The particularity of our study, besides being an unprecedented event in South America, is the way to perform the infusion of fecal microbiota by enteroscopy.
Quezada, Felipe; Castillo, Richard; Villalón, Constanza; Zúñiga, José Miguel; Manterola, Carla; Molina, María Elena; Bellolio, Felipe; Urrejola, Gonzalo
A loop ileostomy with intraoperative anterograde colonic lavage has been described as an alternative to colectomy in the management of cases of Clostridium difficile infection refractory to medical treatment. We report a 69 years old diabetic women admitted with a septic shock. An abdominal CAT scan showed a pan-colitis that seemed to be infectious. A polymerase chain reaction was positive for Clostridium Difficile. Due to the failure to improve after full medical treatment, a derivative loop ileostomy and intra-operatory colonic lavage were performed, leaving a Foley catheter in the proximal colon. In the postoperative period, anterograde colonic instillations of Vancomycin flushes through the catheter were performed every 6 hours. Forty eight hours after surgery, the patient improved. A colonoscopy prior to discharge showed resolution of the pseudomembranous colitis.
Ganc, Arnaldo José; Ganc, Ricardo Leite; Reimão, Sílvia Mansur; Frisoli, Alberto; Pasternak, Jacyr
ABSTRACT Clostridium difficile is the major etiological agent of pseudomembranous colitis and is found in up to 20% of adult inpatients. The recommended treatment is antibiotic therapy with metronidazole and/or vancomycin. However, the recurrence rate may reach up to 25% and it increases in each episode. The newest alternative to treat diarrhea due to recurrent Clostridium difficile is fecal microbiota transplantation. The procedure was performed in 12 patients, with a 6-month follow-up on 10 of them. Of the ten cases, bacterial recurrence was diagnosed in only one patient, after a course of antibiotic to treat urinary tract infection, without presenting with diarrhea. The particularity of our study, besides being an unprecedented event in South America, is the way to perform the infusion of fecal microbiota by enteroscopy. PMID:26154556
Rustandi, Richard R; Wang, Feng; Lancaster, Catherine; Kristopeit, Adam; Thiriot, David S; Heinrichs, Jon H
Ion-exchange (IEX) chromatography is one of many separation techniques that can be employed to analyze proteins. The separation mechanism is based on a reversible interaction between charged amino acids of a protein to the charged ligands attached to a column at a given pH. This interaction depends on both the pI and conformation of the protein being analyzed. The proteins are eluted by increasing the salt concentration or pH gradient. Here we describe the use of this technique to characterize the charge variant heterogeneities and to monitor stability of four protein antigen components of a Clostridium difficile vaccine. Furthermore, the IEX technique can be used to monitor reversion to toxicity for formaldehyde-treated Clostridium difficile toxins.
Cowardin, Carrie A.; Buonomo, Erica L.; Saleh, Mahmoud M.; Wilson, Madeline G.; Burgess, Stacey L.; Kuehne, Sarah A.; Schwan, Carsten; Eichhoff, Anna M.; Koch-Nolte, Friedrich; Lyras, Dena; Aktories, Klaus; Minton, Nigel P.; Petri, William A.
Clostridium difficile is the most common hospital acquired pathogen in the United States, and infection is in many cases fatal. Toxins A and B are its major virulence factors, but increasingly a third toxin may be present, known as C. difficile transferase (CDT). An ADP-ribosyltransferase that causes actin cytoskeletal disruption, CDT is typically produced by the major, hypervirulent strains and has been associated with more severe disease. Here we show that CDT enhances the virulence of two PCR-ribotype 027 strains in mice. The toxin induces pathogenic host inflammation via a Toll-like Receptor 2 (TLR2) dependent pathway, resulting in the suppression of a protective host eosinophilic response. Finally, we show that restoration of TLR2 deficient eosinophils is sufficient for protection from a strain producing CDT. These findings offer an explanation for the enhanced virulence of CDT-expressing C. difficile and demonstrate a mechanism by which this binary toxin subverts the host immune response. PMID:27573114
Brechmann, Thorsten; Swol, Justyna; Knop-Hammad, Veronika; Willert, Jörg; Aach, Mirko; Cruciger, Oliver; Schmiegel, Wolff; Schildhauer, Thomas A; Hamsen, Uwe
A 65-year-old male suffering from acute spinal cord injury leading to incomplete tetraplegia presented with severe recurrent Clostridium difficile (C. difficile) infection subsequent to antibiotic treatment for pneumonia. After a history of ineffective antimicrobial therapies, including metronidazole, vancomycin, fidaxomicin, rifaximin and tigecycline, leading to several relapses, the patient underwent colonoscopic fecal microbiota transplantation from his healthy son. Four days subsequent to the procedure, the patient showed a systemic inflammation response syndrome. Without detecting an infectious cause, the patient received antimicrobial treatment, including tigecycline, metronidazole, vancomycin via polyethylene glycol and an additional enema for a period of seven days, leading to a prompt recovery and no reported C. difficile infection relapse during a 12 wk follow up.
Pruitt, Rory N.; Lacy, D. Borden
Clostridium difficile is a toxin-producing bacterium that is a frequent cause of hospital-acquired and antibiotic-associated diarrhea. The incidence, severity, and costs associated with C. difficile associated disease are substantial and increasing, making C. difficile a significant public health concern. The two primary toxins, TcdA and TcdB, disrupt host cell function by inactivating small GTPases that regulate the actin cytoskeleton. This review will discuss the role of these two toxins in pathogenesis and the structural and molecular mechanisms by which they intoxicate cells. A focus will be placed on recent publications highlighting mechanistic similarities and differences between TcdA, TcdB, and different TcdB variants. PMID:22919620
Duncan, Peter I; Fotopoulos, Grigorios; Pasche, Elisabeth; Porta, Nadine; Masserey Elmelegy, Isabelle; Sanchez-Garcia, Jose-Luis; Bergonzelli, Gabriela E; Corthésy-Theulaz, Irène
Clostridium difficile is responsible for a large proportion of nosocomial cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present study provides evidence that yeast, beef and pork extracts, ingredients commonly used to grow bacteria, can counteract C. difficile toxin A enterotoxicity in vitro and in vivo. In model intestinal epithelial cells the individual extracts could prevent the toxin A-induced decrease in epithelial barrier function and partially prevented actin disaggregation and cell rounding. Mice with ad libitum access to individual extracts for 1 week had almost complete reduction in toxin A-induced fluid secretion in intestinal loops. Concomitantly, the toxin A-induced expression of the essential proinflammatory mediator Cox-2 was normalized. Moreover this protective effect was also seen when mice received only two doses of extract by intragastric gavage within 1 week. These results show that yeast, beef and pork extracts have the potential to counteract the intestinal pathogenesis triggered by C. difficile toxin A.
Tan, Christopher B; Rajan, Dhyan; Shah, Mitanshu; Ahmed, Shadab; Freedman, Lester; Rizvon, Kaleem; Mustacchia, Paul
Pseudomembranous colitis and toxic megacolon (TM) are well-known complications of Clostridium difficile infections. Systemic antibiotic is considered as the major risk factor for the development of C difficile colitis. However, topical antibiotics are rarely associated with the infection. As previously thought, the use of topical antibiotic is capable of systemic absorption in damaged and denuded skin; sufficient enough to suppress the normal bowel flora. Here, we present an unusual case of TM from C difficile infection induced by topical silver sulphadiazine in a 60-year-old man with immune-bullous pemphigus vulgaris. The diagnosis is further complicated by the absence of diarrhoea as the initial presentation. Despite adequate medical and surgical intervention, the patient had an unfavourable outcome.
Oberli, Matthias A; Hecht, Marie-Lyn; Bindschädler, Pascal; Adibekian, Alexander; Adam, Thomas; Seeberger, Peter H
Nosocomial infections with the Gram-positive pathogen Clostridium difficile pose a major risk for hospitalized patients and result in significant costs to health care systems. Here, we present the chemical synthesis of a PS-II hapten of a cell wall polysaccharide of hypervirulent ribotype 027 of C. difficile. Mice were immunized with a conjugate consisting of the synthetic hexasaccharide and the diphtheria toxoid variant CRM(197). The immunogenicity of the glycan repeating unit was demonstrated by the presence of specific IgG antibodies in the serum of immunized mice. Murine monoclonal antibodies interact with the synthetic hexasaccharide, as determined by microarray analysis. Finally, we found that specific IgA antibodies in the stool of hospital patients infected with C. difficile recognize the synthetic PS-II hexasaccharide hapten.
Singh, R; Nieuwdorp, M; ten Berge, I J M; Bemelman, F J; Geerlings, S E
This review gives an outline of the indications for faecal microbiota transplantation (FMT) for diseases other than Clostridium difficile (C. difficile) infection. The remarkable efficacy of FMT against C. difficile infection has already been demonstrated. The use of FMT for other diseases, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and metabolic syndrome, is now being evaluated. The currently available data suggest that FMT might be beneficial for IBD (including ulcerative colitis and, to some extent, Crohn's disease), IBS, and insulin resistance. Several randomized clinical trials are currently being performed, and data are eagerly awaited. A new field of research for the implementation of FMT is the eradication of pathogenic and multiresistant enteric microorganisms. A few animal studies have been performed within this field, but hardly any research data from human studies are available at present.
Songer, J Glenn; Trinh, H T; Dial, Sharon M; Brazier, Jon S; Glock, Robert D
A 14-year-old Quarter Horse with a 48-hr history of colic was euthanized after failure to respond to treatment. At necropsy, cecal and colonic mucosae were congested throughout, and there was segmental edema and significant thickening of the intestinal wall. Excessive numbers of mononuclear cells were found in mucosal lamina propria. Submucosal hemorrhage was diffuse and extensive, and Clostridium difficile toxins A and B were detected. Large numbers of C. difficile were isolated, and genetic characterization revealed them to be North American pulsed-field gel electrophoresis type 1, polymerase chain reaction ribotype 027, and toxinotype III. Genes for the binary toxin were present, and toxin negative-regulator tcdC contained an 18-bp deletion. This genotype comprises the current human "epidemic strain," which is associated with human C. difficile-associated disease of greater than historical severity. The diagnosis was peracute typhlocolitis, with lesions and history typical of those attributed to colitis X.
Mullish, B H; Marchesi, J R; Thursz, M R; Williams, H R T
Faecal microbiome transplantation (FMT) has generated huge recent interest as it presents a potential solution to a significant clinical problem--the increasing incidence of Clostridium difficile infection (CDI). In the short term, however, there remain many practical questions regarding its use, including the optimal selection of donors, material preparation and the mechanics of delivery. In the longer term, enhanced understanding of the mechanisms of action of FMT may potentiate novel therapies, such as targeted manipulation of the microbiome in CDI and beyond.
Fehér, Csaba; Mensa, Josep
Clostridium difficile infection (CDI) is increasingly recognized as an emerging healthcare problem of elevated importance. Prevention and treatment strategies are constantly evolving along with the apperance of new scientific evidence and novel treatment methods, which is well-reflected in the differences among consecutive international guidelines. In this article, we summarize and compare current guidelines of five international medical societies on CDI management, and discuss some of the controversial and currently unresolved aspects which should be addressed by future research.
Woodworth, Michael H; Neish, Emma M; Miller, Nancy S; Dhere, Tanvi; Burd, Eileen M; Carpentieri, Cynthia; Sitchenko, Kaitlin L; Kraft, Colleen S
Fecal microbiota transplantation is an efficacious and inexpensive therapy for recurrent Clostridium difficile infection, yet its safety is thought to depend on appropriate fecal donor screening. FDA guidance for regulation of this procedure is in flux, but screening and manufacture of fecal material from asymptomatic donors present many challenges to clinical laboratories. This minireview summarizes FDA regulatory changes, principles of donor selection, and recommended laboratory screening practices for fecal microbiota transplantation.
Kouzegaran, Samaneh; Ganjifard, Mahmood; Tanha, Amir Saber
Background: Clostridium difficile is the most prevalent cause of antibiotic-associated infectious diarrhea al-around the world. Prevalence of virulent and resistant strains of Clostridium difficile is increasing now a day. The present investigation was carried out to study the prevalence, ribotyping and antibiotic resistance pattern of C. difficile isolated from diarrheic and non-diarrheic pediatrics. Materials and methods: Four-hundred stool specimens were collected from the diarrheic and non-diarrheic pediatrics hospitalized due to the diseases other than diarrhea. Samples were cultured and their positive results were subjected to disk diffusion and PCR-based ribotyping. Results: Thirty-five out of 400 (8.75%) samples were positive for C. difficile. Prevalence of C. difficile in diarrheic and non-diarrheic pediatrics were 11.25% and 4.16%, respectively. Male had the higher prevalence of bacteria than female (P < 0.05). eight to twelve months old pediatrics were the most commonly infected group. R27 (14.28%), R1 (10.71%), R12 (7.14%), R13 (7.14%) and R18 (7.14%) were most commonly detected ribotypes. There were no positive results for studied ribotypes in non-diarrheic pediatrics. C. difficile strains had the highest levels of resistance against tetracycline (71.42%), erythromycin (57.14%), moxifloxacin (48.57%), metronidazole (28.57%) and clindamycin (22.85%) antibiotics. Conclusion: Prescription of antibiotics in diarrheic pediatrics, males and also 8-12 months old pediatrics should be done in a regular and cautious manner. PMID:27999477
Chen, Jonathan H K; Cheng, Vincent C C; Wong, Oi-Ying; Wong, Sally C Y; So, Simon Y C; Yam, Wing-Cheong; Yuen, Kwok-Yung
The clinical workflow of using chromogenic agar and matrix-assisted laser desorption ionization time-of-fight mass spectrometry (MALDI-TOF MS) for Clostridium difficile identification was evaluated. The addition of MALDI-TOF MS identification after the chromID C. difficile chromogenic agar culture could significantly improve the diagnostic accuracy of C. difficile.
Ya-Nan, Guo; Hao-Jun, Zhang; Hong-Bing, Jia; Ping, Li; Xin-Zhu, Liu
Traditional Chinese medicine QPYF has a good effect for treating antibiotic-associated diarrhea in clinical practice. The aim of this study is to test its efficacy to prevent Clostridium difficile associated diarrhea (CDAD) in a mouse model. C57BL/6 mice were infected with Clostridium difficile VPI 10463 after exposure to antimicrobial mixture. QPYF was administered from 7 days prior to Clostridium difficile infection to 20 days after infection, and its effect was compared with no treatment and receiving placebo. The mice were monitored for 20 days and the percent survival, disease activity index, weight loss, colon histopathology, and the levels of toxins in the feces were measured. The expressions of TNF α, MCP-1, NF-κB p65, and phospho-NF-κB p65 in the colon were presented by immunohistochemistry. The survival rate of QPYF group (93.75%) was higher than that of model control group (65%). The mice treated with QPYF had a lower weight loss and disease activity index, compared to the mice with placebo. A significantly lower level of histopathology scores, toxins in the feces, and TNF α, MCP-1, NF-κB p65, and phospho-NF-κB p65 were detected for QPYF-treated mice. Traditional Chinese medicine QPYF showed a good preventive effect for CDAD in a mouse model. PMID:27999606
Agolini, G; Protano, C; Puro, V; Raitano, A; Ferraro, F; Vitali, M
In the last years, Clostridium difficile acquired great interest for public health because of constant increase of Clostridium difficile associated diarrhea (CDAD), especially in nosocomial field and as a consequences of its pathogenicity and virulence. Oro-faecal transmission and great environmental persistence of Clostridium difficile indicate hand hygiene of health care workers and environmental disinfection practices as key interventions for prevention and control of nosocomial CDAD. The current indications relative to the hand hygiene suggest the use of soap and water for hand washing and, to achieve a better compliance of health care workers to this treatment, the alternative use of sodium dichloroisocyanurate or alcohol-based solution or gel waterless. Regard to environmental disinfection, to avoid high concentrations of sodium hypochlorite (in the magnitude of 5.000-6.000 ppm), necessary to reduce microbic load of dirty environment, the most appropriate treatment should consist of 2 phases: preliminary cleaning with water and detergents or polyphenol, followed by treatment with solution containing 1.000 ppm available chlorine, obtained from sodium hypochlorite or sodium dichloroisocyanurate.
Moya, Andrés; Vázquez-Castellanos, Jorge F.; Artacho, Alejandro; Chen, Xinhua; Kelly, Ciaran
ABSTRACT The onset of Clostridium difficile infection (CDI) has been associated with treatment with wide-spectrum antibiotics. Antibiotic treatment alters the activity of gut commensals and may result in modified patterns of immune responses to pathogens. To study these mechanisms during CDI, we separated bacteria with high cellular RNA content (the active bacteria) and their inactive counterparts by fluorescence-activated cell sorting (FACS) of the fecal bacterial suspension. The gut dysbiosis due to the antibiotic treatment may result in modification of immune recognition of intestinal bacteria. The immune recognition patterns were assessed by FACS of bacterial fractions either coated or not with intestinal secretory immunoglobulin A (SIgA). We described the taxonomic distributions of these four bacterial fractions (active versus inactive and SIgA coated versus non-SIgA coated) by massive 16S rRNA gene amplicon sequencing and quantified the proportion of C. difficile toxin genes in the samples. The overall gut microbiome composition was more robustly influenced by antibiotics than by the C. difficile toxins. Bayesian networks revealed that the C. difficile cluster was preferentially SIgA coated during CDI. In contrast, in the CDI-negative group Fusobacterium was the characteristic genus of the SIgA-opsonized fraction. Lactobacillales and Clostridium cluster IV were mostly inactive in CDI-positive patients. In conclusion, although the proportion of C. difficile in the gut is very low, it is able to initiate infection during the gut dysbiosis caused by environmental stress (antibiotic treatment) as a consequence of decreased activity of the protective bacteria. IMPORTANCE C. difficile is a major enteric pathogen with worldwide distribution. Its expansion is associated with broad-spectrum antibiotics which disturb the normal gut microbiome. In this study, the DNA sequencing of highly active bacteria and bacteria opsonized by intestinal secretory immunoglobulin
Girinathan, Brintha P.; Monot, Marc; Boyle, Daniel; McAllister, Kathleen N.; Dupuy, Bruno
ABSTRACT Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin
Background Probiotics have beneficial effects in rodent models of Clostridium difficile (C. diffiicle)-induced colitis. The spore forming probiotic strain Bacillus Coagulans GBI-30, 6086 (BC30) has demonstrated anti-inflammatory and immune-modulating effects in vitro. Our goal was to determine if BC30 improved C. difficile-induced colitis in mice. Starting on study day 0, female C57BL/6 mice were dosed by oro-gastric gavage for 15 days with vehicle (saline) or BC30 (2 × 109 CFU per day). Mice in the C. difficile groups received an antibiotic mixture (study days 5 to 8 in the drinking water), and clindamycin (10 mg/kg, i.p., on study day 10). The C. difficile strain VPI 10463 was given by gavage at 104 CFU to induce colitis on day 11. On day 16, stools and colons were collected for further analyses. Results All mice treated with BC30 survived on study day 13, while two mice treated with vehicle did not survive. On day 12, a significant difference (p = 0.0002) in the percentage of mice with normal stools (66.7%) was found in the BC30/C. difficile group, as compared to the vehicle/C. diffcile group (13.0%). On study day 16, 23.8% of mice treated with BC30 had normal stools, while this value was 0% with vehicle treatment (p value = 0.0187). On this day, the stool consistency score for the BC30/C. difficile group (1.1 ± 0.2) was significantly lower (p < 0.05) than for the vehicle/C. difficile cohort (1.9 ± 0.2). BC30 modestly attenuated the colonic pathology (crypt damage, edema, leukocyte influx) that was present following C. difficile infection. Colonic MIP-2 chemokine contents (pg/2 cm colon) were: 10.2 ± 0.5 (vehicle/no C. difficile), 24.6 ± 9.5 (vehicle/C. difficile) and 16.3 ± 4.3 (BC30/C. difficle). Conclusion The probiotic BC30 improved some parameters of C. difficile-induced colitis in mice. BC30 prolonged the survival of C. diffiicle infected mice. Particularly, this probiotic improved the stool consistency of mice, in this infectious colitis model. PMID
Goldberg, E; Amir, I; Zafran, M; Gophna, U; Samra, Z; Pitlik, S; Bishara, J
We aimed to assess differences in bacterial intensities of Bacteroidetes phylum and different clostridial species in the human intestines with respect to C. difficile infection. Patients with a stool assay for C. difficile toxin were identified via the microbiology laboratory in our institute. Bacterial populations were quantified from stool samples of four groups of patients: Group I-patients with C. difficile associated diarrhea (CDAD); Group II-asymptomatic C. difficile carriers; Group III-patients with non-C. difficile diarrhea; Group IV-patients with no diarrhea and negative stool samples for the C. difficile toxin (control group). Stool was examined for three genes-C. difficile toxin A gene, 16S rRNA gene from Clostridium thermocellum representing other clostridial species, and 16S rRNA gene from Bacteroides fragilis representing the Bacteroidetes phylum. Fifty-nine patients underwent analysis of the stool (CDAD group 14, carriers group 14, non-C. difficile diarrhea group 16, control group 15). C. difficile concentration was highest in the CDAD group, followed by the carriers group. Higher concentrations of both clostridial species and Bacteriodetes were observed in the control and non-C. difficile diarrhea groups compared to the CDAD and carriers groups. We demonstrated an inverse association between infection with C. difficile and the abundance of Bacteroidetes phylum and other clostridial species in human intestines. Studies with larger samples and broader diagnostic procedures are needed in order to better explore and understand this association.
Odo, Chioma; DuPont, Herbert L.
ABSTRACT Clostridium difficile infection (CDI) is responsible for most of the definable cases of antibiotic- and hospital-associated diarrhea worldwide and is a frequent cause of morbidity and mortality in older patients. C. difficile, a multidrug-resistant anaerobic pathogen, causes disease by producing toxins A and B, which are controlled by an accessory gene regulator (Agr) quorum signaling system. Some C. difficile strains encode two Agr loci in their genomes, designated agr1 and agr2. The agr1 locus is present in all of the C. difficile strains sequenced to date, whereas the agr2 locus is present in a few strains. The functional roles of agr1 and agr2 in C. difficile toxin regulation and pathogenesis were unknown until now. Using allelic exchange, we deleted components of both agr loci and examined the mutants for toxin production and virulence. The results showed that the agr1 mutant cannot produce toxins A and B; toxin production can be restored by complementation with wild-type agr1. Furthermore, the agr1 mutant is able to colonize but unable to cause disease in a murine CDI model. These findings have profound implications for CDI treatment because we have uncovered a promising therapeutic target for the development of nonantibiotic drugs to treat this life-threatening emerging pathogen by targeting the toxins directly responsible for disease. PMID:27531912
Connor, Mairéad; Flynn, Padrig B.; Fairley, Derek J.; Marks, Nikki; Manesiotis, Panagiotis; Graham, William G.; Gilmore, Brendan F.; McGrath, John W.
Clostridium difficile is a spore forming bacterium and the leading cause of colitis and antibiotic associated diarrhoea in the developed world. Spores produced by C. difficile are robust and can remain viable for months, leading to prolonged healthcare-associated outbreaks with high mortality. Exposure of C. difficile spores to a novel, non-thermal atmospheric pressure gas plasma was assessed. Factors affecting sporicidal efficacy, including percentage of oxygen in the helium carrier gas admixture, and the effect on spores from different strains representing the five evolutionary C. difficile clades was investigated. Strains from different clades displayed varying resistance to cold plasma. Strain R20291, representing the globally epidemic ribotype 027 type, was the most resistant. However all tested strains displayed a ~3 log reduction in viable spore counts after plasma treatment for 5 minutes. Inactivation of a ribotype 078 strain, the most prevalent clinical type seen in Northern Ireland, was further assessed with respect to surface decontamination, pH, and hydrogen peroxide concentration. Environmental factors affected plasma activity, with dry spores without the presence of organic matter being most susceptible. This study demonstrates that cold atmospheric plasma can effectively inactivate C. difficile spores, and highlights factors that can affect sporicidal activity. PMID:28155914
LaMarche, Matthew J.; Leeds, Jennifer A.; Amaral, Adam; Brewer, Jason T.; Bushell, Simon M.; Deng, Gejing; Dewhurst, Janetta M.; Ding, Jian; Dzink-Fox, JoAnne; Gamber, Gabriel; Jain, Akash; Lee, Kwangho; Lee, Lac; Lister, Troy; McKenney, David; Mullin, Steve; Osborne, Colin; Palestrant, Deborah; Patane, Michael A.; Rann, Elin M.; Sachdeva, Meena; Shao, Jian; Tiamfook, Stacey; Trzasko, Anna; Whitehead, Lewis; Yifru, Aregahegn; Yu, Donghui; Yan, Wanlin; Zhu, Qingming
Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.
Background Clostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria of one strain (PCR ribotype) per animal species per laboratory. Results Altogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates as well as in terms of number of different host species: 078 (14.3% of isolates; 4 hosts), 014/020 (11.6%; 8 hosts); 002 (5.4%; 4 hosts) and 012 (5.4%; 5 hosts). Two animal hosts were best represented; cattle with 31 isolates (20 PCR ribotypes; 7 countries) and pigs with 31 isolates (16 PCR ribotypes; 10 countries). Conclusions This results show that although PCR ribotype 078 is often reported as the major animal C. difficile type, especially in pigs, the variability of strains in pigs and other animal hosts is substantial. Most common human PCR ribotypes (014/020 and 002) are also among most prevalent animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen. PMID:24972659
Norman, Keri N; Harvey, Roger B; Andrews, Kathleen; Hume, Michael E; Callaway, Todd R; Anderson, Robin C; Nisbet, David J
The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America with the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer to humans. The objective of the present study was to investigate the prevalence of C. difficile in retail seafood from grocery stores in College Station, Texas. C. difficile was found in 4.5% (3/67) of shellfish and finfish samples. The positive samples included one each from fresh mussel, frozen salmon and frozen shrimp. The mussel and salmon isolates were characterized as toxinotype V and pulsed-field gel electrophoresis (PFGE) type-NAP7. The shrimp isolate was identified as toxinotype XII, but had an unknown PFGE type. Susceptibilities to 11 antimicrobial agents were identical for the mussel and salmon isolates and were sensitive to eight of 11 antimicrobials (including ampicillin) and intermediate to clindamycin. However, the shrimp isolate was resistant to clindamycin and ampicillin. This study demonstrates that seafood, like other food commodities, can be contaminated by C. difficile.
Batah, Jameel; Denève-Larrazet, Cécile; Jolivot, Pierre-Alain; Kuehne, Sarah; Collignon, Anne; Marvaud, Jean-Christophe; Kansau, Imad
Clostridium difficile has become the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. This has coincided with the appearance of serious cases related to the emergence of hypervirulent strains. The toxins are the main virulence factors and elicit an inflammatory response during C. difficile infection. However, other bacterial components appear to be involved in the inflammatory process. In some pathogens, flagella play a role in pathogenesis through abnormal stimulation of the TLR5-mediated host immune response. To date, few studies have addressed this role for C. difficile flagella. In the current study, we confirm in two different epithelial cell models that C. difficile thanks to its FliC flagellin interacts with TLR5. In addition, thanks to inhibition and transcriptomic studies we demonstrate that the interaction of flagellin and TLR5 predominantly activates the NF-κB and, in a lesser degree, the MAPK pathways, via TLR5, leading to up-regulation of pro-inflammatory gene expression and synthesis of pro-inflammatory mediators. These results suggest a role for C. difficile flagella in contributing to inflammatory response in host intestinal cells.
Xu, Changyun; Salsali, Hamidreza; Weese, Scott; Warriner, Keith
There has been an increase in community-associated Clostridium difficile infections with biosolids derived from wastewater treatment being identified as one potential source. The current study evaluated the efficacy of thermophilic digestion in decreasing levels of C. difficile ribotype 078 associated with sewage sludge. Five isolates of C. difficile 078 were introduced (final density of 5 log CFU/g) into digested sludge and subjected to anaerobic digestion at mesophilic (36 or 42 °C) or thermophilic (55 °C) temperatures for up to 60 days. It was found that mesophilic digestion at 36 °C did not result in a significant reduction in C. difficile spore levels. In contrast, thermophilic sludge digestion reduced endospore levels at a rate of 0.19-2.68 log CFU/day, depending on the strain tested. The mechanism of lethality was indirect - by stimulating germination then inactivating the resultant vegetative cells. Acidification of sludge by adding acetic acid (6 g/L) inhibited the germination of spores regardless of the sludge digestion temperature. In conclusion, thermophilic digestion can be applied to reduce C. difficile in biosolids, thereby reducing the environmental burden of the enteric pathogen.
Connor, Mairéad; Flynn, Padrig B.; Fairley, Derek J.; Marks, Nikki; Manesiotis, Panagiotis; Graham, William G.; Gilmore, Brendan F.; McGrath, John W.
Clostridium difficile is a spore forming bacterium and the leading cause of colitis and antibiotic associated diarrhoea in the developed world. Spores produced by C. difficile are robust and can remain viable for months, leading to prolonged healthcare-associated outbreaks with high mortality. Exposure of C. difficile spores to a novel, non-thermal atmospheric pressure gas plasma was assessed. Factors affecting sporicidal efficacy, including percentage of oxygen in the helium carrier gas admixture, and the effect on spores from different strains representing the five evolutionary C. difficile clades was investigated. Strains from different clades displayed varying resistance to cold plasma. Strain R20291, representing the globally epidemic ribotype 027 type, was the most resistant. However all tested strains displayed a ~3 log reduction in viable spore counts after plasma treatment for 5 minutes. Inactivation of a ribotype 078 strain, the most prevalent clinical type seen in Northern Ireland, was further assessed with respect to surface decontamination, pH, and hydrogen peroxide concentration. Environmental factors affected plasma activity, with dry spores without the presence of organic matter being most susceptible. This study demonstrates that cold atmospheric plasma can effectively inactivate C. difficile spores, and highlights factors that can affect sporicidal activity.
Hong, Huynh A; Hitri, Krisztina; Hosseini, Siamand; Kotowicz, Natalia; Bryan, Donna; Mawas, Fatme; Wilkinson, Anthony J; van Broekhoven, Annie; Kearsey, Jonathan; Cutting, Simon M
Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26-39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26-39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26-39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26-39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI.
Xie, Jinfu; Zorman, Julie; Indrawati, Lani; Horton, Melanie; Soring, Keri; Antonello, Joseph M; Zhang, Yuhua; Secore, Susan; Miezeiewski, Matthew; Wang, Su; Kanavage, Anthony D; Skinner, Julie M; Rogers, Irene; Bodmer, Jean-Luc; Heinrichs, Jon H
Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on labor-intensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.
Donald, Robert G K; Flint, Mike; Kalyan, Narender; Johnson, Erik; Witko, Susan E; Kotash, Cheryl; Zhao, Ping; Megati, Shakuntala; Yurgelonis, Irina; Lee, Phillip Kwok; Matsuka, Yury V; Severina, Elena; Deatly, Anne; Sidhu, Mini; Jansen, Kathrin U; Minton, Nigel P; Anderson, Annaliesa S
The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.
Lawley, Trevor D; Croucher, Nicholas J; Yu, Lu; Clare, Simon; Sebaihia, Mohammed; Goulding, David; Pickard, Derek J; Parkhill, Julian; Choudhary, Jyoti; Dougan, Gordon
Clostridium difficile, a major cause of antibiotic-associated diarrhea, produces highly resistant spores that contaminate hospital environments and facilitate efficient disease transmission. We purified C. difficile spores using a novel method and show that they exhibit significant resistance to harsh physical or chemical treatments and are also highly infectious, with <7 environmental spores per cm(2) reproducibly establishing a persistent infection in exposed mice. Mass spectrometric analysis identified approximately 336 spore-associated polypeptides, with a significant proportion linked to translation, sporulation/germination, and protein stabilization/degradation. In addition, proteins from several distinct metabolic pathways associated with energy production were identified. Comparison of the C. difficile spore proteome to those of other clostridial species defined 88 proteins as the clostridial spore "core" and 29 proteins as C. difficile spore specific, including proteins that could contribute to spore-host interactions. Thus, our results provide the first molecular definition of C. difficile spores, opening up new opportunities for the development of diagnostic and therapeutic approaches.
Gebhart, Dana; Williams, Steven R; Bishop-Lilly, Kimberly A; Govoni, Gregory R; Willner, Kristin M; Butani, Amy; Sozhamannan, Shanmuga; Martin, David; Fortier, Louis-Charles; Scholl, Dean
Clostridium difficile causes one of the leading nosocomial infections in developed countries, and therapeutic choices are limited. Some strains of C. difficile produce phage tail-like particles upon induction of the SOS response. These particles have bactericidal activity against other C. difficile strains and can therefore be classified as bacteriocins, similar to the R-type pyocins of Pseudomonas aeruginosa. These R-type bacteriocin particles, which have been purified from different strains, each have a different C. difficile-killing spectrum, with no one bacteriocin killing all C. difficile isolates tested. We have identified the genetic locus of these "diffocins" (open reading frames 1359 to 1376) and have found them to be common among the species. The entire diffocin genetic locus of more than 20 kb was cloned and expressed in Bacillus subtilis, and this resulted in production of bactericidal particles. One of the interesting features of these particles is a very large structural protein of ~200 kDa, the product of gene 1374. This large protein determines the killing spectrum of the particles and is likely the receptor-binding protein. Diffocins may provide an alternate bactericidal agent to prevent or treat infections and to decolonize individuals who are asymptomatic carriers.
Maldarelli, Grace A.; De Masi, Leon; von Rosenvinge, Erik C.; Carter, Mihaela; Donnenberg, Michael S.
The Gram-positive anaerobe Clostridium difficile is the major cause of nosocomial diarrhea; manifestations of infection include diarrhea, pseudomembranous colitis, and death. Genes for type IV pili, a bacterial nanofiber often involved in colonization and until relatively recently described only in Gram-negatives, are present in all members of the Clostridiales. We hypothesized that any pilins encoded in the C. difficile genome would be immunogenic, as has been shown with pilins from Gram-negative organisms. We describe nine pilin or pilin-like protein genes, for which we introduce a coherent nomenclature, in the C. difficile R20291 genome. The nine predicted pilin or pilin-like proteins have relatively conserved N-terminal hydrophobic regions, but diverge at their C-termini. Analysis of synonymous and nonsynonymous substitutions revealed evidence of diversifying selective pressure in two pilin genes. Six of the nine identified proteins were purified and used to immunize mice. Immunization of mice with each individual protein generated antibody responses that varied in titer and crossreactivity, a notable result given the low amino acid sequence identity among the pilins. Further studies in other small mammals mirrored our results in mice. Our results illuminate components of the C. difficile type IV pilus, and help identify targets for an anti-C. difficile vaccine. PMID:24550179
Arvand, M; Vollandt, D; Bettge-Weller, G; Harmanus, C; Kuijper, E J
After the first outbreak of Clostridium difficile PCR ribotype (RT) 027 in Germany in 2007, no further outbreaks were reported until the recent re-emergence of RT 027 in Hesse, a federal state with 6 million inhabitants located in south-west Germany. We undertook a survey to determine the prevalence of RT 027 and other strains in a prospective study. From January 2011 to July 2013, we analysed 291 specimens from patients diagnosed with C. difficile infection (CDI) in 40 healthcare facilities in Hesse. The mean incidence of CDI in hospitals including at least 10 patients in the survey was 9.9 per 10,000 patient days (range 4.8-22.8) in November 2012. We obtained 214 toxigenic C. difficile isolates. RT 001 was the most prevalent (31.8%). RT 027, the second most common type (26.6%), was prevalent in all hospitals (n=14) from which at least seven isolates were available for typing, but its frequency varied considerably (range: 9.1–70%). The annual frequency of RT 027 increased from 21.4% in 2011 to 30.0% in 2013 (p=0.04). Our study indicates that infections with C. difficile RT 027 are now prevalent in Hesse. It underscores the need for surveillance programmes to analyse the molecular epidemiology of C. difficile.
Soutourina, Olga A.; Monot, Marc; Boudry, Pierre; Saujet, Laure; Pichon, Christophe; Sismeiro, Odile; Semenova, Ekaterina; Severinov, Konstantin; Le Bouguenec, Chantal; Coppée, Jean-Yves; Dupuy, Bruno; Martin-Verstraete, Isabelle
Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA–seq and differential 5′-end RNA–seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA–based regulation of gene expression in this emergent enteropathogen. PMID:23675309
Morgan, Oliver W.; Rodrigues, Boaventura; Elston, Tony; Verlander, Neville Q.; Brown, Derek F. J.; Brazier, Jonathan; Reacher, Mark
Background Clostridium difficile is a leading infectious cause of health care associated diarrhoea. Several industrialised countries have reported increased C. difficile infections and outbreaks, which have been attributed to the emergent PCR ribotype 027 strain. Methods and Findings We conducted a case-case study to compare severity of C. difficile disease for patients with 027 versus non-027 ribotypes. We retrospectively collected clinical information about 123/136 patients with C. difficile infections admitted to hospitals in the East of England region in 2006 and from whom stool isolates were cultured and ribotyped as part of an earlier national survey. We defined severe C. difficile disease as having one or more of shock, paralytic ileus, pseudo membranous colitis or toxic megacolon. Patient median age was 83 years old (range 3 to 98, interquartile range 75 to 89), 86% were prescribed antibiotics in the eight weeks before illness onset, 41% had ribotype 027 and 30-day all cause mortality during hospital admission was 21%. Severe disease occurred in 24% (95%CI 13% to 37%) and 17% (95%CI 9% to 27%) of patients with PCR ribotype 027 and non-027 ribotypes respectively. In a multivariable model, ribotype 027 was not associated with severe disease after adjusting for sex, discharge from hospital prior to 60 days of current admission, gastroenteritis on admission, number of initiator antibiotics for C. difficile disease, and hospital where the patient was admitted. Conclusions Our study found no evidence to support previous assertions that ribotype 027 is more virulent than other PCR ribotypes. This finding raises questions about the contribution of this strain to the recent increase in C. difficile disease throughout North America and Europe. PMID:18350149
Koon, Hon Wai; Ho, Samantha; Hing, Tressia C.; Cheng, Michelle; Chen, Xinhua; Ichikawa, Yoshi; Kelly, Ciarán P.
Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI, and its beneficial effect was associated with fewer recurrent infections in CDI patients. Since other macrolides possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile toxin A-induced ileal inflammation in mice. The ileal loops of anesthetized mice were injected with fidaxomicin (5, 10, or 20 μM), and after 30 min, the loops were injected with purified C. difficile toxin A or phosphate-buffered saline alone. Four hours after toxin A administration, ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements. C. difficile toxin A caused histologic damage, evidenced by increased mean histologic score and ileal interleukin-1β (IL-1β) protein and mRNA expression. Treatment with fidaxomicin (20 μM) or its primary metabolite, OP-1118 (120 μM), significantly inhibited toxin A-mediated histologic damage and reduced the mean histology score and ileal IL-1β protein and mRNA expression. Both fidaxomicin and OP-1118 reduced toxin A-induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of ileal loops with vancomycin (20 μM) and metronidazole (20 μM) did not alter toxin A-induced histologic damage and IL-1β protein expression. In addition to its well known antibacterial effects against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal effects of C. difficile toxins. PMID:24890583
Chilton, C. H.; Crowther, G. S.; Śpiewak, K.; Brindell, M.; Singh, G.; Wilcox, M. H.; Monaghan, T. M.
Objectives Clostridium difficile infection (CDI) is a global healthcare problem. Recent evidence suggests that the availability of iron may be important for C. difficile growth. This study evaluated the comparative effects of iron-depleted (1% Fe3+ saturated) bovine apo-lactoferrin (apo-bLf) and iron-saturated (85% Fe3+ saturated) bovine holo-lactoferrin (holo-bLf) in a human in vitro gut model that simulates CDI. Methods Two parallel triple-stage chemostat gut models were inoculated with pooled human faeces and spiked with C. difficile spores (strain 027 210, PCR ribotype 027). Holo- or apo-bLf was instilled (5 mg/mL, once daily) for 35 days. After 7 days, clindamycin was instilled (33.9 mg/L, four times daily) to induce simulated CDI. Indigenous microflora populations, C. difficile total counts and spores, cytotoxin titres, short chain fatty acid concentrations, biometal concentrations, lactoferrin concentration and iron content of lactoferrin were monitored daily. Results In the apo-bLf model, germination of C. difficile spores occurred 6 days post instillation of clindamycin, followed by rapid vegetative cell proliferation and detectable toxin production. By contrast, in the holo-bLf model, only a modest vegetative cell population was observed until 16 days post antibiotic administration. Notably, no toxin was detected in this model. In separate batch culture experiments, holo-bLf prevented C. difficile vegetative cell growth and toxin production, whereas apo-bLf and iron alone did not. Conclusions Holo-bLf, but not apo-bLf, delayed C. difficile growth and prevented toxin production in a human gut model of CDI. This inhibitory effect may be iron independent. These observations suggest that bLf in its iron-saturated state could be used as a novel preventative or treatment strategy for CDI. PMID:26759363
Bertolo, Lisa; Monteiro, Mario A.; Agellon, Al; Viswanathan, V. K.; Vedantam, Gayatri
Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence. PMID:27741317
Rojo, David; Gosalbes, María J; Ferrari, Rafaela; Pérez-Cobas, Ana E; Hernández, Ester; Oltra, Rosa; Buesa, Javier; Latorre, Amparo; Barbas, Coral; Ferrer, Manuel; Moya, Andrés
Clostridium difficile-associated diarrhoea (CDAD) is caused by C. difficile toxins A and B and represents a serious emerging health problem. Yet, its progression and functional consequences are unclear. We hypothesised that C. difficile can drive major measurable metabolic changes in the gut microbiota and that a relationship with the production or absence of toxins may be established. We tested this hypothesis by performing metabolic profiling on the gut microbiota of patients with C. difficile that produced (n=6) or did not produce (n=4) toxins and on non-colonised control patients (n=6), all of whom were experiencing diarrhoea. We report a statistically significant separation (P-value <0.05) among the three groups, regardless of patient characteristics, duration of the disease, antibiotic therapy and medical history. This classification is associated with differences in the production of distinct molecules with presumptive global importance in the gut environment, disease progression and inflammation. Moreover, although severe impaired metabolite production and biological deficits were associated with the carriage of C. difficile that did not produce toxins, only previously unrecognised selective features, namely, choline- and acetylputrescine-deficient gut environments, characterised the carriage of toxin-producing C. difficile. Additional results showed that the changes induced by C. difficile become marked at the highest level of the functional hierarchy, namely the metabolic activity exemplified by the gut microbial metabolome regardless of heterogeneities that commonly appear below the functional level (gut bacterial composition). We discuss possible explanations for this effect and suggest that the changes imposed by CDAD are much more defined and predictable than previously thought.
Yang, Paul; Hash, Sara; Park, Katherine; Wong, Charlene; Doraisamy, Loganathan; Petterson, Jonas; Petti, Cathy A; Ward, Pamela M; Lee, Seung Heon; Menon, Suresh; She, Rosemary C
We evaluated the performance of an early prototype core molecular mirroring nuclear magnetic resonance detection platform (Mentor-100) to detect toxigenic Clostridium difficile from stool. This technology uses customized nanoparticles bound to target specific oligonucleotide probes that form binaries in the presence of nucleic acid from the target microorganism. Liquid patient stool specimens were seeded with C. difficile or other Clostridium species to determine the analytical sensitivity and specificity. Samples underwent nucleic acid extraction and target amplification with probes conjugated with iron nanoparticles. Signal from nuclear magnetic resonance spin-spin relaxation time was measured to detect the presence or absence of toxigenic C. difficile. The limit of detection was <180 colony forming units per reaction of toxigenic C. difficile. No cross-reactivity was observed with nontoxigenic C. difficile, Clostridium sordellii, Clostridium perfringens, Bacillus subtilis, or Paenibacillus polymyxa at 10(8) colony forming units/mL. Correlation studies using frozen stool samples yielded a sensitivity of 88.4% (61 of 69) and a specificity of 87.0% (40 of 46) as compared with a commercial PCR assay for C. difficile. The area under the curve in the receiver operating characteristic curve analysis was 0.922. The prototype molecular mirroring platform showed promising performance for pathogen detection from clinical specimens. The platform design has the potential to offer a novel, low-cost alternative to currently available nucleic acid-based tests.
Saxton, Katie; Baines, Simon D; Freeman, Jane; O'Connor, Rachael; Wilcox, Mark H
The incidence of Clostridium difficile infection is increasing, with reports implicating fluoroquinolone use. A three-stage chemostat gut model was used to study the effects of three fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) on the gut microbiota and two epidemic C. difficile strains, strains of PCR ribotypes 027 and 001, in separate experiments. C. difficile total viable counts, spore counts, and cytotoxin titers were determined. The emergence of C. difficile isolates with reduced antibiotic susceptibility was monitored with fluoroquinolone-containing medium, and molecular analysis of the quinolone resistance-determining region was performed. C. difficile spores were quiescent in the absence of fluoroquinolones. Instillation of each fluoroquinolone led to C. difficile spore germination and high-level cytotoxin production. High-level toxin production occurred after detectable spore germination in all experiments except those with C. difficile PCR ribotype 027 and moxifloxacin, in which marked cytotoxin production preceded detectable germination, which coincided with isolate recovery on fluoroquinolone-containing medium. Three C. difficile PCR ribotype 027 isolates and one C. difficile PCR ribotype 001 isolate from fluoroquinolone-containing medium exhibited elevated MICs (80 to > or =180 mg/liter) and possessed mutations in gyrA or gyrB. These in vitro results suggest that all fluoroquinolones have the propensity to induce C. difficile infection, regardless of their antianaerobe activities. Resistant mutants were seen only following moxifloxacin exposure.
Fimlaid, Kelly A.; Jensen, Owen; Donnelly, M. Lauren; Francis, Michael B.; Sorg, Joseph A.; Shen, Aimee
Clostridium difficile is a Gram-positive spore-forming pathogen and a leading cause of nosocomial diarrhea. C. difficile infections are transmitted when ingested spores germinate in the gastrointestinal tract and transform into vegetative cells. Germination begins when the germinant receptor CspC detects bile salts in the gut. CspC is a subtilisin-like serine pseudoprotease that activates the related CspB serine protease through an unknown mechanism. Activated CspB cleaves the pro-SleC zymogen, which allows the activated SleC cortex hydrolase to degrade the protective cortex layer. While these regulators are essential for C. difficile spores to outgrow and form toxin-secreting vegetative cells, the mechanisms controlling their function have only been partially characterized. In this study, we identify the lipoprotein GerS as a novel regulator of C. difficile spore germination using targeted mutagenesis. A gerS mutant has a severe germination defect and fails to degrade cortex even though it processes SleC at wildtype levels. Using complementation analyses, we demonstrate that GerS secretion, but not lipidation, is necessary for GerS to activate SleC. Importantly, loss of GerS attenuates the virulence of C. difficile in a hamster model of infection. Since GerS appears to be conserved exclusively in related Peptostreptococcaeace family members, our results contribute to a growing body of work indicating that C. difficile has evolved distinct mechanisms for controlling the exit from dormancy relative to B. subtilis and other spore-forming organisms. PMID:26496694
Mori, Nobuaki; Yoshizawa, Sadako; Saga, Tomoo; Ishii, Yoshikazu; Murakami, Hinako; Iwata, Morihiro; Collins, Deirdre A; Riley, Thomas V; Tateda, Kazuhiro
Physicians often fail to suspect Clostridium difficile infection (CDI) and many microbiology laboratories use suboptimal diagnostic techniques. To estimate the extent of and reasons for incorrect diagnosis of CDI in Japan, we investigated toxigenic C. difficile isolated from all stool culture samples and clinical course. Over a 12-month period in 2010, all stool culture samples (n = 975) submitted from inpatients in a university hospital in Japan were cultured for C. difficile and routine microbiological testing was conducted. In total, 177 C. difficile isolates were recovered, and 127 isolates were toxigenic. Among the toxin-A-positive/toxin-B-positive isolates, 12 were also positive for the binary toxin gene. However, clinically important ribotypes, such as 027 and 078, were not identified. A total of 58 (45.7%) cases with toxigenic C. difficile had unformed stool, and the incidence CDI was 1.6 cases per 10,000 patient-days. Of these 58 cases, 40 were not diagnosed in routine testing due to a lack of clinical suspicion (24.1%, 14/58) or a negative C. difficile toxin assay result (44.8%, 26/58). A stool toxin assay was performed in 54 patients (78.2%, 54/69) who did not have unformed stool. The present study demonstrated that a significant number of CDI cases in Japan might be overlooked or misdiagnosed in clinical practice due to a lack of clinical suspicion and limitations of microbiological testing for CDI in Japan. Providing education to promote awareness of CDI among physicians is important to improve the accuracy of diagnosis in Japan.
Vargas, Edwin; Apewokin, Senu; Madan, Rajat
Clostridium difficile is the leading cause of healthcare-associated infections in the United States. Clinically, C. difficile-associated disease can present as asymptomatic colonization, self-limited diarrheal illness or severe colitis (that may result in death). This variability in disease course and outcomes suggests that host factors play an important role as key determinants of disease severity. Currently, there are several scoring indices to estimate severity of C. difficile-associated disease. Leukocytosis and renal failure are considered to be the most important predictors of C. difficile disease severity in hosts with a normal immune system. The degree of leukocytosis which is considered significant for severe disease and how it is scored vary amongst scoring indices. None of the scores have been prospectively validated, and while total WBC count is useful to estimate the magnitude of the host response in most patient populations, in immune-compromised patients like those receiving chemotherapy, solid organ transplant patients or hematopoietic stem cell transplants the WBC response can be variable or even absent making this marker of severity difficult to interpret. Other cellular subsets like neutrophils, eosinophils and lymphocytes provide important information about the host immune status and play an important role in the immune response against C. difficile infection. However, under the current scoring systems the role of these cellular subsets have been underestimated and only total white blood cell counts are taken into account. In this review we highlight the role of host leukocyte response to C. difficile challenge in the normal and immunocompromised host, and propose possible ways that would allow for a better representation of the different immune cell subsets (neutrophils, lymphocytes and eosinophils) in the current scoring indices.
Bobo, Linda D.; El Feghaly, Rana E.; Chen, Yee-Shiuan; Dubberke, Erik R.; Han, Zhuolin; Baker, Alexandra H.; Li, Jinmei; Burnham, Carey-Ann D.
Clostridium difficile infection (CDI) results in toxin-induced epithelial injury and marked intestinal inflammation. Fecal markers of intestinal inflammation correlate with CDI disease severity, but regulation of the inflammatory response is poorly understood. Previous studies demonstrated that C. difficile toxin TcdA activates p38 kinase in tissue culture cells and mouse ilium, resulting in interleukin-8 (IL-8) release. Here, we investigated the role of phosphorylated mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2 kinase, pMK2), a key mediator of p38-dependent inflammation, in CDI. Exposure of cultured intestinal epithelial cells to the C. difficile toxins TcdA and TcdB resulted in p38-dependent MK2 activation. Toxin-induced IL-8 and GROα release required MK2 activity. We found that p38 and MK2 are activated in response to other actin-disrupting agents, suggesting that toxin-induced cytoskeleton disruption is the trigger for kinase-dependent cytokine response. Phosphorylated MK2 was detected in the intestines of C. difficile-infected hamsters and mice, demonstrating for the first time that the pathway is activated in infected animals. Furthermore, we found that elevated pMK2 correlated with the presence of toxigenic C. difficile among 100 patient stool samples submitted for C. difficile testing. In conclusion, we find that MK2 kinase is activated by TcdA and TcdB and regulates the expression of proinflammatory cytokines. Activation of p38-MK2 in infected animals and humans suggests that this pathway is a key driver of intestinal inflammation in patients with CDI. PMID:23264053
Bhansali, Suraj G.; Mullane, Kathleen; Ting, Lillian S. L.; Leeds, Jennifer A.; Dabovic, Kristina; Praestgaard, Jens
Clostridium difficile infection causes diarrheal disease with potentially fatal complications. Although treatments are available, including vancomycin, metronidazole, and fidaxomicin, the recurrence of disease after therapy remains a problem. LFF571 is a novel thiopeptide antibacterial that shows in vitro potency against C. difficile that is comparable to or greater than that of other clinically used antibiotics. Here, we compare the pharmacokinetics (PK) of LFF571 and vancomycin in patients with C. difficile infection as part of an early efficacy study. This multicenter, randomized, evaluator-blind, and active-controlled study evaluated the safety, efficacy, and pharmacokinetics of LFF571 in adults with primary episodes or first relapses of moderate C. difficile infections. Patients were randomized to receive 200 mg of LFF571 or 125 mg of vancomycin four times daily for 10 days. The PK parameters were calculated from drug concentrations measured in serum and fecal samples. The systemic exposure following oral administration of 200 mg of LFF571 four times per day for 10 days in patients with C. difficile infection was limited. The highest LFF571 serum concentration observed was 41.7 ng/ml, whereas the levels in feces at the end of treatment were between 107 and 12,900 μg/g. In comparison, the peak vancomycin level observed in serum was considerably higher, at 2.73 μg/ml; the levels of vancomycin in feces were not measured. Similar to healthy volunteers, patients with C. difficile infections exhibited high fecal concentrations and low serum levels of LFF571. These results are consistent with the retention of LFF571 in the lumen of the gastrointestinal tract. (This study has been registered at ClinicalTrials.gov under registration no. NCT01232595.) PMID:25534724
Nerandzic, Michelle M.; Rackaityte, Elze; Jury, Lucy A.; Eckart, Kevin; Donskey, Curtis J.
Background Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe®) would reduce the burden of spores on skin. Methods Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths. Results Spore removal from hands was enhanced with Vashe soak (>2.5 log10 reduction) versus soap and water wash or soak (~2.0 log10 reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log10 spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin. Conclusions Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin. PMID:23844234
Asojo, Oluwatoyin A; Nelson, Sarah K; Mootien, Sara; Lee, Yashang; Rezende, Wanderson C; Hyman, Daniel A; Matsumoto, Monica M; Reiling, Scott; Kelleher, Alan; Ledizet, Michel; Koski, Raymond A; Anthony, Karen G
Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.
Francis, Michael B.; Allen, Charlotte A.
ABSTRACT Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway. IMPORTANCE Clostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant
Moon, Hee-Won; Kim, Hyeong Nyeon; Hur, Mina; Shim, Hee Sook; Kim, Heejung; Yun, Yeo-Min
Since every single test has some limitations for detecting toxigenic Clostridium difficile, multistep algorithms are recommended. This study aimed to compare the current, representative diagnostic algorithms for detecting toxigenic C. difficile, using VIDAS C. difficile toxin A&B (toxin ELFA), VIDAS C. difficile GDH (GDH ELFA, bioMérieux, Marcy-l'Etoile, France), and Xpert C. difficile (Cepheid, Sunnyvale, California, USA). In 271 consecutive stool samples, toxigenic culture, toxin ELFA, GDH ELFA, and Xpert C. difficile were performed. We simulated two algorithms: screening by GDH ELFA and confirmation by Xpert C. difficile (GDH + Xpert) and combined algorithm of GDH ELFA, toxin ELFA, and Xpert C. difficile (GDH + Toxin + Xpert). The performance of each assay and algorithm was assessed. The agreement of Xpert C. difficile and two algorithms (GDH + Xpert and GDH+ Toxin + Xpert) with toxigenic culture were strong (Kappa, 0.848, 0.857, and 0.868, respectively). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of algorithms (GDH + Xpert and GDH + Toxin + Xpert) were 96.7%, 95.8%, 85.0%, 98.1%, and 94.5%, 95.8%, 82.3%, 98.5%, respectively. There were no significant differences between Xpert C. difficile and two algorithms in sensitivity, specificity, PPV and NPV. The performances of both algorithms for detecting toxigenic C. difficile were comparable to that of Xpert C. difficile. Either algorithm would be useful in clinical laboratories and can be optimized in the diagnostic workflow of C. difficile depending on costs, test volume, and clinical needs.
Schalk, Enrico; Bohr, Ulrich R M; König, Brigitte; Scheinpflug, Katrin; Mohren, Martin
Diarrhoea occurs frequently in neutropenic patients with acute leukaemia receiving chemotherapy and may be caused by either infection- or drug-induced cytotoxicity. Since Clostridium difficile is the most common cause of nosocomial infectious diarrhoea in non-haematologic patients, we were interested in its incidence in patients with acute myeloid leukaemia (AML). In this retrospective study, we analysed 134 patients with AML receiving a total of 301 chemotherapy courses. Diarrhoea occurred during 33% of all courses in 58 patients. C. difficile-associated diarrhoea (CDAD) occurred in 18% of all patients and 9% of all treatment courses. Almost one third of diarrhoea episodes were caused by C. difficile. CDAD was associated with older age (58 vs. 50 years), number of antibiotics administered (2 vs. 1), duration of antibiotic therapy (7 vs. 4 days), ceftazidime as the antibiotic of choice (75% vs. 54%) and duration of neutropenia (12 vs. 7 days) prior to onset of diarrhoea. An increased risk for CDAD was seen for prolonged neutropenia. CDAD responded well to oral metronidazole and/or vancomycin and no patient died of this complication. In conclusion, CDAD is common in patients with AML receiving chemotherapy. C. difficile enterotoxin testing of stool specimens should be included in all symptomatic patients.
Young, Karen W H; Munro, Ian C; Taylor, Steve L; Veldkamp, Peter; van Dissel, Jaap T
A whey protein concentrate prepared from the milk of cows that have been immunized against Clostridium difficile (C. difficile) and its toxins, toxin A and toxin B, is produced for use as a medical food for the dietary management of patients with C. difficile-associated diarrhea (CDAD) to prevent a relapse of the infection. The safety of anti-C. difficile whey protein concentrate (anti-CD WPC) is supported by analytical data comparing the composition of raw milk from immunized cows versus that from non-immunized cows, and the composition of anti-CD WPC versus that of regular whey protein concentrate. Additionally, a prospective clinical study was conducted in 77 patients with CDAD to demonstrate the safety of consuming anti-CD WPC to prevent relapse of the infection. This study, which included adverse event monitoring, physical examinations, and extensive hematological and biochemical assessments, showed that anti-CD WPC is safe to consume by patients with CDAD. The available analytical and clinical evidence demonstrate that anti-CD WPC is safe for use by individuals with CDAD, under the described conditions of use.
Bradshaw, William J.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi
Two structures of Cwp84, a cysteine protease from the S-layer of C. difficile, are presented after propeptide cleavage. They reveal the movement of three loops, two in the active-site groove and one on the surface of the lectin-like domain, exposing a hydrophobic pocket. In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely to be of significant importance to host–pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket.
Hargreaves, K. R.; Colvin, H. V.; Patel, K. V.; Clokie, J. J. P.
Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in health care settings across the world. Despite its pathogenic capacity, it can be carried asymptomatically and has been found in terrestrial and marine ecosystems outside hospital environments. Little is known about these environmental strains, and few studies have been conducted on estuarine systems. Although prophage abundance and diversity are known to occur within clinical strains, prophage carriage within environmental strains of C. difficile has not previously been explored. In this study, we isolated C. difficile from sites sampled in two consecutive years in an English estuarine system. Isolates were characterized by PCR ribotype, antibiotic resistance, and motility. The prevalence and diversity of prophages were detected by transmission electron microscopy (TEM) and a phage-specific PCR assay. We show that a dynamic and diverse population of C. difficile exists within these sediments and that it includes isolates of ribotypes which are associated with severe clinical infections and those which are more frequently isolated from outside the hospital environment. Prophage carriage was found to be high (75%), demonstrating that phages play a role in the biology of these strains. PMID:23913427
Kurtz, C B; Cannon, E P; Brezzani, A; Pitruzzello, M; Dinardo, C; Rinard, E; Acheson, D W; Fitzpatrick, R; Kelly, P; Shackett, K; Papoulis, A T; Goddard, P J; Barker, R H; Palace, G P; Klinger, J D
GT160-246, a high-molecular-weight soluble anionic polymer, was tested in vitro and in vivo for neutralization of Clostridium difficile toxin A and B activities. Five milligrams of GT160-246 per ml neutralized toxin-mediated inhibition of protein synthesis in Vero cells induced by 5 ng of toxin A per ml or 1.25 ng of toxin B per ml. In ligated rat ileal loops, 1 mg of GT160-246 neutralized fluid accumulation caused by 5 microg of toxin A. At doses as high as 80 mg/loop, cholestyramine provided incomplete neutralization of fluid accumulation caused by 5 microg of toxin A. GT160-246 protected 80% of the hamsters from mortality caused by infection with C. difficile, whereas cholestyramine protected only 10% of animals. Treatment of C. difficile-infected hamsters with metronidazole initially protected 100% of the hamsters from mortality, but upon removal of treatment, 80% of the hamsters had relapses and died. In contrast, removal of GT160-246 treatment did not result in disease relapse in the hamsters. GT160-246 showed no antimicrobial activity in tests with a panel of 16 aerobic bacteria and yeast and 22 anaerobic bacteria and did not interfere with the in vitro activities of most antibiotics. GT160-246 offers a novel, nonantimicrobial treatment of C. difficile disease in humans.
Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.
Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385
Guillemin, Isabelle; Marrel, Alexia; Beriot-Mathiot, Axelle; Doucet, Carole; Kazoglou, Odysseas; Luxemburger, Christine; Reygrobellet, Camille; Arnould, Benoit
This qualitative study explored the impact of Clostridium difficile infections on nurses' everyday work in the hospital. Twelve nurses (six in France and six in the United States) were interviewed in depth using a semi-structured interview guide. Thematic analysis of the interviews was performed. Managing diarrhoea and taking precautionary measures for infection control were the two most inconvenient aspects nurses reported with C. difficile patient management. Precautions included contact isolation, hand hygiene and reorganization/coordination of nursing care and ward. Precautions were time consuming and significantly increased nurses' workload when combined with caring for patients with uncontrollable, frequent bouts of diarrhoea. Management of C. difficile infection is extremely burdensome for nurses in their everyday work and disruptive to hospital organizations as a whole. Prevention of C. difficile infections, together with coordinated team work and communication, would therefore contribute to decreasing nurses' workload and the burden to health-care facilities associated with caring for these patients.
Sekulovic, Ognjen; Ospina Bedoya, Maicol; Fivian-Hughes, Amanda S; Fairweather, Neil F; Fortier, Louis-Charles
Bacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. Clostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell-surface protein CwpV provides antiphage protection in C. difficile. This protein, for which the expression is phase-variable, is classified into five types, each differing in their repeat-containing C-terminal domain. When expressed constitutively from a plasmid or the chromosome of locked 'ON' cells of C. difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10(-7) for phages ϕCD38-2, ϕCD111 and ϕCD146. Protection against the myophages ϕMMP01 and ϕCD52 was weaker, with EOP values between 9.0 × 10(-3) and 1.1 × 10(-1). The C-terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host-encoded and phase-variable antiphage system in C. difficile.
Wu, Dan; Joyee, Antony George; Nandagopal, Saravanan; Lopez, Marianela; Ma, Xiuli; Berry, Jody; Lin, Francis
Bacterial products such as toxins can interfere with a variety of cellular processes, leading to severe human diseases. Clostridium difficile toxins, TcdA and TcdB are the primary contributing factors to the pathogenesis of C. difficile-associated diseases (CDAD). While the mechanisms for TcdA and TcdB mediated cellular responses are complex, it has been shown that these toxins can alter chemotactic responses of neutrophils and intestinal epithelial cells leading to innate immune responses and tissue damages. The effects of C. difficile toxins on the migration and trafficking of other leukocyte subsets, such as T lymphocytes, are not clear and may have potential implications for adaptive immunity. We investigated here the direct and indirect effects of TcdA and TcdB on the migration of human blood T cells using conventional cell migration assays and microfluidic devices. It has been found that, although both toxins decrease T cell motility, only TcdA but not TcdB decreases T cell chemotaxis. Similar effects are observed in T cell migration toward the TcdA- or TcdB-treated human epithelial cells. Our study demonstrated the primary role of TcdA (compared to TcdB) in altering T cell migration and chemotaxis, suggesting possible implications for C. difficile toxin mediated adaptive immune responses in CDAD.
Peterfreund, Gregory L; Vandivier, Lee E; Sinha, Rohini; Marozsan, Andre J; Olson, William C; Zhu, Jun; Bushman, Frederic D
Antibiotic disruption of the intestinal microbiota may cause susceptibility to pathogens that is resolved by progressive bacterial outgrowth and colonization. Succession is central to ecological theory but not widely documented in studies of the vertebrate microbiome. Here, we study succession in the hamster gut after treatment with antibiotics and exposure to Clostridium difficile. C. difficile infection is typically lethal in hamsters, but protection can be conferred with neutralizing antibodies against the A and B toxins. We compare treatment with neutralizing monoclonal antibodies (mAb) to treatment with vancomycin, which prolongs the lives of animals but ultimately fails to protect them from death. We carried out longitudinal deep sequencing analysis and found distinctive waves of succession associated with each form of treatment. Clindamycin sensitization prior to infection was associated with the temporary suppression of the previously dominant Bacteroidales and the fungus Saccinobaculus in favor of Proteobacteria. In mAb-treated animals, C. difficile proliferated before joining Proteobacteria in giving way to re-expanding Bacteroidales and the fungus Wickerhamomyces. However, the Bacteroidales lineages returning by day 7 were different from those that were present initially, and they persisted for the duration of the experiment. Animals treated with vancomycin showed a different set of late-stage lineages that were dominated by Proteobacteria as well as increased disparity between the tissue-associated and luminal cecal communities. The control animals showed no change in their gut microbiota. These data thus suggest different patterns of ecological succession following antibiotic treatment and C. difficile infection.
Shirvan, Ali Nazari; Aitken, Robert
Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.
Freeman, J; Vernon, J; Morris, K; Nicholson, S; Todhunter, S; Longshaw, C; Wilcox, M H
Clostridium difficile infection remains a major healthcare burden. Until the recent introduction of fidaxomicin, antimicrobial treatments were limited to metronidazole and vancomycin. The emergence of epidemic C. difficile PCR ribotype 027 and its potential link to decreased antibiotic susceptibility highlight the lack of large-scale antimicrobial susceptibility and epidemiological data available. We report results of epidemiological and antimicrobial susceptibility investigations of C. difficile isolates collected prior to fidaxomicin introduction, establishing important baseline data. Thirty-nine sites in 22 countries submitted a total of 953 C. difficile isolates for PCR ribotyping, toxin testing, and susceptibility testing to metronidazole, vancomycin, fidaxomicin, rifampicin, moxifloxacin, clindamycin, imipenem, chloramphenicol, and tigecycline. Ninety-nine known ribotypes were identified. Ribotypes 027, 014, 001/072, and 078 were most frequently isolated in line with previous European studies. There was no evidence of resistance to fidaxomicin, and reduced susceptibility to metronidazole and vancomycin was also scarce. Rifampicin, moxifloxacin, and clindamycin resistance (13%, 40%, and 50% of total isolates, respectively) were evident in multiple ribotypes. There was a significant correlation between lack of ribotype diversity and greater antimicrobial resistance (measured by cumulative resistance score). Well-known epidemic ribotypes 027 and 001/072 were associated with multiple antimicrobial resistance, but high levels of resistance were also observed, particularly in 018 and closely related emergent ribotype 356 in Italy. This raises the possibility of antimicrobial exposure as the underlying reason for their appearance, and highlights the need for ongoing epidemiological and antimicrobial resistance surveillance.
Antunes, Ana; Camiade, Emilie; Monot, Marc; Courtois, Emmanuelle; Barbut, Frédéric; Sernova, Natalia V.; Rodionov, Dmitry A.; Martin-Verstraete, Isabelle; Dupuy, Bruno
The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (creCD motif), that is, ‘RRGAAAANGTTTTCWW’. Binding of purified CcpA protein to 19 target creCD sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators. PMID:22989714
Wu, Dan; Joyee, Antony George; Nandagopal, Saravanan; Lopez, Marianela; Ma, Xiuli; Berry, Jody; Lin, Francis
Bacterial products such as toxins can interfere with a variety of cellular processes, leading to severe human diseases. Clostridium difficile toxins, TcdA and TcdB are the primary contributing factors to the pathogenesis of C. difficile-associated diseases (CDAD). While the mechanisms for TcdA and TcdB mediated cellular responses are complex, it has been shown that these toxins can alter chemotactic responses of neutrophils and intestinal epithelial cells leading to innate immune responses and tissue damages. The effects of C. difficile toxins on the migration and trafficking of other leukocyte subsets, such as T lymphocytes, are not clear and may have potential implications for adaptive immunity. We investigated here the direct and indirect effects of TcdA and TcdB on the migration of human blood T cells using conventional cell migration assays and microfluidic devices. It has been found that, although both toxins decrease T cell motility, only TcdA but not TcdB decreases T cell chemotaxis. Similar effects are observed in T cell migration toward the TcdA- or TcdB-treated human epithelial cells. Our study demonstrated the primary role of TcdA (compared to TcdB) in altering T cell migration and chemotaxis, suggesting possible implications for C. difficile toxin mediated adaptive immune responses in CDAD. PMID:23645153
Fraga, Edmir Geraldo; Nicodemo, Antonio Carlos; Sampaio, Jorge Luiz Mello
Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125μg/mL for both antimicrobials. The MIC90 were 4μg/mL and 2μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil.
Sandolo, Chiara; Péchiné, Séverine; Le Monnier, Alban; Hoys, Sandra; Janoir, Claire; Coviello, Tommasina; Alhaique, Franco; Collignon, Anne; Fattal, Elias; Tsapis, Nicolas
We have designed an oral vaccine against Clostridium difficile infection. The virulent factor Cwp84, that is a cystein protease highly immunogenic in patients with C. difficile-associated disease, was entrapped within pectin beads. Beads encapsulating Cwp84 were shown to be stable in the simulated intestinal medium and to release the cystein protease once in the simulated colonic medium. Three groups of hamsters were immunized, the first receiving pectin beads encapsulating Cwp84, the second unloaded beads and the third one free Cwp84. After three immunizations by the intragastric route, all groups received clindamycine. Post-challenge survival with a strain of C. difficile showed that 2 days after infection, all hamsters treated with unloaded beads and all hamsters treated with free Cwp84 have deceased after 7 days, whereas about 40% of hamsters administered with Cwp84-loaded beads survived 10 days after challenge, proving that oral vaccination provides partial protection. These first data obtained with an oral vaccine against C. difficile appear promising for preventing this infection.
Karczewski, Jerzy; Bodmer, Jean-Luc; Cook, James C; Xoconostle, Rachel F; Nahas, Debbie D; Joyce, Joseph G; Heinrichs, Jon H; Secore, Susan
Clostridium difficile is a gram-positive bacterium responsible for a large proportion of nosocomial infections in the developed world. C. difficile secretes toxins A and B (TcdA and TcdB) and both toxins act synergistically to induce a spectrum of pathological responses in infected individuals ranging from pseudomembranous colitis to C. difficile-associated diarrhea. Toxins A and B have been actively investigated as components of prophylactic vaccine as well as targets for therapeutic intervention with antibodies. Expression of such toxins by recombinant technology is often difficult and may require special handling and adherence to strict safety regulations during the manufacturing process due to the inherent toxicity of the proteins. Both toxins are large proteins (308 kDa and 270 kDa, respectively) and contain distinct domains mediating cell attachment, cellular translocation, and enzymatic (glucosidase) activity. Here we describe methods to produce fragments of Toxin B for their subsequent evaluation as components of experimental C. difficile vaccines. Methods presented include selection of fragments encompassing distinct functional regions of Toxin B, purification methods to yield high quality proteins, and analytical evaluation techniques. The approach presented focuses on Toxin B but could be applied to the other component, Toxin A, and/or to any difficult to express or toxic protein.
Kasendra, Magdalena; Barrile, Riccardo; Leuzzi, Rosanna; Soriani, Marco
The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been studied extensively, but their impact on bacterial colonization remains unclear. By setting up 2- and 3-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the mucosal barrier are substantially enhanced in poorly polarized or ethylene glycol tetraacetic acid-treated cells, indicating that bacteria bind preferentially to the basolateral (BL) cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to BL receptors, leading to a successful colonization of the colonic mucosa.
Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M
Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.
Vuotto, Claudia; Moura, Ines; Barbanti, Fabrizio; Donelli, Gianfranco; Spigaglia, Patrizia
Resistance mechanism to metronidazole is still poorly understood, even if the number of reports on Clostridium difficile strains with reduced susceptibility to this antibiotic is increasing. In this study, we investigated the ability of the C. difficile strains 7032994, 7032985 and 7032989, showing different susceptibility profiles to metronidazole but all belonging to the PCR ribotype 010, to form biofilm in vitro in presence and absence of subinhibitory concentrations of metronidazole. The quantitative biofilm production assay performed in presence of metronidazole revealed a significant increase in biofilm formation in both the susceptible strain 7032994 and the strain 7032985 exhibiting a reduced susceptibility to this antibiotic, while antibiotic pressure did not affect the biofilm-forming ability of the stable-resistant strain 7032989. Moreover, confocal microscopy analysis showed an abundant biofilm matrix production by the strains 7032994 and 7032885, when grown in presence of metronidazole, but not in the stable-resistant one. These results seem to demonstrate that subinhibitory concentrations of metronidazole are able to enhance the in vitro biofilm production of the above-mentioned PCR ribotype 010 C. difficile strains, susceptible or with reduced susceptibility to this antibiotic, suggesting a possible role of biofilm formation in the multifactorial mechanism of metronidazole resistance developed by C. difficile.
Martin, Melissa J.; Clare, Simon; Goulding, David; Faulds-Pain, Alexandra; Barquist, Lars; Browne, Hilary P.; Pettit, Laura; Dougan, Gordon; Lawley, Trevor D.
The transcriptional regulator AgrA, a member of the LytTR family of proteins, plays a key role in controlling gene expression in some Gram-positive pathogens, including Staphylococcus aureus and Enterococcus faecalis. AgrA is encoded by the agrACDB global regulatory locus, and orthologues are found within the genome of most Clostridium difficile isolates, including the epidemic lineage 027/BI/NAP1. Comparative RNA sequencing of the wild type and otherwise isogenic agrA null mutant derivatives of C. difficile R20291 revealed a network of approximately 75 differentially regulated transcripts at late exponential growth phase, including many genes associated with flagellar assembly and function, such as the major structural subunit, FliC. Other differentially regulated genes include several involved in bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) synthesis and toxin A expression. C. difficile 027 R20291 agrA mutant derivatives were poorly flagellated and exhibited reduced levels of colonization and relapses in the murine infection model. Thus, the agr locus likely plays a contributory role in the fitness and virulence potential of C. difficile strains in the 027/BI/NAP1 lineage. PMID:23772065
Hell, M; Bernhofer, C; Stalzer, P; Kern, J M; Claassen, E
In the past two years an enormous amount of molecular, genetic, metabolomic and mechanistic data on the host-bacterium interaction, a healthy gut microbiota and a possible role for probiotics in Clostridium difficile infection (CDI) has been accumulated. Also, new hypervirulent strains of C. difficile have emerged. Yet, clinical trials in CDI have been less promising than in antibiotic associated diarrhoea in general, with more meta-analysis than primary papers on CDI-clinical-trials. The fact that C. difficile is a spore former, producing at least three different toxins has not yet been incorporated in the rational design of probiotics for (recurrent) CDI. Here we postulate that the plethora of effects of C. difficile and the vast amount of data on the role of commensal gut residents and probiotics point towards a multistrain mixture of probiotics to reduce CDI, but also to limit (nosocomial) transmission and/or endogenous reinfection. On the basis of a retrospective chart review of a series of ten CDI patients where recurrence was expected, all patients on adjunctive probiotic therapy with multistrain cocktail (Ecologic®AAD/OMNiBiOTiC® 10) showed complete clinical resolution. This result, and recent success in faecal transplants in CDI treatment, are supportive for the rational design of multistrain probiotics for CDI.
Ross, Caná L; Spinler, Jennifer K; Savidge, Tor C
Alteration of the gut microbial community structure and function through antibiotic use increases susceptibility to colonization by Clostridium difficile and other enteric pathogens. However, the mechanisms that mediate colonization resistance remain elusive. As the leading definable cause of infectious diarrhea, toxigenic C. difficile represents a burden for patients and health care systems, underscoring the need for better diagnostics and treatment strategies. Next-generation sequence data has increased our understanding of how the gut microbiota is influenced by many factors including diet, disease, aging and drugs. However, a microbial-based biomarker differentiating C. difficile infection from antibiotic-associated diarrhea has not been identified. Metabolomics profiling, which is highly responsive to changes in physiological conditions, have shown promise in differentiating subtle disease phenotypes that exhibit a nearly identical microbiome community structure, suggesting metabolite-based biomarkers may be an ideal diagnostic for identifying patients with CDI. This review focuses on the current understanding of structural and functional changes to the gut microbiota during C. difficile infection obtained from studies assessing the microbiome and metabolome of samples from patients and murine models.
Quesada-Gómez, Carlos; Mulvey, Michael R; Vargas, Pablo; Gamboa-Coronado, María del Mar; Rodríguez, César; Rodríguez-Cavillini, Evelyn
We isolated a regional toxigenic genotype of Clostridium difficile, previously found in human infection in 4 of 200 (2%) samples of retail meats for human consumption: 1 of 67 samples of beef, 2 of 66 of pork, and 1 of 67 of poultry meat. These four isolates were positive for the tcdA and tcdB genes but negative for deletion of the tcdC and cdtB genes. All strains induced cytopathic effects in HeLa cells. However, they were susceptible to some antibiotics to which clinical isolates are often resistant. All strains were susceptible to vancomycin, metronidazole, moxifloxacin, and rifampicin but resistant to clindamycin and ciprofloxacin. This first report of isolation of C. difficile in foodstuff from Latin America lends support to the notion that animal products serve as a reservoir for clinical strains of this pathogen in the community.
López-Ureña, Diana; Quesada-Gómez, Carlos; Miranda, Erick; Fonseca, Mercedes; Rodríguez-Cavallini, Evelyn
The rate and severity of Clostridium difficile infection (CDI) have been linked to the emergence and spread of the hypervirulent toxigenic strain NAP1/027. This strain has been responsible for large outbreaks in healthcare facilities in North America and Europe and most recently in Latin America. This is the first report of the NAP1 strain in Panama. It suggests that the spread of C. difficile NAP1 throughout Latin America could be a possibility as evidenced in the following case reports. Five isolates typed as NAP1 had tcdA, tcdB, binary toxin gene cdtB and tcdC deletion. All isolates were resistant to clindamycin, fluoroquinolones and rifampicin. Under this scenario, surveillance programmes for CDI should be implemented in public health facilities in Latin America and diagnosis of CDI should be considered, especially in patients with predisposing factors.
Edmond, Michael B
The human gut is colonized with 200 to 1,000 bacterial species. Administration of antibiotics reduces the diversity of the intestinal microbiota, reduces colonization resistance, and can lead to infection with Clostridium difficile. These infections have become more prevalent and increasingly patients are experiencing multiple recurrences that are incurable with standard treatment. Although fecal microbiota transplantation (FMT) has been used for centuries in human and veterinary medicine, only recently has it be shown to be highly effective for recurrent C. difficile infection. The goal of FMT is to re-introduce a complete, stable community of gut microorganisms to repair or replace the disrupted native microbiota. FMT can be delivered via nasoenteric tube, colonoscopy, or enema. Despite a cure rate approximating 90%, many barriers to FMT have limited its availability to patients. The recent development of a not-for-profit stool bank has helped to make this therapy more accessible. Additional indications for FMT are currently under investigation.
Dutta, Sudhir K; Girotra, Mohit; Garg, Shashank; Dutta, Anand; von Rosenvinge, Erik C; Maddox, Cynthia; Song, Yang; Bartlett, John G; Vinayek, Rakesh; Fricke, W Florian
The prevalence of recurrent Clostridium difficile infection (RCDI) is increasing; fecal microbiota transplantation (FMT) is an effective therapy. However, there have been no studies of the efficacy of a single session of combined enteral and colonic FMT or characterizations of changes in the microbiota between donors and recipients. We performed a study of 27 patients with RCDI who were given a fixed volume of processed fecal filtrate via enteroscopy and colonoscopy in a single session. Patients were closely monitored, and fecal samples were collected from 2 patient-donor pairs for 16S rRNA analysis. All patients had reduced stool frequency, abdominal pain, white blood cell counts, and elimination of fecal C difficile toxin (P < .05). FMT increased microbial diversity, increasing proportions of Lachnospiraceae (phylum Firmicutes) and reducing proportions of Enterobacteriaceae. FMT was associated with marked changes in the composition of fecal microbiota in 2 patients with RCDI.
Marukawa, Yohei; Komura, Takuya; Kagaya, Takashi; Ohta, Hajime; Unoura, Masashi
In July 2012, metronidazole was approved for the treatment of Clostridium difficile infection (CDI). To clarify the selection criteria for the drug in terms of CDI severity, we established a diagnostic and therapeutic algorithm with reference to the SHEA-IDSA Clinical Practice Guidelines. We compared patients whose treatments were guided by the algorithm (29 cases, October 2012-September 2013) with patients treated prior to the development of the algorithm (37 cases, October 2011-September 2012). All cases treated with reference to the algorithm were diagnosed using enzyme immunoassay of C. difficile toxins A and B and glutamate dehydrogenase;an appropriate drug was prescribed in 93.1% of the cases. We found no significant between-group differences in the cure, recurrence, or complication rates. However, drug costs in cases wherein treatments were guided by the algorithm were markedly reduced. We have, thus, shown that algorithm-guided treatment is efficacious and cost-effective.
Brauch, Rebecca; Cherabuddi, Kartikeya
The clinical spectrum of Clostridium difficile infection can range from benign gastrointestinal colonization to mild diarrhea and life threatening conditions such as pseudomembranous colitis and toxic megacolon. Extraintestinal manifestations of C. difficile are rare. Here, we report a patient with a history of an endovascular aortic aneurysm repair (EVAR) presenting with an endovascular leak complicated by C. difficile bacteremia and a mycotic aneurysm. He was successfully treated with an explant of the EVAR, an aorto-left renal bypass, and aorto-bi-iliac bypass graft placement along with a six-week duration of intravenous vancomycin and oral metronidazole. PMID:28348903
Kassam, Zain; Fabersunne, Camila Cribb; Smith, Mark B.; Alm, Eric J.; Kaplan, Gilaad G.; Nguyen, Geoffrey C.; Ananthakrishnan, Ashwin N.
Background Clostridium difficile infection (CDI) is public health threat and associated with significant mortality. However, there is a paucity of objectively derived CDI severity scoring systems to predict mortality. Aims To develop a novel CDI risk score to predict mortality entitled: Clostridium difficile Associated Risk of Death Score (CARDS). Methods We obtained data from the United States 2011 Nationwide Inpatient Sample (NIS) database. All CDI-associated hospitalizations were identified using discharge codes (ICD-9-CM, 008.45). Multivariate logistic regression was utilized to identify independent predictors of mortality. CARDS was calculated by assigning a numeric weight to each parameter based on their odds ratio in the final logistic model. Predictive properties of model discrimination were assessed using the c-statistic and validated in an independent sample using the 2010 NIS database. Results We identified 77,776 hospitalizations, yielding an estimate of 374,747 cases with an associated diagnosis of CDI in the United States, 8% of whom died in the hospital. The 8 severity score predictors were identified on multivariate analysis: age, cardiopulmonary disease, malignancy, diabetes, inflammatory bowel disease, acute renal failure, liver disease and ICU admission, with weights ranging from −1 (for diabetes) to 5 (for ICU admission). The overall risk score in the cohort ranged from 0 to 18. Mortality increased significantly as CARDS increased. CDI-associated mortality was 1.2% with a CARDS of 0 compared to 100% with CARDS of 18. The model performed equally well in our validation cohort. Conclusion CARDS is a promising simple severity score to predict mortality among those hospitalized with CDI. PMID:26849527
Edwards, Adrianne N.; Karim, Samiha T.; Pascual, Ricardo A.; Jowhar, Lina M.; Anderson, Sarah E.; McBride, Shonna M.
Clostridium difficile is a Gram-positive, sporogenic and anaerobic bacterium that causes a potentially fatal colitis. C. difficile enters the body as dormant spores that germinate in the colon to form vegetative cells that secrete toxins and cause the symptoms of infection. During transit through the intestine, some vegetative cells transform into spores, which are more resistant to killing by environmental insults than the vegetative cells. Understanding the inherent resistance properties of the vegetative and spore forms of C. difficile is imperative for the development of methods to target and destroy the bacterium. The objective of this study was to define the chemical and environmental resistance properties of C. difficile vegetative cells and spores. We examined vegetative cell and spore tolerances of three C. difficile strains, including 630Δerm, a 012 ribotype and a derivative of a past epidemic strain; R20291, a 027 ribotype and current epidemic strain; and 5325, a clinical isolate that is a 078 ribotype. All isolates were tested for tolerance to ethanol, oxygen, hydrogen peroxide, butanol, chloroform, heat and sodium hypochlorite (household bleach). Our results indicate that 630Δerm vegetative cells (630 spo0A) are more resistant to oxidative stress than those of R20291 (R20291 spo0A) and 5325 (5325 spo0A). In addition, 5325 spo0A vegetative cells exhibited greater resistance to organic solvents. In contrast, 630Δerm spores were more sensitive than R20291 or 5325 spores to butanol. Spores from all three strains exhibited high levels of resistance to ethanol, hydrogen peroxide, chloroform and heat, although R20291 spores were more resistant to temperatures in the range of 60–75°C. Finally, household bleach served as the only chemical reagent tested that consistently reduced C. difficile vegetative cells and spores of all tested strains. These findings establish conditions that result in vegetative cell and spore elimination and illustrate the
Camacho-Ortiz, Adrián; López-Barrera, Daniel; Hernández-García, Raúl; Galván-De los Santos, Alejandra M.; Flores-Treviño, Samantha M.; Llaca-Díaz, Jorge M.; Garza, Héctor J. Maldonado; Bosques-Padilla, Francisco J.; Garza-González, Elvira
Background and Objective Clostridium difficile NAP1/ribotype 027 is associated with severe disease and high mortality rates. Our aim was to determine the prevalence of NAP1/ribotype 027 among C. difficile isolates in a tertiary care hospital, and review the main clinical data. Methods We included 106 stool samples from 106 patients. Samples were tested for A&B toxins and were cultured on CCFA agar. The genes tcdA, tcdB, tcdC, cdtA, and cdtB were amplified using PCR in clinical isolates. The tcdA 3’-end deletion analysis, PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) were also performed. Stool samples that were positive for culture were tested by the GeneXpert C. difficile assay. Clinical data were collected. Results Thirty-six patients tested positive for A&B toxins; and 22 patients had positive culture for C. difficile, 14 of which tested positive for the A&B toxins and all 22 patients tested positive by the GeneXpert C. difficile assay. Risk factors included an average hospital stay of 16.1 days prior to toxin detection, average antibiotic use for 16.2 days, and a median of 3 antibiotics used. The 30-day crude mortality rate was 8.4%. Six of the 22 patients died, and 3 of those deaths were directly attributed to C. difficile infection. The majority of isolates, 90.9% (20/22), carried genes tcdB, tcdA, cdtA, and cdtB; and these strains carried the corresponding downregulator gene tcdC, with an 18-bp deletion. PFGE was performed on 17 isolates, and one main pattern was observed. Analysis of the ribotyping data showed similar results. Conclusion The above findings represent the clonal spread of C. difficile in our institution, which mainly includes the NAP1/027 strain. This is the first report of C. difficile ribotype NAP1/027 in Mexico. PMID:25915544
Clare-Pascoe, N; Lee, MG; Murphy, T; Nicholson, A; Ferguson, TS
ABSTRACT Objectives: This study examined the frequency of Clostridium difficile infection (CDI) among hospital admission and diarrhoeal stool samples over a six-year period. Methods: A review of all suspected cases of C difficile positive patients from 2007 to 2012 at the University Hospital of the West Indies (UHWI), Jamaica, was performed. Clostridium difficile infection was confirmed by clinical features and a positive enzyme-linked immunosorbent assay (ELISA) stool test for Clostridium Toxins A and B. The demographics, clinical features, risk factors, treatment and outcomes were also collated. Results: There were 56 patients reviewed. The most commonly affected age group was 40–59 years of age. The proportion of CDI cases per total stool samples increased from 0.5% in 2007 to 5.9% in 2010 then fell to 2.2% in 2011 but increased again to 4.3% in 2012. The proportion of cases per total UHWI admissions also increased from 0.12 cases per 1000 admissions in 2007 to 1.16 in 2010 and 1.36 in 2012 (p < 0.001). Most CDI cases were nosocomial (76% males, 48.6% females). Co-morbidities included hypertension and end-stage renal disease. Ceftazidime was the most common antibiotic associated with the development of CDI. Resolution occurred in 62.5% of patients. Duration of hospital stay was longer in males than females (≥ 21 versus < 7 days) and males had more adverse outcomes, with death in 23.8% versus 11.4%. Conclusion: There has been an increase in the frequency of CDI at UHWI with a greater than expected frequency of community acquired CDI. Increased awareness is needed of the increasing risk for CDI and measures must be taken to prevent the disease, especially in hospitalized patients. PMID:26624597
Hammitt, Melissa C; Bueschel, Dawn M; Keel, M Kevin; Glock, Robert D; Cuneo, Peder; DeYoung, Donald W; Reggiardo, Carlos; Trinh, Hien T; Songer, J Glenn
Clostridium difficile was investigated as a possible cause of enteritis in calves. The organism and its toxins (TcdA and TcdB), respectively, were found in 25.3% and 22.9% of stool samples from diarrheic calves. Culture positive samples were more likely than culture negative samples to be toxin positive. However, toxin positive stools were more common among nondiarrheic calves, but diarrheic calves were nearly twice as likely to be culture positive. Ribotype 078 was dominant among isolates. Salmonella sp. was isolated from both diarrheic and nondiarrheic calves, but large numbers of E. coli were found more commonly in diarrheic calves than in nondiarrheic animals. Prevalence rates for coronavirus and Cryptosporidium sp. were substantially higher in nondiarrheic calves than in diarrheic, but rates of detection of rotavirus and Giardia sp. were more nearly equal between groups. Lesions in naturally infected calves included superficial mucosal erosion with associated fibrinous exudates. Neutrophils and eosinophils infiltrated lamina propria. Large Gram-positive rods morphologically compatible with C. difficile were abundant in the colonic lumen and the organism was isolated by bacteriologic culture. Toxins were found throughout the colon. Purified toxins A and B (individually and conjointly) caused comparable lesions, as well as fluid accumulation, in ligated intestinal loops. Our findings are in substantial agreement with those of others [Rodriguez-Palacios, A., Stampfli, H.R., Duffield, T., Peregrine, A.S., Trotz-Williams, L.A., Arroyo, L.G., Brazier, J.S., Weese, J.S., 2006. Clostridium difficile PCR ribotypes in calves, Canada. Emerg. Infect. Dis. 12, 1730-1736; Porter, M.C., Reggiardo, C., Bueschel, D.M., Keel, M.K., Songer, J.G., 2002. Association of Clostridium difficile with bovine neonatal diarrhea. Proc. 45th Ann. Mtg. Amer. Assoc. Vet. Lab. Diagn., St. Louis, MO, U.S.A.] and add strength to a working hypothesis that C. difficile infection and the
Neemann, K; Eichele, D D; Smith, P W; Bociek, R; Akhtari, M; Freifeld, A
We present a case of severe Clostridium difficile infection (CDI) in a non-neutropenic allogeneic hematopoietic stem cell transplant recipient who was treated successfully with fecal microbiota therapy after standard pharmacologic therapy had failed. Following naso-jejunal instillation of donor stool, the patient's symptoms resolved within 48 h. Bowel resection was averted. This is the first case in the literature, to our knowledge, to describe fecal microbiota therapy in a profoundly immunocompromised host with severe CDI. We propose that fecal microbiota therapy be considered as a therapeutic option in immunosuppressed patients with refractory severe CDI.
Karjalainen, Tuomo; Waligora-Dupriet, Anne-Judith; Cerquetti, Marina; Spigaglia, Patrizia; Maggioni, Andrea; Mauri, Pierluigi; Mastrantonio, Paola
The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79–685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79–685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins. PMID:11292772
Clostridium difficile-associated diarrhea (CDAD) is the most common cause of healthcare-associated diarrhea. The current recommended treatment regimens of metronidazole and vancomycin have not changed in nearly 25 years. Fidaxomicin, an exceedingly narrow spectrum macrolide antibiotic, was recently approved for the treatment of CDAD. In phase III clinical trials, fidaxomicin was noninferior to vancomycin in achieving clinical cure of CDAD. Furthermore, fidaxomicin was associated with fewer recurrences of CDAD compared with vancomycin in clinical trials. These results, combined with the ease of administration and a good safety profile, make fidaxomicin an attractive treatment option for treating CDAD. PMID:23152733
Dang, Uyen T.; Zamora, Idalia; Hevener, Kirk E.; Adhikari, Sudip; Wu, Xiaoqian
We characterized clinically occurring and novel mutations in the β subunit of RNA polymerase in Clostridium difficile (CdRpoB), conferring rifamycin (including rifaximin) resistance. The Arg505Lys substitution did not impose an in vitro fitness cost, which may be one reason for its dominance among rifamycin-resistant clinical isolates. These observations were supported through the structural modeling of CdRpoB. In general, most mutations lacked in vitro fitness costs, suggesting that rifamycin resistance may in some cases persist in the clinic. PMID:27381389
Mullany, Peter; Allan, Elaine; Roberts, Adam P.
Approximately 11% the Clostridium difficile genome is made up of mobile genetic elements which have a profound effect on the biology of the organism. This includes transfer of antibiotic resistance and other factors that allow the organism to survive challenging environments, modulation of toxin gene expression, transfer of the toxin genes themselves and the conversion of non-toxigenic strains to toxin producers. Mobile genetic elements have also been adapted by investigators to probe the biology of the organism and the various ways in which these have been used are reviewed. PMID:25576774
Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B
Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We
Bassis, Christine M.; Young, Vincent B.
Antibiotics can play dual roles in Clostridium difficile infection (CDI); antibiotic treatment increases the risk of CDI, and antibiotics are used to treat CDI. The glycylcycline antibiotic tigecycline has broad antimicrobial activity, yet it is rarely associated with the development of CDI, presumably due to its activity against C. difficile. In this study, we investigated how tigecycline treatment affects the structure of the gut microbiota and susceptibility to CDI by treating mice with tigecycline (n = 20) or saline (n = 8) for 10 days. A sequence analysis of the bacterial 16S rRNA gene amplicons was used to monitor changes in the fecal microbiota. A subset of the mice was followed for 5 weeks after the end of treatment. The remaining mice were challenged with C. difficile strain VPI 10463 spores 2 days after the tigecycline treatment ended. Tigecycline treatment resulted in major shifts in the gut microbiota, including large decreases in Bacteroidetes levels and large increases in Proteobacteria levels. Mice with tigecycline-altered microbial communities were susceptible to challenge with C. difficile spores and developed clinical signs of severe CDI. Five weeks after the cessation of tigecycline treatment, the recovery of the bacterial community was incomplete and diversity was lower than in the untreated controls. Antibiotics with intrinsic activity against C. difficile can still alter the microbiota in a way that leads to susceptibility to CDI after discontinuation of the drug. These results indicate that microbiotic dynamics are key in the development of CDI, and a better understanding of these dynamics may lead to better strategies to prevent and treat this disease. PMID:24590475
Tian, Tian-tian; Zhao, Jian-hong; Yang, Jing; Qiang, Cui-xin; Li, Zhi-rong; Chen, Jing; Xu, Kai-yue; Ciu, Qing-qing; Li, Ru-xin
Clostridium difficile is a spore-forming, gram-positive, anaerobic bacillus that can cause C. difficile infection (CDI). However, only a few studies on the prevalence and antibiotic resistance of C. difficile in healthy individuals in China have been reported. We employed a spore enrichment culture to screen for C. difficile in the stool samples of 3699 healthy Chinese individuals who were divided into 4 groups: infants younger than 2 years of age and living at home with their parents; children aged 1 to 8 years of age and attending three different kindergarten schools; community-dwelling healthy adult aged 23–60 years old; and healthcare workers aged 28–80 years old. The C. difficile isolates were analyzed for the presence of toxin genes and typed by PCR ribotyping and multilocus sequence typing (MLST). The minimum inhibitory concentration of 8 antimicrobial agents was determined for all of the isolates using the agar dilution method. The intestinal carriage rate in the healthy children was 13.6% and ranged from 0% to 21% depending on age. The carriage rates in the 1654 community-dwelling healthy adults and 348 healthcare workers were 5.5% and 6.3%, respectively. Among the isolates, 226 were toxigenic (225 tcdA+/tcdB+ and 1 tcdA+/tcdB+ ctdA+/ctdB+). Twenty-four ribotypes were found, with the dominant type accounting for 29.7% of the isolates. The toxigenic isolates were typed into 27 MLST genotypes. All of the strains were susceptible to vancomycin, metronidazole, fidaxomicin, and rifaximin. High resistance to levofloxacin and ciprofloxacin at rates of 39.8% and 98.3%, respectively, were observed. ST37 isolates were more resistant to levofloxacin than the other STs. The PCR ribotypes and sequence types from the healthy populations were similar to those from the adult patients. PMID:27011211
Quesada-Gómez, Carlos; López-Ureña, Diana; Acuña-Amador, Luis; Villalobos-Zúñiga, Manuel; Du, Tim; Freire, Rosemayre; Guzmán-Verri, Caterina; Gamboa-Coronado, María del Mar; Lawley, Trevor D.; Moreno, Edgardo; Mulvey, Michael R.; Brito, Gerly Anne de Castro; Rodríguez-Cavallini, Evelyn; Rodríguez, César
The prevalence of Clostridium difficile infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This C. difficile variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAPCR1. Whole-genome sequencing and ribotyping indicated that the NAPCR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAPCR1 strains are resistant to fluoroquinolones due to a mutation in gyrA, and they possess an 18-bp deletion in tcdC that is characteristic of the epidemic, evolutionarily distinct, C. difficile NAP1 variant. NAPCR1 genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAPCR1 was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAPCR1 strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging C. difficile variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA. PMID:25653402
Kumar, Nitin; Miyajima, Fabio; He, Miao; Roberts, Paul; Swale, Andrew; Ellison, Louise; Pickard, Derek; Smith, Godfrey; Molyneux, Rebecca; Dougan, Gordon; Parkhill, Julian; Wren, Brendan W.; Parry, Christopher M.; Pirmohamed, Munir; Lawley, Trevor D.
Background. Accurate tracking of Clostridium difficile transmission within healthcare settings is key to its containment but is hindered by the lack of discriminatory power of standard genotyping methods. We describe a whole-genome phylogenetic-based method to track the transmission of individual clones in infected hospital patients from the epidemic C. difficile 027/ST1 lineage, and to distinguish between the 2 causes of recurrent disease, relapse (same strain), or reinfection (different strain). Methods. We monitored patients with C. difficile infection in a UK hospital over a 2-year period. We performed whole-genome sequencing and phylogenetic analysis of 108 strains isolated from symptomatic patients. High-resolution phylogeny was integrated with in-hospital transfers and contact data to create an infection network linking individual patients and specific hospital wards. Results. Epidemic C. difficile 027/ST1 caused the majority of infections during our sampling period. Integration of whole-genome single nucleotide polymorphism (SNP) phylogenetic analysis, which accurately discriminated between 27 distinct SNP genotypes, with patient movement and contact data identified 32 plausible transmission events, including ward-based contamination (66%) or direct donor–recipient contact (34%). Highly contagious donors were identified who contributed to the persistence of clones within distinct hospital wards and the spread of clones between wards, especially in areas of intense turnover. Recurrent cases were identified between 4 and 26 weeks, highlighting the limitation of the standard <8-week cutoff used for patient diagnosis and management. Conclusions. Genome-based infection tracking to monitor the persistence and spread of C. difficile within healthcare facilities could inform infection control and patient management. PMID:26683317
Bruxelle, J-F; Mizrahi, A; Hoys, S; Collignon, A; Janoir, C; Péchiné, S
Clostridium difficile is an opportunistic pathogen causing gut inflammation generally associated with an intestinal dysbiosis due to antibiotics. Several virulence factors have been identified as playing a key role in gut colonization. The surface-layer proteins, comprised of two proteins, the high molecular weight SlpA (HMW-SLP) and the low molecular weight SlpA (LMW-SLP), are the most abundant proteins on the C. difficile surface. These two proteins are derived from the Cwp84-mediated cleavage of a single precursor protein SlpA. In this study, we assessed the immunogenic properties of a recombinant SlpA precursor derived from a toxigenic C. difficile strain (630) and its protective effect as a vaccine antigen co-administered with the cholera toxin as an adjuvant in both hamster and mouse models. First, we confirmed the immunogenicity of SlpA in humans. Sera from patients with C. difficile infection were analyzed by ELISA. Patients with CDI have a greater number of SlpA antibodies than healthy patients, confirming the immunogenicity of this protein during the pathogenic process. Then, rectal vaccination assays were performed in both conventional hamsters and mice. The animals' sera were sampled before and after vaccination, and were analyzed by ELISA. In addition, in the mouse model, feces were sampled after vaccination and IgA directed against SlpA were detected by ELISA. In both models, the intestinal colonization was evaluated by fecal bacterial count after challenge. Intra-rectal vaccination with SlpA and cholera toxin as an adjuvant induced a local and systemic humoral immune response in mice and hamsters potentially responsible for the weak decrease of C. difficile colonization in mice and the partial protection observed in a lethal-hamster model.
Kelly, Sean G; Yarrington, Michael; Zembower, Teresa R; Sutton, Sarah H; Silkaitis, Christina; Postelnick, Michael; Mikolajczak, Anessa; Bolon, Maureen K
BACKGROUND The nationally reported metric for Clostridium difficile infection (CDI) relies solely on laboratory testing, which can result in overreporting due to asymptomatic C. difficile colonization. OBJECTIVE To review the clinical scenarios of cases of healthcare facility-onset CDI (HO-CDI) and to determine the appropriateness of C. difficile testing on the basis of presence of symptomatic diarrhea in order to identify areas for improvement. DESIGN Retrospective cohort study. SETTING Northwestern Memorial Hospital, a large, tertiary academic hospital in Chicago, Illinois. PATIENTS The cohort included all patients with a positive C. difficile test result who were reported to the National Healthcare Safety Network as HO-CDI during a 1-year study period. METHODS We reviewed the clinical scenario of each HO-CDI case. On the basis of documentation and predefined criteria, appropriateness of C. difficile testing was determined; cases were deemed appropriate, inappropriate, or indeterminate. Statistical analysis was performed to compare demographic and clinical parameters among the categories of testing appropriateness. RESULTS Our facility reported 168 HO-CDI cases to NHSN during the study period. Of 168 cases, 33 (19.6%) were judged to be appropriate tests, 25 (14.8%) were considered inappropriate, and 110 (65.5%) were indeterminate. Elimination of inappropriate testing would have improved our facility's standardized infection ratio from 0.962 to 0.819. CONCLUSION Approximately 15% of HO-CDI cases were judged to be tested inappropriately. Testing only patients with clinically significant diarrhea would more accurately estimate CDI incidence, reduce unnecessary antibiotic use, and improve facilities' performance of reportable CDI metrics. Improved documentation could facilitate targeted interventions. Infect Control Hosp Epidemiol 2016;1395-1400.
Calderón, Gloria M.; Torres-López, Javier; Lin, Tong-Jun; Chavez, Bibiana; Hernández, Manuel; Muñoz, Onofre; Befus, A. Dean; Enciso, J. Antonio
Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-α). In contrast, 1 μg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 μg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response to C. difficile infection by releasing TNF-α upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficile toxins. PMID:9596744
Donnelly, M. Lauren; Li, William; Li, Yong-qing; Hinkel, Lauren; Setlow, Peter
ABSTRACT Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG), a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies. PMID:28096487
Nath, Swapan K; Salama, Suzette; Persaud, Devia; Thornley, James H; Smith, Ian; Foster, Gary; Rotstein, Coleman
A case-control study was undertaken to identify and quantify antimicrobial and nonantimicrobial drug risk factors associated with a sustained outbreak of Clostridium difficile diarrhea on two medical (teaching and nonteaching) units and an oncology unit. In total, 80 cases associated with an endemic clone of toxigenic C difficile were compared with controls. Eighty controls were selected from a group of 290 controls randomly chosen from the outbreak period. The controls were matched to cases according to age, admitting diagnosis and unit of admission. Seventy (88%) patients in the case group received at least one antibiotic before diarrhea, compared with 37 (46%) patients in the control group. Major risk factors implicated in the development of C difficile diarrhea in hospitalized patients were the following antimicrobial agents: ceftazidime (adjusted odds ratio [aor]=26.01, 95% ci 5.67 to 119.19, P=0.0001); cefuroxime (aor=5.17, ci 1.86 to 14.36, P=0.005); ciprofloxacin (aor=3.81, ci 1.05 to 13.79, P=0.04); and clindamycin (aor=15.16, ci 2.93 to 78.44, P=0.004). This is the first time that the use of ciprofloxacin has been linked to the development of C difficile diarrhea. Use of gastrointestinal drugs (ranitidine, famotidine, cimetidine, omeprazole and sucralfate) was also an added risk (aor=3.20, ci 1.39 to 7.34, P=0.01); however, antineoplastic therapy was not significant (P<0.53). Recognition of the specific high risk drugs may spur more restricted use of these agents, which may help in controlling C difficile diarrhea in hospitalized patients. PMID:22346513
Kiser, Casey J; Urish, Kenneth L; Boateng, Henry A
Clostridium myonecrosis or gas gangrene is a life-threatening infection characterized by either traumatic or atraumatic etiology. It has been widely described in patients with traumatic open wounds and in immunocompromised patients, including malignancy. A third source can result from natural flora in the gastrointestinal tract after bowel ischemia. This is a rare occurrence and is even less commonly described in the pediatric population. We present a pediatric patient who developed Clostridium septicum myonecrosis as an iatrogenic complication from clindamycin-induced Clostridium difficile ischemic colitis.
Purcell, Erin B.; McKee, Robert W.; Bordeleau, Eric; Burrus, Vincent
ABSTRACT The intestinal pathogen Clostridium difficile is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States. C. difficile rates of recurrence and mortality have increased in recent years due to the emergence of so-called “hypervirulent” epidemic strains. A great deal of the basic biology of C. difficile has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for C. difficile gene regulation. However, the function(s) of TFP in C. difficile remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of C. difficile. We demonstrate that TFP contribute to C. difficile biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in C. difficile. The expression and regulation of the pilA1 pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in pilA1 expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic C. difficile strains may contribute to the different infection outcomes presented by these strains. IMPORTANCE How Clostridium difficile establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of C. difficile are likely relevant during infection, representing possible interactions between the bacterium and the
Buchan, Blake W; Mackey, Tami-Lea A; Daly, Judy A; Alger, Garrison; Denys, Gerald A; Peterson, Lance R; Kehl, Sue C; Ledeboer, Nathan A
We compared the Portrait Toxigenic C. difficile Assay, a new semiautomated sample-to-result molecular test, to a toxigenic bacterial culture/cell cytotoxin neutralization assay (TBC/CCNA) for the detection of toxigenic Clostridium difficile in 549 stool specimens. Stool specimens were also tested by one of three alternative FDA-cleared molecular tests for toxigenic C. difficile (Xpert C. difficile, Illumigene C. difficile, or GeneOhm Cdiff). The sensitivities and specificities of the molecular tests compared to TBC/CCNA were as follows: 98.2% and 92.8% for the Portrait assay, 100% and 91.7% for the Xpert assay, 93.3% and 95.1% for the Illumigene assay, and 97.4% and 98.5% for the GeneOhm assay, respectively. The majority of Portrait false-positive results (20/31; 64.5%) were also positive for C. difficile by an alternative molecular test, suggesting an increased sensitivity compared to the culture-based "gold standard" method. The Portrait test detected an assay input of 30 CFU in 100% of spiked samples and detected an input of 10 CFU in 96.7% of samples tested.
Aptekorz, Małgorzata; Szczegielniak, Anna; Wiechuła, Barbara; Harmanus, Celine; Kuijper, Ed; Martirosian, Gayane
Clostridium difficile is an important healthcare-associated pathogen, responsible for a broad spectrum of diarrheal diseases. The aim of this prospective study was to determine the occurrence of C. difficile infection (CDI), to characterize cultured C. difficile strains and to investigate the association of fecal lactoferrin with CDI. Between January 2013 and June 2014, 148 stool samples were obtained from adult diarrheal patients (C. difficile as a suspected pathogen) hospitalized in different healthcare facilities of 15 Silesian hospitals. Out of 134 isolated C. difficile strains, 108 were ribotyped: 82.4% belonged to Type 027, 2.8% to Type 176, 2.8% to Type 014, 1.9% to Type 010 and 0.9% to Types 001, 018, 020 and 046 each. In total, 6.5% non-typable strains were identified. All Type 027 isolates contained both toxin genes tcdA & tcdB, and binary toxin genes (cdtA &cdtB). Susceptibility testing revealed that all Type 027 isolates were sensitive to metronidazole and vancomycin and resistant to moxifloxacin, ciprofloxacin, imipenem and erythromycin. Of 89 Type 027 strains, 16 had a ermB (688 bp) gene coinciding with high levels of erythromycin resistance (MIC >256 μg/mL). Of 16 ermB positive strains, 14 demonstrated also high level of resistance to clindamycin (>256 μg/mL). A significant difference (p = 0.004) in lactoferrin level was found between C. difficile toxin-positive (n = 123; median 185.9 μg/mL; IQR 238.8) and toxin-negative (n = 25; median 22.4 μg/mL; IQR 141.7) fecal samples. Stool samples from n = 89 patients with CDI caused by Type 027 demonstrated significantly higher (p = 0.03) lactoferrin level (median 173.0 μg/mL; IQR 237.3) than from patients with CDI caused by other ribotypes and non-typable C. difficile strains (median 189.4 μg/mL; IQR 190.8).
Prior, A R; Kevans, D; McDowell, L; Cudmore, S; Fitzpatrick, F
Adherence to Clostridium difficile infection treatment guidelines is associated with lower recurrence rates and mortality as well as cost savings. This survey of Irish clinicians indicates that patients are managed using a variety of approaches. Faecal microbiota transplantation is potentially underused despite its recommendation in national and European guidelines.
Pantosti, A; Luzzi, I; Cardines, R; Gianfrilli, P
The in vitro activity of teicoplanin was compared with that of vancomycin against fecal isolates of Clostridium difficile. All strains were susceptible to both antibiotics, but teicoplanin was fourfold more active than vancomycin. Cholestyramine was found to bind teicoplanin almost completely, reducing its activity to nondetectable levels. PMID:2935077
Blaich, Annette; Frei, Reno; Castellano, Carine; Kiessling, Christine; Geschke, Angelika; Rentsch, Katharina M; Egli, Adrian
A novel immunoassay for Clostridium difficile glutamate dehydrogenase (GDH) and toxin A&B (LIAISON, DiaSorin) was compared to another GDH assay (Alere), PCR and toxigenic culture. The GDH-DiaSorin is slightly more sensitive than the GDH-Alere. Sensitivity of the Toxin-Diasorin test is in accordance to the sensitivity of other immunoassays in literature.
Fawley, Warren N; Wilcox, Mark H
Of 53 potential Clostridium difficile infection (CDI) case clusters/outbreaks, affecting 2 to 41 patients in 27 institutions, 19% comprised unrelated isolates and 34% had highly related and distinct isolates as shown by multilocus variable-number tandem-repeat analysis, despite sharing a common ribotype. These findings emphasize the value of enhanced fingerprinting to confirm or refute suspected CDI case clusters.
Polgreen, Philip M; Yang, Ming; Kuntz, Jennifer L; Laxminarayan, Ramanan; Cavanaugh, Joseph E
Sales for a drug may be correlated with the prevalence of a condition treated by the drug. We found that state data revealed a strong spatial association and national data a strong temporal association between Clostridium difficile and oral vancomycin prescription sales, suggesting a new "signal" for detecting disease activity.
Clostridium difficile can be a major problem in hospitals because the bacterium primarily affects individuals with an altered gut flora, which largely occurs through prolonged antibiotic use. Proposed sources of increased community-acquired infections are food animals and retail meats. The objecti...
Moure, Raquel; Cañizares, Ángeles; Muíño, María; Lobato, Margarita; Fernández, Ana; Rodríguez, María; Gude, Maria José; Tomás, Maria; Bou, Germán
The new cobas® Cdiff and cobas® MRSA/SA tests were compared with conventional methods for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus. The final concordance between cobas Cdiff Test and GDH/toxin gene screening was 97.62% and between cobas MRSA/SA Test and chromogenic culture, 91.30%, respectively.
Donskey, Curtis J.
Exposing Clostridium difficile spores to germinants in a quaternary ammonium matrix was an effective method to reduce environmental contamination by sensitizing the spores, leaving them susceptible to ambient conditions and enhancing killing by acid, high-intensity visible light, and radiation. PMID:28066792
Nölke, Thilo; Schwan, Carsten; Lehmann, Friederike; Østevold, Kristine; Pertz, Olivier; Aktories, Klaus
Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule–membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton. PMID:27339141
Zhang, Ting; Lin, Qian-Yun; Fei, Jia-Xi; Zhang, Yan; Lin, Min-Yi; Jiang, Shuang-Hong; Wang, Pu; Chen, Ye
The prevalence of Clostridium difficile infection (CDI) in patients suffering from inflammatory bowel disease (IBD) has increased rapidly over the past several decades in North America and Europe. However, the exact global epidemiology remains unclear because of insufficient data from developing countries. A total of 646 hospitalized adult IBD patients were enrolled; and their fresh stool specimens were obtained and used for Clostridium difficile detection. The incidence of CDI in Crohn’s disease (CD) patients (12.7%) was significantly lower than that in Ulcerative disease (UC) patients (19.3%). Among the toxin types, A+B+ strain was the most common. Length of stay, hospitalization frequency and bowel surgery rate were significantly higher in the CDI than in the non-CDI group in CD or UC patients. More patients in CDI-CD group were still in active and even clinical moderate or severe CD stage than non-CDI-CD group after 2 years of following-up. Fistula, antibiotics and infliximab usage likely increased the CDI rate in CD patients, Infliximab treatment was considered a risk factor in UC patients. CDI is an exacerbating public health issue that may influence IBD course, increase expenditures, and delay the remission of IBD patients. IBD patients with CDI require urgent attention. PMID:27417996
Nagaro, Kristin J; Phillips, S Tyler; Cheknis, Adam K; Sambol, Susan P; Zukowski, Walter E; Johnson, Stuart; Gerding, Dale N
Nontoxigenic Clostridium difficile (NTCD) has been shown to prevent fatal C. difficile infection in the hamster model when hamsters are challenged with standard toxigenic C. difficile strains. The purpose of this study was to determine if NTCD can prevent C. difficile infection in the hamster model when hamsters are challenged with restriction endonuclease analysis group BI C. difficile strains. Groups of 10 hamsters were given oral clindamycin, followed on day 2 by 10(6) CFU of spores of NTCD strain M3 or T7, and were challenged on day 5 with 100 CFU of spores of BI1 or BI6. To conserve animals, results for control hamsters challenged with BI1 or BI6 from the present study and controls from previous identical experiments were combined for statistical comparisons. NTCD strains M3 and T7 achieved 100% colonization and were 100% protective against challenge with BI1 (P ≤ 0.001). M3 colonized 9/10 hamsters and protected against BI6 challenge in the colonized hamsters (P = 0.0003). T7 colonized 10/10 hamsters, but following BI6 challenge, cocolonization occurred in 5 hamsters, 4 of which died, for protection of 6/10 animals (P = 0.02). NTCD colonization provides protection against challenge with toxigenic BI group strains. M3 is more effective than T7 in preventing C. difficile infection caused by the BI6 epidemic strain. Prevention of C. difficile infection caused by the epidemic BI6 strain may be more challenging than that of infections caused by historic BI1 and non-BI C. difficile strains.
LaFrance, Michelle E.; Farrow, Melissa A.; Chandrasekaran, Ramyavardhanee; Sheng, Jinsong; Rubin, Donald H.; Lacy, D. Borden
Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection. PMID:26038560
Chen, Rong; Feng, Yu; Wang, Xiaohui; Yang, Jingyu; Zhang, Xiaoxia; Lü, Xiaoju; Zong, Zhiyong
Clostridium difficile consists of six clades but studies on Clade 3 are limited. Here, we report genome sequences of three Clade 3 C. difficile strains carrying genes encoding toxin A and B and the binary toxin. Isolates 103 and 133 (both of ST5) and isolate 106 (ST285) were recovered from three ICU patients. Whole genome sequencing using HiSeq 2500 revealed 4.1-Mb genomes with 28-29% GC content. There were ≥1,104 SNP between the isolates, suggesting they were not of a single clone. The toxin A and B gene-carrying pathogenicity locus (PaLoc) of the three isolates were identical and had the insertion of the transposon Tn6218. The genetic components of PaLoc among Clade 3 strains were the same with only a few nucleotide mutations and deletions/insertions, suggesting that the Tn6218 insertion might have occurred before the divergence within Clade 3. The binary toxin-genes carrying CDT locus (CdtLoc) of the three isolates were identical and were highly similar to those of other Clade 3 strains, but were more divergent from those of other clades. In conclusion, Clade 3 has an unusual clade-specific PaLoc characteristic of a Tn6218 insertion which appears to be the main feature to distinguish Clade 3 from other C. difficile.
Pant, Chaitanya; Sferra, Thomas J; Deshpande, Abhishek; Minocha, Anil
The rising incidence of Clostridium difficile (C. difficile) infection or CDI is now a problem of pandemic proportions. The NAP1 hypervirulent strain of C. difficile is responsible for a majority of recent epidemics and the widespread use of fluoroquinolone antibiotics may have facilitated the selective proliferation of this strain. The NAP1 strain also is more likely to cause severe and fulminant colitis characterized by marked leukocytosis, renal failure, hemodynamic instability, and toxic megacolon. No single test suffices to diagnose severe CDI, instead; the clinician must rely on a combination of clinical acumen, laboratory testing, and radiologic and endoscopic modalities. Although oral vancomycin and metronidazole are considered standard therapies in the medical management of CDI, recently it has been demonstrated that vancomycin is the more effective antibiotic in cases of severe disease. Moreover, early surgical consultation is necessary in patients who do not respond to medical therapy or who demonstrate rising white blood cell counts or hemodynamic instability indicative of fulminant colitis. Subtotal colectomy with end ileostomy is the procedure of choice for fulminant colitis. When applied to select patients in a judicious and timely fashion, surgery can be a life-saving intervention. In addition to these therapeutic approaches, several investigational treatments including novel antibiotics, fecal bacteriotherapy and immunotherapy have shown promise in the care of patients with severe CDI.
Corthier, G; Muller, M C
In previous studies, we showed that diet composition or Saccharomyces boulardii ingestion could protect gnotobiotic mice against lethal Clostridium difficile infection. Using an original method, we detected nontoxinogenic clones from feces of protected mice challenged with a toxinogenic clone of C. difficile. These clones became established at the same level as the toxinogenic one after about 30 days. In these protected mice bearing nontoxinogenic clones, no enterotoxin production could be detected and cytotoxin titers were highly reduced. These nontoxinogenic clones were genetically stable because nontoxinogenic clones and clones that produce intermediate levels of toxins in vivo did not revert to toxin production, even after repeated culture in vitro. Furthermore, the nontoxinogenic clones were shown to arise from a single toxinogenic clone and were identical to that clone in metabolic patterns and antibiotic sensitivity tests. When mice fed a nonprotective diet were challenged with a nontoxinogenic or intermediate clone, they remained healthy and no toxin production could be detected in their feces. Moreover, these mice were protected against further infections with toxinogenic strains of C. difficile, and a strong antagonism between nontoxinogenic and toxinogenic clones was observed. PMID:3372017
Kali, Arunava; Charles, Marie Victor Pravin; Srirangaraj, Srirangaraj
Clostridium difficile infection (CDI) is a potential life-threatening consequence of antibiotic therapy. Although the risk increases with duration of treatment, it can also occur after a short treatment course. In addition to broad-spectrum antibiotics, anti-neoplastic agents, proton pump inhibitors, H(2) blockers, and several other drugs have been reported to induce intestinal dysbiosis, which is central to the pathogenesis of CDI. There is an increase in incidence and mortality attributed to CDI globally. Moreover, the epidemiology of C. difficile-associated diseases has changed significantly with an increasing occurrence of community-acquired CDI. Metronidazole and oral vancomycin are the first-line antibiotics used to treat CDI. However, metronidazole has limited effectiveness in severe cases and vancomycin use is associated with increasing risk of vancomycin resistance among Enterococcus spp. Cadazolid, a novel oxazolidinone antibiotic, has recently shown potent antimicrobial activity against C. difficile and has a lower propensity to induce resistance. The implications of its use in treating CDI have been reviewed based on current evidence.
Nam, Seung Taek; Hwang, Jung Hwan; Kim, Dae Hong; Lu, Li Fang; Hong, Ji; Zhang, Peng; Yoon, I Na; Hwang, Jae Sam; Chung, Hyo Kyun; Shong, Minho; Lee, Chul-Ho; Kim, Ho
Clostridium difficile toxin A causes acute gut inflammation in animals and humans. It is known to downregulate the tight junctions between colonic epithelial cells, allowing luminal contents to access body tissues and trigger acute immune responses. However, it is not yet known whether this loss of the barrier function is a critical factor in the progression of toxin A-induced pseudomembranous colitis. We previously showed that NADH:quinone oxidoreductase 1 (NQO1) KO (knockout) mice spontaneously display weak gut inflammation and a marked loss of colonic epithelial tight junctions. Moreover, NQO1 KO mice exhibited highly increased inflammatory responses compared with NQO1 WT (wild-type) control mice when subjected to DSS-induced experimental colitis. Here, we tested whether toxin A could also trigger more severe inflammatory responses in NQO1 KO mice compared with NQO1 WT mice. Indeed, our results show that C. difficile toxin A-mediated enteritis is significantly enhanced in NQO1 KO mice compared with NQO1 WT mice. The levels of fluid secretion, villus disruption, and epithelial cell apoptosis were also higher in toxin A-treated NQO1 KO mice compared with WT mice. The previous and present results collectively show that NQO1 is involved in the formation of tight junctions in the small intestine, and that defects in NQO1 enhance C. difficile toxin A-induced acute inflammatory responses, presumably via the loss of epithelial cell tight junctions.
Chen, Rong; Feng, Yu; Wang, Xiaohui; Yang, Jingyu; Zhang, Xiaoxia; Lü, Xiaoju; Zong, Zhiyong
Clostridium difficile consists of six clades but studies on Clade 3 are limited. Here, we report genome sequences of three Clade 3 C. difficile strains carrying genes encoding toxin A and B and the binary toxin. Isolates 103 and 133 (both of ST5) and isolate 106 (ST285) were recovered from three ICU patients. Whole genome sequencing using HiSeq 2500 revealed 4.1-Mb genomes with 28–29% GC content. There were ≥1,104 SNP between the isolates, suggesting they were not of a single clone. The toxin A and B gene-carrying pathogenicity locus (PaLoc) of the three isolates were identical and had the insertion of the transposon Tn6218. The genetic components of PaLoc among Clade 3 strains were the same with only a few nucleotide mutations and deletions/insertions, suggesting that the Tn6218 insertion might have occurred before the divergence within Clade 3. The binary toxin-genes carrying CDT locus (CdtLoc) of the three isolates were identical and were highly similar to those of other Clade 3 strains, but were more divergent from those of other clades. In conclusion, Clade 3 has an unusual clade-specific PaLoc characteristic of a Tn6218 insertion which appears to be the main feature to distinguish Clade 3 from other C. difficile. PMID:28262711
Romano, Vincenza; Pasquale, Vincenzo; Krovacek, Karel; Mauri, Federica; Demarta, Antonella; Dumontet, Stefano
The occurrence of Clostridium difficile in nine wastewater treatment plants in the Ticino Canton (southern Switzerland) was investigated. The samples were collected from raw sewage influents and from treated effluents. Forty-seven out of 55 characterized C. difficile strains belonged to 13 different reference PCR ribotypes (009, 010, 014, 015, 039, 052, 053, 066, 070, 078, 101, 106, and 117), whereas 8 strains did not match any of those available in our libraries. The most frequently isolated ribotype (40%) was 078, isolated from six wastewater treatment plants, whereas ribotype 066, a toxigenic emerging ribotype isolated from patients admitted to hospitals in Europe and Switzerland, was isolated from the outgoing effluent of one plant. The majority of the isolates (85%) were toxigenic. Forty-nine percent of them produced toxin A, toxin B, and the binary toxin (toxigenic profile A(+) B(+) CDT(+)), whereas 51% showed the profile A(+) B(+) CDT(-). Interestingly, eight ribotypes (010, 014, 015, 039, 066, 078, 101, and 106) were among the riboprofiles isolated from symptomatic patients admitted to the hospitals of the Ticino Canton in 2010. Despite the limitation of sampling, this study highlights that toxigenic ribotypes of C. difficile involved in human infections may occur in both incoming and outgoing biological wastewater treatment plants. Such a finding raises concern about the possible contamination of water bodies that receive wastewater treatment plant effluents and about the safe reuse of treated wastewater.
Kuijper, E J; Coignard, B; Brazier, J S; Suetens, C; Drudy, D; Wiuff, C; Pituch, H; Reichert, P; Schneider, F; Widmer, A F; Olsen, K E; Allerberger, F; Notermans, D W; Barbut, F; Delmée, M; Wilcox, M; Pearson, A; Patel, B C; Brown, D J; Frei, R; Akerlund, T; Poxton, I R; Tüll, P
Recent outbreaks of Clostridium difficile-associated diarrhoea (CDAD) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America, Japan and Europe. Definitions have been proposed by the European Centre of Disease Prevention and Control (ECDC) to identify severe cases of CDAD and to differentiate community-acquired cases from nosocomial CDAD (http://www.ecdc.europa.eu/documents/pdf/Cl_dif_v2.pdf). CDAD is mainly known as a healthcare-associated disease, but it is also increasingly recognised as a community-associated disease. The emerging strain is referred to as North American pulsed-field type 1 (NAP1) and PCR ribotype 027. Since 2005, individual countries have developed surveillance studies to monitor the spread of this strain. C. difficile type 027 has caused outbreaks in England and Wales, Ireland, the Netherlands, Belgium, Luxembourg, and France, and has also been detected in Austria, Scotland, Switzerland, Poland and Denmark. Preliminary data indicated that type 027 was already present in historical isolates collected in Sweden between 1997 and 2001.
Dawson, Lisa F.; Valiente, Esmeralda; Donahue, Elizabeth H.; Birchenough, George; Wren, Brendan W.
The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings. PMID:22039420
Dawson, Lisa F; Valiente, Esmeralda; Donahue, Elizabeth H; Birchenough, George; Wren, Brendan W
The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings.
Bouza, E; Padilla, B; Catalán, P; Sánchez-Carrillo, C; Blázquez, R; Peláez, T
Clostridium difficile is considered the most common cause of nosocomial acquired diarrhoea, with frequencies differing widely from one institution to another. So far, it is a scarcely reported condition in Spain. In the present study 129 episodes of Clostridium difficile associated diarrhoea (CDAD) occurred in 120 patients in a 2,000-bed hospital in 1994 is reported. All cases were diagnosed by demonstrating cytotoxicity on cellular lines (MRC-5) from feces or from the strain isolated from a culture medium (CCFA). The overall incidence was 2.4 episodes every 1,000 admissions. Twenty-eight out of the 120 patients (23%) were HIV-positive patients, that is, an incidence of 30 episodes every 1,000 admissions. No significant differences were observed regarding the presentation and clinical course between HIV-positive and HIV-negative patients, with the exception of the antimicrobial agents used previously. Forty-two percent of patients had undergone surgery and 97% had received antimicrobials in the 8 weeks before the CDAD episode, with an average of 3.3 antibiotics per patient. Out of the 129 episodes, 72.8% were treated correctly. A total of 11.7% of patients responded exclusively to the discontinuation of the antimicrobials that were being administered. Eighty-three patients were treated with specific antibiotics, 59 with oral vancomycin, and 24 with metronidazole. Seventy-six patients (91.5%) responded to the initial therapy, 5 relapsed (6%), and 2 (2.5%) failed. The associated mortality rate was 0.7%. C. difficile can be a relevant cause of nosocomial diarrhoea in our setting, particularly in HIV-positive patients, but also in other patients. Its early diagnosis and appropriate therapy can contribute to decrease a relevant cause of morbidity in inpatients.
Pérez-Cobas, Ana E.; Artacho, Alejandro; Ott, Stephan J.; Moya, Andrés; Gosalbes, María J.; Latorre, Amparo
Antibiotic therapy is a causative agent of severe disturbances in microbial communities. In healthy individuals, the gut microbiota prevents infection by harmful microorganisms through direct inhibition (releasing antimicrobial compounds), competition, or stimulation of the host's immune defenses. However, widespread antibiotic use has resulted in short- and long-term shifts in the gut microbiota structure, leading to a loss in colonization resistance in some cases. Consequently, some patients develop Clostridium difficile infection (CDI) after taking an antibiotic (AB) and, at present, this opportunistic pathogen is one of the main causes of antibiotic-associated diarrhea in hospitalized patients. Here, we analyze the composition and functional differences in the gut microbiota of C. difficile infected (CDI) vs. non-infected patients, both patient groups having been treated with AB therapy. To do so we used 16S rRNA gene and metagenomic 454-based pyrosequencing approaches. Samples were taken before, during and after AB treatment and were checked for the presence of the pathogen. We performed different analyses and comparisons between infected (CD+) vs. non-infected (CD−) samples, allowing proposing putative candidate taxa and functions that might protect against C. difficile colonization. Most of these potentially protective taxa belonged to the Firmicutes phylum, mainly to the order Clostridiales, while some candidate protective functions were related to aromatic amino acid biosynthesis and stress response mechanisms. We also found that CDI patients showed, in general, lower diversity and richness than non-infected, as well as an overrepresentation of members of the families Bacteroidaceae, Enterococcaceae, Lactobacillaceae and Clostridium clusters XI and XIVa. Regarding metabolic functions, we detected higher abundance of genes involved in the transport and binding of carbohydrates, ions, and others compounds as a response to an antibiotic environment. PMID
Ghavidel, M.; Salari Sedigh, H.; Razmyar, J.
The aim of this study was to isolate Clostridium difficile from dogs’ faeces, and to study the frequency of its virulence genes. A total of 151 samples of dogs’ faeces were collected. The isolation of C. difficile was performed by using the bacterial culture methods followed by DNA extraction using boiling method. Multiplex PCR method was performed for identification of tcdA, tcdB, cdtA and cdtB genes and single method was carried out for detection of tcdC. Twelve samples (7.9%) were positive in bacteriological assay and based on molecular assay, 66.7% of the isolates (8 of 12 C. difficile isolated) had shown tcdA+, tcdB+ profile. This is the first investigation on molecular assay of C. difficile in Iran’s dog population. PMID:28224013
Theriot, Casey M.; Koenigsknecht, Mark J.; Carlson, Paul E.; Hatton, Gabrielle E.; Nelson, Adam M.; Li, Bo; Huffnagle, Gary B.; Li, Jun; Young, Vincent B.
Antibiotics can have significant and long lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile. Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids, and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including primary bile acid taurocholate for germination, and carbon sources mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favors C. difficile germination and growth. PMID:24445449
Nale, Janet Y.; Spencer, Janice; Hargreaves, Katherine R.; Buckley, Anthony M.; Trzepiński, Przemysław
The microbiome dysbiosis caused by antibiotic treatment has been associated with both susceptibility to and relapse of Clostridium difficile infection (CDI). Bacteriophage (phage) therapy offers target specificity and dose amplification in situ, but few studies have focused on its use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. While it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of seven C. difficile phages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage, lysing 18 and 62 of the ribotypes and strains tested, respectively. Single-phage treatments of ribotype 076, 014/020, and 027 strains showed an initial reduction in the bacterial load followed by the emergence of phage-resistant colonies. However, these colonies remained susceptible to infection with an unrelated phage. In contrast, specific phage combinations caused the complete lysis of C. difficile in vitro and prevented the appearance of resistant/lysogenic clones. Using a hamster model, the oral delivery of optimized phage combinations resulted in reduced C. difficile colonization at 36 h postinfection. Interestingly, free phages were recovered from the bowel at this time. In a challenge model of the disease, phage treatment delayed the onset of symptoms by 33 h compared to the time of onset of symptoms in untreated animals. These data demonstrate the therapeutic potential of phage combinations to treat CDI. PMID:26643348
Elliott, Briony; Dingle, Kate E; Didelot, Xavier; Crook, Derrick W; Riley, Thomas V
The symptoms of Clostridium difficile infection are caused by two closely related toxins, TcdA and TcdB, which are encoded by the 19.6 kb Pathogenicity Locus (PaLoc). The PaLoc is variably present among strains, and in this respect it resembles a mobile genetic element. The C. difficile population structure consists mainly of five phylogenetic clades designated 1-5. Certain genotypes of clade 5 are associated with recently emergent highly pathogenic strains causing human disease and animal infections. The aim of this study was to explore the evolutionary history of the PaLoc in C. difficile clade 5. Phylogenetic analyses and annotation of clade 5 PaLoc variants and adjoining genomic regions were undertaken using a representative collection of toxigenic and nontoxigenic strains. Comparison of the core genome and PaLoc phylogenies obtained for clade 5 and representatives of the other clades identified two distinct PaLoc acquisition events, one involving a toxin A(+)B(+) PaLoc variant and the other an A(-)B(+) variant. Although the exact mechanism of each PaLoc acquisition is unclear, evidence of possible homologous recombination with other clades and between clade 5 lineages was found within the PaLoc and adjacent regions. The generation of nontoxigenic variants by PaLoc loss via homologous recombination with PaLoc-negative members of other clades was suggested by analysis of cdu2, although none is likely to have occurred recently. A variant of the putative holin gene present in the clade 5 A(-)B(+) PaLoc was likely acquired via allelic exchange with an unknown element. Fine-scale phylogenetic analysis of C. difficile clade 5 revealed the extent of its genetic diversity, consistent with ancient evolutionary origins and a complex evolutionary history for the PaLoc.
Yuan, Pengfei; Zhang, Hongmin; Cai, Changzu; Zhu, Shiyou; Zhou, Yuexin; Yang, Xiaozhou; He, Ruina; Li, Chan; Guo, Shengjie; Li, Shan; Huang, Tuxiong; Perez-Cordon, Gregorio; Feng, Hanping; Wei, Wensheng
As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.
Karczewski, Jerzy; Zorman, Julie; Wang, Su; Miezeiewski, Matthew; Xie, Jinfu; Soring, Keri; Petrescu, Ioan; Rogers, Irene; Thiriot, David S; Cook, James C; Chamberlin, Mihaela; Xoconostle, Rachel F; Nahas, Debbie D; Joyce, Joseph G; Bodmer, Jean-Luc; Heinrichs, Jon H; Secore, Susan
Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability.
Andersen, Kasper Krogh; Strokappe, Nika M.; Hultberg, Anna; Truusalu, Kai; Smidt, Imbi; Mikelsaar, Raik-Hiio; Mikelsaar, Marika; Verrips, Theo; Hammarström, Lennart
Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA− TcdB+ strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients. PMID:26573738
Mackin, Kate E.; Carter, Glen P.; Howarth, Pauline; Rood, Julian I.; Lyras, Dena
Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility. PMID:24236153
Sadighi Akha, Amir A; McDermott, Andrew J; Theriot, Casey M; Carlson, Paul E; Frank, Charles R; McDonald, Roderick A; Falkowski, Nicole R; Bergin, Ingrid L; Young, Vincent B; Huffnagle, Gary B
Our previous work has shown the significant up-regulation of Il22 and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the mucosal inflammatory response to Clostridium difficile infection in mice. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes interleukin-22 (IL-22) and CD160-mediated components. The current study sought to determine the potential role(s) of IL-22 and/or CD160 in the mucosal response to C. difficile infection. Clostridium difficile-infected mice treated with anti-IL-22, anti-CD160 or a combination of the two showed significantly reduced STAT3 phosphorylation in comparison to C. difficile-infected mice that had not received either antibody. In addition, C. difficile-infected mice treated with anti-IL-22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile-infected mice. The affected genes included pro-inflammatory chemokines and cytokines, and anti-microbial peptides. Furthermore, histopathological and flow cytometric assessments both showed a significantly reduced influx of neutrophils in C. difficile-infected mice treated with anti-IL-22/CD160. These data demonstrate that IL-22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL-22 and CD160 in mediating both the pro-inflammatory and pro-survival aspects of the host mucosal response in this infection.
Knight, Daniel R.; Androga, Grace O.; Ballard, Susan A.; Howden, Benjamin P.
ABSTRACT In the last decade, Clostridium difficile infection (CDI) has reached an epidemic state with increasing incidence and severity in both health care and community settings. Vancomycin is an important first-line therapy for CDI, and the emergence of resistance would have significant clinical consequences. In this study, we describe for the first time a vanB2 vancomycin resistance operon in C. difficile, isolated from an Australian veal calf at slaughter. The operon was carried on an ~42-kb element showing significant homology and synteny to Tn1549, a conjugative transposon linked with the emergence and global dissemination of vancomycin-resistant enterococci (VRE). Notably, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly as a result of an aberrant vanRB gene. As observed for other anaerobic species of the animal gut microbiota, C. difficile may be a reservoir of clinically important vancomycin resistance genes. IMPORTANCE In an era when the development of new antimicrobial drugs is slow, vancomycin remains the preferred antimicrobial therapy for Clostridium difficile infection (CDI), the most important health care-related infection in the world today. The emergence of resistance to vancomycin would have significant consequences in relation to treating patients with CDI. In this paper, we describe for the first time a complete set of vancomycin resistance genes in C. difficile. The genes were very similar to genes found in vancomycin-resistant enterococci (VRE) that were associated with the emergence and global dissemination of this organism. Fortunately, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly because of a small difference in one gene. However, this observation signals that we may be very close to seeing a fully vancomycin-resistant strain of C. difficile. PMID:27536735
Leslie, Jhansi L.; Huang, Sha; Opp, Judith S.; Nagy, Melinda S.; Kobayashi, Masayuki; Young, Vincent B.
Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile. PMID:25312952
Seiler, Peter; Chen, Xinhua; Schroeder, Susanne; Pfaff, Philippe; Enderlin, Michel; Klenk, Axel; Fournier, Elvire; Hubschwerlen, Christian; Ritz, Daniel; Kelly, Ciaran P.; Keck, Wolfgang
Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 μg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD. PMID:24277020
Locher, Hans H; Seiler, Peter; Chen, Xinhua; Schroeder, Susanne; Pfaff, Philippe; Enderlin, Michel; Klenk, Axel; Fournier, Elvire; Hubschwerlen, Christian; Ritz, Daniel; Kelly, Ciaran P; Keck, Wolfgang
Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 μg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.
Rodriguez, C; Taminiau, B; Avesani, V; Van Broeck, J; Delmée, M; Daube, G
Clostridium difficile has been isolated from food animals and meat, specially ground pork and ground beef. The recovered isolates were closely related to C. difficile human strains, indicating that animals and food are possible transmission routes of human C. difficile infection. The main objective of this study was to characterize C. difficile isolates from retail meat and to compare with human isolates recovered from hospital patients in Belgium. Raw meat (beef and pork) was obtained from the retail trade. C. difficile was recovered from 2.3% of the beef samples and from 4.7% of the pork samples. A total of 4 different PCR-ribotypes were identified with a large percentage of types 078 and 014. Resistance to moxifloxacin and erythromycin was detected. The multi-locus sequence typing (MLST) analysis showed that meat and human isolates cluster in the same lineage. This study reveals the presence of toxigenic C. difficile in retail meat in Belgium with predominance PCR-ribotypes 078 and 014, which are among the four most prevalent ribotypes of C. difficile isolated from humans in Europe.
Knetsch, C W; Connor, T R; Mutreja, A; van Dorp, S M; Sanders, I M; Browne, H P; Harris, D; Lipman, L; Keessen, E C; Corver, J; Kuijper, E J; Lawley, T D
Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n = 19), asymptomatic farmers (n = 15) and hospitalised patients (n = 31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.
Leslie, Jhansi L; Huang, Sha; Opp, Judith S; Nagy, Melinda S; Kobayashi, Masayuki; Young, Vincent B; Spence, Jason R
Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile.
Abt, Michael C.; Lewis, Brittany B.; Caballero, Silvia; Xiong, Huizhong; Carter, Rebecca A.; Sušac, Bože; Ling, Lilan; Leiner, Ingrid; Pamer, Eric G.
Summary 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. PMID:26159718
Bhattacharjee, Disha; Francis, Michael B.; Ding, Xicheng; McAllister, Kathleen N.; Shrestha, Ritu
ABSTRACT Clostridium difficile spore germination is essential for colonization and disease. The signals that initiate C. difficile spore germination are a combination of taurocholic acid (a bile acid) and glycine. Interestingly, the chenodeoxycholic acid class (CDCA) bile acids competitively inhibit taurocholic acid-mediated germination, suggesting that compounds that inhibit spore germination could be developed into drugs that prophylactically prevent C. difficile infection or reduce recurring disease. However, a recent report called into question the utility of such a strategy to prevent infection by describing C. difficile strains that germinated in the apparent absence of bile acids or germinated in the presence of the CDCA inhibitor. Because the mechanisms of C. difficile spore germination are beginning to be elucidated, the mechanism of germination in these particular strains could yield important information on how C. difficile spores initiate germination. Therefore, we quantified the interaction of these strains with taurocholic acid and CDCA, the rates of spore germination, the release of DPA from the spore core, and the abundance of the germinant receptor complex (CspC, CspB, and SleC). We found that strains previously observed to germinate in the absence of taurocholic acid correspond to more potent 50% effective concentrations (EC50 values; the concentrations that achieve a half-maximum germination rate) of the germinant and are still inhibited by CDCA, possibly explaining the previous observations. By comparing the germination kinetics and the abundance of proteins in the germinant receptor complex, we revised our original model for CspC-mediated activation of spore germination and propose that CspC may activate spore germination and then inhibit downstream processes. IMPORTANCE Clostridium difficile forms metabolically dormant spores that persist in the health care environment. In susceptible hosts, C. difficile spores germinate in response to certain
Bertolo, Lisa; Boncheff, Alexander G; Ma, Zuchao; Chen, Yu-Han; Wakeford, Terra; Friendship, Robert M; Rosseau, Joyce; Weese, J Scott; Chu, Michele; Mallozzi, Michael; Vedantam, Gayatri; Monteiro, Mario A
Clostridium difficile is responsible for severe diarrhea in humans that may cause death. Spores are the infectious form of C. difficile, which germinate into toxin-producing vegetative cells in response to bile acids. Recently, we discovered that C. difficile cells possess three complex polysaccharides (PSs), named PSI, PSII, and PSIII, in which PSI was only associated with a hypervirulent ribotype 027 strain, PSII was hypothesized to be a common antigen, and PSIII was a water-insoluble polymer. Here, we show that (i) C. difficile spores contain, at least in part, a D-glucan, (ii) PSI is not a ribotype 027-unique antigen, (iii) common antigen PSII may in part be present as a low molecular weight lipoteichoic acid, (iv) selective hydrolysis of PSII yields single PSII repeat units, (v) the glycosyl diester-phosphate linkage affords high flexibility to PSII, and (vi) that PSII is immunogenic in sows. Also, with the intent of creating a dual anti-diarrheal vaccine against C. difficile and enterotoxin Escherichia coli (ETEC) infections in humans, we describe the conjugation of PSII to the ETEC-associated LTB enterotoxin.
Siddiqui, Farida; O'Connor, Jennifer R; Nagaro, Kristin; Cheknis, Adam; Sambol, Susan P; Vedantam, Gayatri; Gerding, Dale N; Johnson, Stuart
Toxin A has historically been regarded as the primary virulence determinant in Clostridium difficile infection, but naturally occurring toxin A-negative, toxin B-positive (A-/B+) C. difficile strains are known to be virulent. To determine the role of toxin B in these strains, we immunized hamsters with a toxoid prepared from purified toxin B to determine whether they would be protected from lethal challenge with an A-/B+ strain of C. difficile.
Diversity of Clostridium difficile PCR ribotypes in Europe: results from the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013.
Davies, Kerrie A; Ashwin, Helen; Longshaw, Christopher M; Burns, David A; Davis, Georgina L; Wilcox, Mark H
Clostridium difficile infection (CDI) is the major cause of infective diarrhoea in healthcare environments. As part of the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), the largest C. difficile epidemiological study of its type, PCR ribotype distribution of C. difficile isolates in Europe was investigated. PCR ribotyping was performed on 1,196 C. difficile isolates from diarrhoeal samples sent to the European coordinating laboratory in 2012-13 and 2013 (from two sampling days) by 482 participating hospitals from 19 European countries. A total of 125 ribotypes were identified, of which ribotypes 027 (19%, n =222), 001/072 (11%, n = 134) and 014/020 (10%, n = 119) were the most prevalent. Distinct regional patterns of ribotype distribution were noted. Of 596 isolates from patients with toxin-positive stools (CDI cases), ribotype 027 accounted for 22% (32/144) of infections in cases aged from 18 to less than 65 years, but the prevalence decreased in those aged ≥ 65 years (14% (59/412)) and further decreased in those aged ≥ 81 years (9% (18/195)). The prevalence of ribotype 027 and 176, but not other epidemic strains, was inversely proportional to overall ribotype diversity (R(2) = 0.717). This study highlights an increased diversity of C. difficile ribotypes across Europe compared with previous studies, with considerable intercountry variation in ribotype distribution. Continuous surveillance programmes are necessary to monitor the changing epidemiology of C. difficile.
Stripling, Joshua; Kumar, Ranjit; Baddley, John W.; Nellore, Anoma; Dixon, Paula; Howard, Donna; Ptacek, Travis; Lefkowitz, Elliot J.; Tallaj, Jose A.; Benjamin, William H.; Morrow, Casey D.; Rodriguez, J. Martin
We report the use of fecal microbiota transplantation in a single heart-kidney transplant recipient with recurrent Clostridium difficile, vancomycin-resistant Enterococcus (VRE) fecal dominance, and recurrent VRE infections. Fecal microbiota transplantation resulted in the reconstruction of a diverse microbiota with (1) reduced relative abundance of C difficile and VRE and (2) positive clinical outcome. PMID:26180828
Heinsen, Femke Anouska; Knecht, Carolin; Schilhabel, Anke; Schmitz, Ruth A.; Zimmermann, Alexandra; dos Santos, Vitor Martins; Ferrer, Manuel; Rosenstiel, Philip C.; Schreiber, Stefan; Friedrichs, Anette K.; Ott, Stephan J.
Clostridium difficile infections are an emerging health problem in the modern hospital environment. Severe alterations of the gut microbiome with loss of resistance to colonization against C. difficile are thought to be the major trigger, but there is no clear concept of how C. difficile infection evolves and which microbiological factors are involved. We sequenced 16S rRNA amplicons generated from DNA and RNA/cDNA of fecal samples from three groups of individuals by FLX technology: (i) healthy controls (no antibiotic therapy); (ii) individuals receiving antibiotic therapy (Ampicillin/Sulbactam, cephalosporins, and fluoroquinolones with subsequent development of C. difficile infection or (iii) individuals receiving antibiotic therapy without C. difficile infection. We compared the effects of the three different antibiotic classes on the intestinal microbiome and the effects of alterations of the gut microbiome on C. difficile infection at the DNA (total microbiota) and rRNA (potentially active) levels. A comparison of antibiotic classes showed significant differences at DNA level, but not at RNA level. Among individuals that developed or did not develop a C. difficile infection under antibiotics we found no significant differences. We identified single species that were up- or down regulated in individuals receiving antibiotics who developed the infection compared to non-infected individuals. We found no significant differences in the global composition of the transcriptionally active gut microbiome associated with C. difficile infections. We suggest that up- and down regulation of specific bacterial species may be involved in colonization resistance against C. difficile providing a potential therapeutic approach through specific manipulation of the intestinal microbiome. PMID:24586762
Jump, Robin L P; Li, Yuejin; Pultz, Michael J; Kypriotakis, Georgios; Donskey, Curtis J
Tigecycline is a broad-spectrum glycylcycline antibiotic with potent in vitro activity against Clostridium difficile. We used a mouse model to test the hypothesis that tigecycline has a low propensity to promote colonization and toxin production by C. difficile due to inhibitory activity in the colon. Mice (5 to 8 per group) received subcutaneous injections of tigecycline (low and high doses) alone or in combination with clindamycin for 6 days. Growth of and toxin production by 3 strains of C. difficile (tigecycline MICs ≤ 0.012 μg/ml) were measured in cecal contents collected 6 h or 3 days after the final antibiotic dose. Antibiotic concentrations were measured using a bioassay, and concentrations of total anaerobes and Bacteroides spp. were measured. The effects of tigecycline on rendering mice susceptible to colonization with and reducing the burden of C. difficile were also examined. In comparison to saline controls, clindamycin promoted the growth of C. difficile (P < 0.001) in cecal contents, whereas tigecycline did not. Tigecycline did not suppress total anaerobes or Bacteroides spp. in comparison to saline controls. Concurrent administration of tigecycline prevented clindamycin-induced promotion of C. difficile in cecal contents collected 6 h or 3 days (high dose only) after the final antibiotic dose. Tigecycline did not promote the establishment of colonization in mice, yet it did not reduce concentrations of C. difficile in animals with established colonization. In summary, tigecycline did not promote the growth of or toxin production by C. difficile, probably due to inhibitory activity against C. difficile and relative sparing of indigenous anaerobic microflora.
Muzslay, Monika; Wilson, Peter
The horizontal transmission of Clostridium difficile in the hospital environment is difficult to establish. Current methods to detect C. difficile spores on surfaces are not quantitative, lack sensitivity, and are protracted. We propose a novel rapid method to detect and quantify C. difficile contamination on surfaces. Sponge swabbing was compared to contact plate sampling to assess the in vitro recovery of C. difficile ribotype 027 contamination (∼100, 101, or 102 CFU of spores) from test surfaces (a bed rail, a stainless steel sheet, or a polypropylene work surface). Sponge swab contents were concentrated by vacuum filtration, and the filter membrane was plated onto selective agar. The efficacy of each technique for the recovery of C. difficile from sites in the clinical environment that are touched at a high frequency was evaluated. Contact plates recovered 19 to 32% of the total contamination on test surfaces, whereas sponge swabs recovered 76 to 94% of the total contamination, and contact plates failed to detect C. difficile contamination below a detection limit of 10 CFU/25 cm2 (0.4 CFU/cm2). In use, contact plates failed to detect C. difficile contamination (0/96 contact plates; 4 case wards), while sponge swabs recovered C. difficile from 29% (87/301) of the surfaces tested in the clinical environment. Approximately 74% (36/49) of the area in the vicinity of the patient was contaminated (∼1.34 ± 6.88 CFU/cm2 C. difficile spores). Reservoirs of C. difficile extended to beyond the areas near the patient: a dirty utility room sink (2.26 ± 5.90 CFU/cm2), toilet floor (1.87 ± 2.40 CFU/cm2), and chair arm (1.33 ± 4.69 CFU/cm2). C. difficile was present on floors in ∼90% of case wards. This study highlights that sampling with a contact plate may fail to detect C. difficile contamination and result in false-negative reporting. Our sponge sampling technique permitted the rapid and quantitative measurement of C. difficile contamination on surfaces with a
Ali, Shanom; Muzslay, Monika; Wilson, Peter
The horizontal transmission of Clostridium difficile in the hospital environment is difficult to establish. Current methods to detect C. difficile spores on surfaces are not quantitative, lack sensitivity, and are protracted. We propose a novel rapid method to detect and quantify C. difficile contamination on surfaces. Sponge swabbing was compared to contact plate sampling to assess the in vitro recovery of C. difficile ribotype 027 contamination (∼10(0), 10(1), or 10(2) CFU of spores) from test surfaces (a bed rail, a stainless steel sheet, or a polypropylene work surface). Sponge swab contents were concentrated by vacuum filtration, and the filter membrane was plated onto selective agar. The efficacy of each technique for the recovery of C. difficile from sites in the clinical environment that are touched at a high frequency was evaluated. Contact plates recovered 19 to 32% of the total contamination on test surfaces, whereas sponge swabs recovered 76 to 94% of the total contamination, and contact plates failed to detect C. difficile contamination below a detection limit of 10 CFU/25 cm(2) (0.4 CFU/cm(2)). In use, contact plates failed to detect C. difficile contamination (0/96 contact plates; 4 case wards), while sponge swabs recovered C. difficile from 29% (87/301) of the surfaces tested in the clinical environment. Approximately 74% (36/49) of the area in the vicinity of the patient was contaminated (∼1.34 ± 6.88 CFU/cm(2) C. difficile spores). Reservoirs of C. difficile extended to beyond the areas near the patient: a dirty utility room sink (2.26 ± 5.90 CFU/cm(2)), toilet floor (1.87 ± 2.40 CFU/cm(2)), and chair arm (1.33 ± 4.69 CFU/cm(2)). C. difficile was present on floors in ∼90% of case wards. This study highlights that sampling with a contact plate may fail to detect C. difficile contamination and result in false-negative reporting. Our sponge sampling technique permitted the rapid and quantitative measurement of C. difficile contamination on
Lawley, Trevor D.; Stares, Mark D.; Connor, Thomas R.; Raisen, Claire; Goulding, David; Rad, Roland; Schreiber, Fernanda; Brandt, Cordelia; Deakin, Laura J.; Pickard, Derek J.; Duncan, Sylvia H.; Flint, Harry J.; Clark, Taane G.; Parkhill, Julian; Dougan, Gordon
Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis. PMID:23133377
Sanhueza, Carlos A; Cartmell, Jonathan; El-Hawiet, Amr; Szpacenko, Adam; Kitova, Elena N; Daneshfar, Rambod; Klassen, John S; Lang, Dean E; Eugenio, Luiz; Ng, Kenneth K-S; Kitov, Pavel I; Bundle, David R
A focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction. Our approach combines modular fragment-based design with in silico screening of synthetically feasible compounds and lays the groundwork for future efforts in development of composite bifunctional ligands for large clostridial toxins.
Nagy, Gergely György; Várvölgyi, Csaba; Balogh, Zoltán; Orosi, Piroska; Paragh, György
The incidence of Clostridium difficile associated enteral disease shows dramatic increase worldwide, with appallingly high treatment costs, mortality figures, recurrence rates and treatment refractoriness. It is not surprising, that there is significant interest in the development and introduction of alternative therapeutic strategies. Among these only stool transplantation (or faecal bacteriotherapy) is gaining international acceptance due to its excellent cure rate (≈92%), low recurrence rate (≈6%), safety and cost-effectiveness. Unfortunately faecal transplantation is not available for most patients, although based on promising international results, its introduction into the routine clinical practice is well justified and widely expected. The authors would like to facilitate this process, by presenting a detailed faecal transplantation protocol prepared in their Institution based on the available literature and clinical rationality. Officially accepted national methodological guidelines will need to be issued in the future, founded on the expert opinion of relevant professional societies and upcoming advances in this field.
Rafiullah, Fnu; Kanwal, Sunil; Majeed, Usman M; Korsten, Mark A; Cheema, Faisal H; Luthra, Munish; Sohail, Muhammad Rizwan
A 78-year-old, retired Caucasian male presented in emergency room with 3 days history of progressive watery diarrhoea. Two weeks earlier, he received intravenous levofloxacin for community acquired pneumonia. The patient was diagnosed as severe Clostridium difficile infection based on clinical presentation, labs and imaging studies. The patient was initially treated with intravenous metronidazole and oral vancomycin. While awaiting subacute rehabilitation placement, the patient relapsed twice. After first recurrence the patient was treated with 2 weeks of oral nitazoxanide. After second recurrence, the patient was treated 2 weeks of nitazoxanide followed by tapering dose of vancomycin. The patient was followed and no relapse was reported at 1 year follow-up visit. PMID:22674696
Pant, Chaitanya; Sferra, Thomas J; Ondrade, Christina; Bass, Pat F; Deshpande, Abhishek; Burton, Cary V
Clostridium difficile infection (CDI) has emerged as the leading cause of nosocomial diarrhea in the developed world. The prompt recognition of severe CDI is essential in providing early aggressive therapy. Though previous studies have identified leukocytosis, azotemia, and hypoalbuminemia as markers to differentiate severe from non-severe CDI in the general patient population, there is little data in immunosuppressed patients. We conducted a retrospective chart review of immunosuppressed patients with CDI to identify serum markers associated with severe CDI. Twenty-nine immunosuppressed patients with CDI (nine with severe disease) were identified. Those with severe disease were older and had evidence of renal dysfunction. The white blood cell count, platelet, and albumin levels were the same in the severe and non-severe immunosuppressed CDI patients. Therefore, recognized serum markers of severe CDI are not universally useful in immunosuppressed patients. Moreover, the clinician must be aware that immunosuppressed patients can develop severe CDI while remaining leukopenic.
Costello, Samuel P; Tucker, Emily C; La Brooy, Justin; Schoeman, Mark N; Andrews, Jane M
Recurrent or refractory Clostridium difficile infection (CDI) has become an increasing problem in the past decade. Fecal microbiota transplant (FMT) is a highly efficacious treatment for recurrent CDI; however, a number of technical, logistical, and regulatory issues have hampered the development of an FMT capability at many hospitals. The development of a frozen stool bank of screened donor stool is an important step in the standardization of the procedure. This gives clinicians rapid access to thoroughly screened donor stool when needed, without the ethical and logistical problems associated with patient-selected donors. We describe the practicalities of establishing such a service using a stool bank of prescreened donor stool including detail regarding donor recruitment and screening, stool preparation, and delivery of the FMT.
Zidaric, Valerija; Rupnik, Maja
Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains.
Greenberg, Richard N; Marbury, Thomas C; Foglia, Ginamarie; Warny, Michel
Fifty healthy adult (18-55 years) and 48 elderly (≥ 65 years) volunteers were randomized to receive a candidate Clostridium difficile toxoid vaccine (2 μg, 10 μg, or 50 μg) or placebo on Days 0, 28, and 56. No volunteer receiving placebo seroconverted. For toxin A, seroconversion by Day 56 (post-dose 2) was observed in 100% of volunteers aged 18-55 years in all dose groups and in 50%, 89%, and 100% of elderly participants in the 2 μg, 10 μg, and 50 μg dose groups, respectively. For both age groups, seroconversion for toxin B was lower than toxin A. There were no safety concerns.
Spencer, Janice; Leuzzi, Rosanna; Buckley, Anthony; Irvine, June; Candlish, Denise; Scarselli, Maria; Douce, Gillian R
Clostridium difficile is a major cause of antibiotic associated diarrhea. Recently, we have shown that effective protection can be mediated in hamsters through the inclusion of specific recombinant fragments from toxin A and B in a systemically delivered vaccine. Interestingly while neutralizing antibodies to the binding domains of both toxin A and B are moderately protective, enhanced survival is observed when fragments from the glucosyltransferase region of toxin B replace those from the binding domain of this toxin. In this addendum, we discuss additional information that has been derived from such vaccination studies. This includes observations on efficacy and cross-protection against different ribotypes mediated by these vaccines and the challenges that remain for a vaccine which prevents clinical symptoms but not colonization. The use and value of vaccination both in the prevention of infection and for treatment of disease relapse will be discussed.
Lancaster, Catherine; Rustandi, Richard R; Pannizzo, Paola; Ha, Sha
High-performance size-exclusion chromatography (HPSEC or SEC) is a method that can be applied to measure size distribution of proteins, including aggregates, monomers, and fragments. In the biopharmaceutical industry the quantitation of aggregates contained in biotherapeutics and protein-based vaccines is critical given the potential impact on safety, immunogenicity, and efficacy. Hence, aggregation analysis of therapeutic proteins or protein-based vaccine products is almost always a requirement of regulatory agencies. SEC, also referred to as gel-filtration chromatography, separates molecules by size through a porous resin stationary phase. Under isocratic flow small molecules are retained on the column longer than large molecules. Here we describe the use of this SEC technique to characterize aggregation levels for four different protein antigens for a Clostridium difficile vaccine.
Liu, Runhui; Suárez, Jose M; Weisblum, Bernard; Gellman, Samuel H; McBride, Shonna M
Nylon-3 polymers (poly-β-peptides) have been investigated as synthetic mimics of host-defense peptides in recent years. These polymers are attractive because they are much easier to synthesize than are the peptides themselves, and the polymers resist proteolysis. Here we describe in vitro analysis of selected nylon-3 copolymers against Clostridium difficile, an important nosocomial pathogen that causes highly infectious diarrheal disease. The best polymers match the human host-defense peptide LL-37 in blocking vegetative cell growth and inhibiting spore outgrowth. The polymers and LL-37 were effective against both the epidemic 027 ribotype and the 012 ribotype. In contrast, neither vancomycin nor nisin inhibited outgrowth for the 012 ribotype. The best polymer was less hemolytic than LL-37. Overall, these findings suggest that nylon-3 copolymers may be useful for combatting C. difficle.
IV, Edward C Oldfield; III, Edward C Oldfield; Johnson, David A
Clostridium difficile infection (CDI) presents a rapidly evolving challenge in the battle against hospital-acquired infections. Recent advances in CDI diagnosis and management include rapid changes in diagnostic approach with the introduction of newer tests, such as detection of glutamate dehydrogenase in stool and polymerase chain reaction to detect the gene for toxin production, which will soon revolutionize the diagnostic approach to CDI. New medications and multiple medical society guidelines have introduced changing concepts in the definitions of severity of CDI and the choice of therapeutic agents, while rapid expansion of data on the efficacy of fecal microbiota transplantation heralds a revolutionary change in the management of patients suffering multiple relapses of CDI. Through a comprehensive review of current medical literature, this article aims to offer an intensive review of the current state of CDI diagnosis, discuss the strengths and limitations of available laboratory tests, compare both current and future treatments options and offer recommendations for best practice strategies. PMID:24729930
Rea, Mary C; Alemayehu, Debebe; Ross, R Paul; Hill, Colin
Clostridium difficile infection (CDI) is a major cause of morbidity and mortality among hospitalized patients and imposes a considerable financial burden on health service providers in both Europe and the USA. The incidence of CDI has dramatically increased in recent years, partly due to the emergence of a number of hypervirulent strains. The most commonly documented risk factors associated with CDIs are antibiotic usage leading to alterations of the gut microbiota, age >65 years and long-term hospital stay. Since standard therapies for antibiotic-associated diarrhoea and CDI have limited efficacy, there is now an urgent need for alternative therapeutics. In this review, we outline the current state of play with regard to the potential of gut-derived bacteriocins, probiotics and phage to act as antimicrobial agents against CDI in the human gut.
Fuentes, Susana; van Nood, Els; Tims, Sebastian; Heikamp-de Jong, Ineke; ter Braak, Cajo J F; Keller, Josbert J; Zoetendal, Erwin G; de Vos, Willem M
Recurrent Clostridium difficile infection (CDI) can be effectively treated by infusion of a healthy donor faeces suspension. However, it is unclear what factors determine treatment efficacy. By using a phylogenetic microarray platform, we assessed composition, diversity and dynamics of faecal microbiota before, after and during follow-up of the transplantation from a healthy donor to different patients, to elucidate the mechanism of action of faecal infusion. Global composition and network analysis of the microbiota was performed in faecal samples from nine patients with recurrent CDI. Analyses were performed before and after duodenal donor faeces infusion, and during a follow-up of 10 weeks. The microbiota data were compared with that of the healthy donors. All patients successfully recovered. Their intestinal microbiota changed from a low-diversity diseased state, dominated by Proteobacteria and Bacilli, to a more diverse ecosystem resembling that of healthy donors, dominated by Bacteroidetes and Clostridium groups, including butyrate-producing bacteria. We identified specific multi-species networks and signature microbial groups that were either depleted or restored as a result of the treatment. The changes persisted over time. Comprehensive and deep analyses of the microbiota of patients before and after treatment exposed a therapeutic reset from a diseased state towards a healthy profile. The identification of microbial groups that constitute a niche for C. difficile overgrowth, as well as those driving the reinstallation of a healthy intestinal microbiota, could contribute to the development of biomarkers predicting recurrence and treatment outcome, identifying an optimal microbiota composition that could lead to targeted treatment strategies.
Bandelj, Petra; Golob, Majda; Ocepek, Matjaz; Zdovc, Irena; Vengust, Modest
A significant risk factor for developing Clostridium difficile infection (CDI) in humans and animals is associated with the antimicrobial use. It has often been hypothesized that farm animals could be the source for human infection with Clostridium difficile (CD). In the European Union, family-run dairy farms are the predominant farming model, which are more interlinked within the community compared to large-scale intensive dairy or beef farms. Therefore, it is important to investigate antimicrobial susceptibility patterns of CD in such environment. A total of 159 CD isolates from 20 family dairy farms were tested with a customized broth microdilution plate for their antimicrobial resistance. Seventeen antimicrobials were selected (amoxicillin, ceftriaxone, clindamycin, daptomycin, erythromycin, fusidic acid, imipenem, levofloxacin, linezolid, metronidazole, moxifloxacin, oxacillin, rifampicin, tetracycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin), which are commonly used for treatment of CDI in veterinary and human medicine, or were previously applied in CD epidemiological studies. Antimicrobials, which are used for treatment of CDI in humans (metronidazole, vancomycin, fusidic acid, tigecycline,