The antibacterial activity of immune-related peptides, identified by a differential gene
expression analysis, was investigated to suggest novel antibacterial peptides. A cDNA encoding a defensin-like peptide, Coprisin, was isolated from bacteria-immunized dung beetle, Copris tripartitus, by using differential dot blot hybridization. Northern blot
analysis showed that Coprisin mRNA was up-regulated from 4 hours after bacteria injection and its expression level was reached a peak at 16 hours. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with a predicted molecular weight of 8.6 kDa and a pI of 8.7. The amino acid sequence of mature Coprisin was found to be 79.1% and 67.4% identical to those of defensin-like peptides of Anomala cuprea and Allomyrina dichotoma, respectively. We also investigated active sequences of Coprisin by using amino acid modification. The result showed that the 9-mer peptide, LLCIALRKK-NH2, exhibited potent antibacterial activities against Escherichia coli and Staphylococcus aureus.
Background—Many individuals have serum antibodies
against Clostridium difficile toxins. Those with an
impaired antitoxin response may be susceptible to recurrent, prolonged,
or severe C difficile diarrhoea and colitis.
Aims—To examine whether treatment with intravenous
immunoglobulin might be effective in patients with severe
pseudomembranous colitis unresponsive to standard antimicrobial therapy.
Patients—Two patients with pseudomembranous
colitis not responding to metronidazole and vancomycin were given
normal pooled human immunoglobulin intravenously (200-300 mg/kg).
Methods—Antibodies against C
difficile toxins were measured in nine immunoglobulin
preparations by ELISA and by cytotoxin neutralisation assay.
Results—Both patients responded quickly as shown
by resolution of diarrhoea, abdominal tenderness, and distension. All
immunoglobulin preparations tested contained IgG against C
difficile toxins A and B by ELISA and neutralised the cytotoxic
activity of C difficile toxins in vitro at IgG
concentrations of 0.4-1.6 mg/ml.
Conclusion—Passive immunotherapy with
intravenous immunoglobulin may be a useful addition to antibiotic
therapy for severe, refractory C difficile colitis. IgG
antitoxin is present in standard immunoglobulin preparations and
C difficile toxin neutralising activity is evident at IgG
concentrations which are readily achieved in the serum by intravenous
Clostridium difficile; toxin; diarrhoea; IgG; immunotherapy; antibiotic
Clostridium difficile (C. difficile) is a common causative agent of pseudomembranous colitis (PMC). C. difficile-associated diarrhea (CDAD) ranges from mild diarrhea to life threatening PMC. Recently, a highly virulent strain of C. difficile polymerase chain reaction ribotype 027 was found in North America, Europe, and Japan. A 52-yr-old woman with anti-tuberculosis medication and neurogenic bladder due to traffic accident experienced five episodes of C. difficile PMC after taking antibiotics for pneumonia along with septic shock and acute renal failure. She was readmitted to the intensive care unit and treated with oral vancomycin with refractory of oral metronidazole, inotropics and probiotics for over 60 days. C. difficile isolated both at the first and the last admission was identified as C. difficile ribotype 027 by ribotyping, toxinotyping, and tcdC gene sequencing, which turned out the same pathogen as the epidemic hypervirulent B1/NAP1 strain. This is the first case of C. difficile PCR ribotype 027 in Korea. After discharge, she was maintained on probiotics and rifaximin for 3 weeks. She had no relapse for 6 months.
Enterocolitis, Pseudomembranous; Clostridium difficile; Ribotype 027
Background & Aims
Clostridium difficile (C.difficile) is the leading cause of nosocomial infectious diarrhea. Increasing incidence, antibiotic resistance and more virulent strains have dramatically increased the number of C.difficile-related deaths worldwide. The innate host response mechanisms to C.difficile are not resolved; however, we hypothesize that hypoxia-inducible factor (HIF-1) plays an innate protective role in C.difficile colitis. Thus, we assessed the impact of C.difficile toxins on the regulation of HIF-1 and evaluated the role of HIF-1α in C.difficile-mediated injury/inflammation.
In vitro studies assessed HIF-1α mRNA, protein levels and DNA binding events in human mucosal biopsies and Caco-2 cells exposed to C.difficile toxins. In vivo studies employed the murine ileal loop model of C.difficile toxin-induced intestinal injury. Mice with targeted deletion of HIF-1α in the intestinal epithelium were used to assess the impact of HIF-1α signaling in response to C.difficile toxin.
Mucosal biopsies and Caco-2 cells exposed to C.difficile toxin displayed a significant increase in HIF-1α transcription and protein levels. Toxin-induced DNA binding was also observed in Caco-2 cells. Toxin-induced HIF-1α accumulation was attenuated by nitric oxide synthase inhibitors. In vivo, deletion of intestinal epithelial HIF-1α resulted in more severe toxin-induced intestinal injury and inflammation. In contrast, stabilization of HIF-1α, with dimethyloxallyl glycine, attenuated toxin-induced injury and inflammation. This was associated with an induction of HIF-1-regulated protective factors including VEGFa, CD73 and intestinal trefoil factor and down-regulation of proinflammatory molecules TNF and KC.
Our study is the first to describe the innate protective role for HIF-1α in response to C.difficile toxins. Harnessing the innate protective actions of HIF-1α in response to C.difficile toxins may represent a novel form of therapy for C.difficile-associated disease.
HIF-1; Clostridium difficile; dimethyloxallyl glycine; epithelial barrier
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.
We have used the hamster model of antibiotic-induced Clostridium difficile intestinal disease to evaluate nitazoxanide (NTZ), a nitrothiazole benzamide antimicrobial agent. The following in vitro and in vivo activities of NTZ in the adult hamster were examined and compared to those of metronidazole and vancomycin: (i) MICs and minimum bactericidal concentrations (MBCs) against C. difficile, (ii) toxicity, (iii) ability to prevent C. difficile-associated ileocecitis, and (iv) propensity to induce C. difficile-associated ileocecitis. The MICs and MBCs of NTZ against 15 toxigenic strains of C. difficile were comparable to those of vancomycin or metronidazole. C. difficile-associated ileocecitis was induced with oral clindamycin and toxigenic C. difficile in a group of 60 hamsters. Subgroups of 10 hamsters were given six daily intragastric treatments of NTZ (15, 7.5, and 3.0 mg/100 g of body weight [gbw]), metronidazole (15 mg/100 gbw), vancomycin (5 mg/100 gbw), or saline (1 ml/100 gbw). Animals receiving saline died 3 days post-C. difficile challenge. During the treatment period, NTZ (≥7.5 mg/100 gbw), like metronidazole and vancomycin, prevented outward manifestations of clindamycin-induced C. difficile intestinal disease. Six of ten hamsters on a scheduled dose of 3.0 mg of NTZ/100 gbw survived for the complete treatment period. Of these surviving animals, all but three died of C. difficile disease by between 3 and 12 days following discontinuation of antibiotic therapy. Another group of hamsters received six similar daily doses of the three antibiotics, followed by an inoculation with toxigenic C. difficile. All of the NTZ-treated animals survived the 15-day postinfection period. Upon necropsy, all hamsters appeared normal: there were no gross signs of toxicity or C. difficile intestinal disease, nor was C. difficile detected in the cultures of the ceca of these animals. By contrast, vancomycin and metronidazole treatment induced fatal C. difficile intestinal disease in 20 and 70% of recipients, respectively.
Clostridium difficile is a spore-forming, anaerobic, gram-positive bacillus that releases two main virulence factors: toxins A and B. Toxin A plays an important pathogenic role in antibiotic-induced diarrhea and pseudomembranous colitis, a condition characterized by intense mucosal inflammation and secretion. Agonist activity at A2A adenosine receptors attenuates inflammation and damage in many tissues. This study evaluated the effects of a new selective A2A adenosine receptor agonist (ATL 313) on toxin A-induced injury in murine ileal loops. ATL 313 (0.5 to 5 nM) and/or the A2A adenosine receptor antagonist (ZM241385; 5 nM) or phosphate-buffered saline (PBS) were injected into ileal loops immediately prior to challenge with toxin A (1 to 10 μg/loop) or PBS. Intestinal fluid volume/length and weight/length ratios were calculated 3 h later. Ileal tissues were collected for the measurement of myeloperoxidase, adenosine deaminase activity, tumor necrosis factor alpha (TNF-α) production, histopathology, and detection of cell death by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) method. Toxin A significantly increased volume/length and weight/length ratios in a dose-dependent fashion. ATL 313 treatment significantly (P < 0.05) reduced toxin A-induced secretion and edema, prevented mucosal disruption, and neutrophil infiltration as measured by myeloperoxidase activity. ATL 313 also reduced the toxin A-induced TNF-α production and adenosine deaminase activity and prevented toxin A-induced cell death. These protective effects of ATL 313 were reversed by ZM241385. In conclusion, the A2A adenosine receptor agonist, ATL 313, reduces tissue injury and inflammation in mice with toxin A-induced enteritis. The finding of increased ileal adenosine deaminase activity following the administration of toxin A is new and might contribute to the pathogenesis of the toxin A-induced enteritis by deaminating endogenous adenosine.
The neuropeptide neurotensin mediates several intestinal functions, including chloride secretion, motility, and cellular growth. However, whether this peptide participates in intestinal inflammation is not known. Toxin A, an enterotoxin from Clostridium difficile, mediates pseudomembranous colitis in humans. In animal models, toxin A causes an acute inflammatory response characterized by activation of sensory neurons and intestinal nerves and immune cells of the lamina propria. Here we show that neurotensin and its receptor are elevated in the rat colonic mucosa following toxin A administration. Pretreatment of rats with the neurotensin receptor antagonist SR-48,692 inhibits toxin A–induced changes in colonic secretion, mucosal permeability, and histologic damage. Exposure of colonic explants to toxin A or neurotensin causes mast cell degranulation, which is inhibited by SR-48,692. Because substance P was previously shown to mediate mast cell activation, we examined whether substance P is involved in neurotensin-induced mast cell degranulation. Our results show that neurotensin-induced mast cell degranulation in colonic explants is inhibited by the substance P (neurokinin-1) receptor antagonist CP-96,345, indicating that colonic mast activation in response to neurotensin involves release of substance P. We conclude that neurotensin plays a key role in the pathogenesis of C. difficile–induced colonic inflammation and mast cell activation.
Clostridium difficile is a major nosocomial pathogen responsible for pseudomembranous colitis and many cases of antibiotic-associated diarrhea. Because of potential relapse of disease with current antimicrobial therapy protocols, there is a need for additional and/or alternative antimicrobial agents for the treatment of disease caused by C. difficile. We have synthesized a systematic series of 14 structurally simple bismuth compounds and assessed their biological activities against C. difficile and four other gastrointestinal species, including Helicobacter pylori. Here, we report on the activities of six compounds that exhibit antibacterial activities against C. difficile, and some of the compounds have MICs of less than 1 μg/ml. Also tested, for comparison, were the activities of bismuth subcitrate and ranitidine bismuth citrate obtained from commercial sources. C. difficile and H. pylori were more sensitive both to the synthetic bismuth compounds and to the commercial products than were Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis, and the last three species were markedly resistant to the commercial bismuth salts. Testing with human foreskin fibroblast cells revealed that some of the synthetic compounds were more cytotoxic than others. Killing curves for C. difficile treated with the more active compounds revealed rapid death, and electron microscopy showed that the bismuth of these compounds was rapidly incorporated by C. difficile. Energy dispersive spectroscopy X-ray microanalysis of C. difficile cells containing electron-dense material confirmed the presence of internalized bismuth. Internalized bismuth was not observed in C. difficile treated with synthetic bismuth compounds that lacked antimicrobial activity, which suggests that the uptake of the metal is required for killing activity. The nature of the carrier would seem to determine whether bismuth is transported into susceptible bacteria like C. difficile.
A toxin produced by Clostridium difficile has been implicated in the pathogenesis of antibiotic-associated colitis in humans and experimental animals. This study was undertaken in order to define the sequential evolution of caecal mucosal lesions in the hamster and to relate those lesions directly to the clostridial toxin. Sterile filtrates from a culture of C. difficile and from caecal contents of clindamycin-treated hamsters were studied with respect to their effects on the caecal mucosa and on cultured cell monolayers. The toxic filtrates both produced cellular swelling in vitro, and appeared to have a similar cytotoxic effect on caecal epithelial cells in vivo. Cellular damage was followed by extensive epithelial desquamation and the evolution of an acute pseudomembranous typhlitis. The pathogenetic sequence produced by the filtrates was identical with that previously described after direct clindamycin treatment. These findings demonstrate that intraluminal clostridial toxin can mediate development of the characteristic antibiotic-associated mucosal lesions.
Diarrhea is a common side effect of chemotherapy. Pseudomembranous colitis is a well known complication of antibiotic treatment that can also be observed, albeit rarely, with certain chemotherapeutic agents. We present four cases of severe colitis in patients undergoing treatment with taxane-based chemotherapy for pancreatic, lung and breast cancer. None of them had recently received antibiotics. One patient presented with a bowel perforation and three had endoscopic findings of pseudomembranous colitis. Two of these three patients had negative stool toxin assays for Clostridium difficile. In the patient presenting with perforation, an emergency left hemicolectomy was performed and the pathological findings in the colon were acute inflammation and ischemic necrosis; the other three patients were treated with oral vancomycin and/or oral or intravenous metronidazole leading to complete resolution of the symptoms. Apart from pseudomembranous colitis, we describe patients presenting with neutropenic enterocolitis as well as ischemic colitis after docetaxel use. These cases provide some insight into the spectrum and varied clinical presentations of severe colitis associated with taxane-based chemotherapy.
Pseudomembranous colitis; Chemotherapy; Clostridium difficile; Docetaxel
Clostridium difficile is the most frequently identified enteric pathogen in patients with nosocomially acquired, antibiotic-associated diarrhea. The drugs most commonly used to treat diseases associated with C. difficile are metronidazole and vancomycin. Most clinical laboratories assume that all C. difficile isolates are susceptible to metronidazole and vancomycin. We report on the antimicrobial susceptibilities of 415 C. difficile isolates to metronidazole and vancomycin over an 8-year period (1993 to 2000). The overall rate of resistance to metronidazole at the critical breakpoint (16 μg/ml) was 6.3%. Although full resistance to vancomycin was not observed, the overall rate of intermediate resistance was 3.1%. One isolate had a combination of resistance to metronidazole and intermediate resistance to vancomycin. Rates of resistance to metronidazole and vancomycin were higher among isolates from human immunodeficiency virus-infected patients. Molecular typing methods proved the absence of clonality among the isolates with decreased susceptibilities to the antimicrobials tested.
Clostridium difficile is the major causative agent of nosocomial antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of C. difficile is closely related to the production of toxins A and B. Toxigenic C. difficile detection by a tissue culture cytotoxin assay is often considered the “gold standard.” However, this assay is time consuming, as it implies an incubation period of at least 24 h. We have developed a rapid real-time fluorescence-based multiplex PCR assay targeting the C. difficile toxin genes tcdA and tcdB, with the Smart Cycler. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon type. The analytical sensitivity of the assay was around 10 genome copies for all nine C. difficile strains tested, representing the 6 most common toxinotypes. The specificity was demonstrated by the absence of amplification with DNA purified from bacterial species other than C. difficile (n = 14), including Clostridium sordellii for which the lethal toxin gene sequence is closely related to the toxin genes of C. difficile. Following a rapid (15 min) and simple fecal sample preparation protocol, both tcdA and tcdB were efficiently amplified from 28 of 29 cytotoxin-positive feces samples. There was no amplification observed with all 27 cytotoxin-negative feces samples tested. This is the first real-time PCR assay for the detection of C. difficile. It is rapid, sensitive, and specific and allows detection of C. difficile directly from feces samples.
OBJECTIVE: To review the basic microbiology, pathogenesis of disease, and diagnosis of the nosocomial pathogen Clostridium difficile and to examine therapies recommended by the Canadian Task Force on Preventive Health Care. QUALITY OF EVIDENCE MEDLINE: was searched using MeSH headings. Controlled trials for therapy were sought, but case-control studies and observational reviews were included. MAIN MESSAGE: Clostridium difficile causes approximately 20% of cases of diarrhea associated with antibiotics, including clindamycin and the second- and third-generation cephalosporins. Diarrhea is usually mild, but can be severe; extreme cases develop toxic megacolon. Diagnosis is dependent on demonstrating presence of clostridial toxin in stool specimens or of pseudomembranes through sigmoidoscopy. First-line therapy for C. difficile diarrhea is restricted to metronidazole. Second-line therapy for treatment failure is vancomycin. For relapse, a second course of metronidazole is recommended; tapering courses of vancomycin and probiotics are used for multiple recurrences. CONCLUSION: Clostridium difficile is an important nosocomial pathogen requiring prudent use of antibiotics and strict infection-control policies to prevent large health care costs.
Fecal specimens from 223 subjects were evaluated for the presence of Clostridium difficile by use of a selective medium developed in our laboratory and for the presence of C. difficile cytotoxin. C. difficile and cytotoxin were detected in 89 and 83%, respectively, of patients with antimicrobial agent-associated pseudomembranous colitis (PMC). In patients in whom PMC was not documented, C. difficile and cytotoxin were present in only 37 and 21%, respectively. C. difficile and cytotoxin were also recovered from the feces of 6 and 3, respectively, of 13 antimicrobial recipients who did not have diarrhea. Although C. difficile appears to be a major cause of PMC, it is not responsible for at least some two-thirds of cases of antimicrobial agent-associated diarrhea in which PMC is not documented. Neither the recovery of C. difficile nor the detection of its cytotoxin should be considered diagnostic for C. difficile-induced disease.
Clostridium difficile mediates intestinal inflammation by releasing toxin A (TxA), a potent enterotoxin. Cathelicidins (Camp as gene name, LL-37 peptide in humans and mCRAMP peptide in mice) are antibacterial peptides that also posses anti-inflammatory properties.
To determine the role of cathelicidins in models of Clostridium difficile infection and TxA-mediated ileal inflammation and cultured human primary monocytes.
Wild-type (WT) and mCRAMP-deficient (Camp−/−) mice were treated with an antibiotic mixture and infected orally with C difficile. Some mice were intracolonically given mCRAMP daily for 3 days. Ileal loops were also prepared in WT mice and treated with either saline or TxA and incubated for 4 h, while some TxA-treated loops were injected with mCRAMP.
Intracolonic mCRAMP administration to C difficile-infected WT mice showed significantly reduced colonic histology damage, apoptosis, tissue myeloperoxidase (MPO) and tumour necrosis factor (TNF)α levels. Ileal mCRAMP treatment also significantly reduced histology damage, tissue apoptosis, MPO and TNFα levels in TxA-exposed ileal loops. WT and Camp−/− mice exhibited similar intestinal responses in both models, implying that C difficile/TxA-induced endogenous cathelicidin may be insufficient to modulate C difficile/TxA-mediated intestinal inflammation. Both LL-37 and mCRAMP also significantly reduced TxA-induced TNFα secretion via inhibition of NF-κB phosphorylation. Endogenous cathelicidin failed to control C difficile and/or toxin A-mediated inflammation and even intestinal cathelicidin expression was increased in humans and mice.
Exogenous cathelicidin modulates C difficile colitis by inhibiting TxA-associated intestinal inflammation. Cathelicidin administration may be a new anti-inflammatory treatment for C difficile toxin-associated disease.
Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI), C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s) to efficiently persist in the host colonic environment.
In this work, we provide evidence that C. difficile spores are well suited to survive the host’s innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells’ ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1.
These results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells.
Unrecognized severe pseudomembranous colitis may become life threatening. A typical Clostridium difficile infection is associated with involvement of the colon; however, small bowel disease has also been described. Here, we present a case of a 48-year-old man with Clostridium difficile colitis of an isolated segment in the descending colon treated by a novel catheter intraluminal antibiotic irrigation. The intraluminal antibiotic irrigation was performed through a Foley catheter inserted into the isolated mucus fistula. The patient recovered after three weeks of intraluminal vancomycin (250 mg diluted in 150 ml of normal saline x Q6) and metronidazole (500 mg x Q8). Both antibiotics were given into the mucus fistula over 30 min. The patient was discharged from the unit four weeks after admission. This novel technique, in which the antibiotic was administered through an inserted intraluminal Foley urinary catheter, may be an efficient and safe alternative when conventional routes cannot be implemented.
Clostridium difficile has been recognized as the cause of antibiotic-associated pseudomembranous colitis and of less severe diarrheal diseases associated with the use of antimicrobial agents. However, healthy carriers of this microorganism have been found, particularly healthy neonates and small children. Various typing systems have been used to clarify the epidemiology of C. difficile. We used the electrophoretic patterns of EDTA-extracted proteins to characterize C. difficile strains from various sources. Altogether, 110 strains were studied, including 2 reference strains, and 21 different protein profiles were obtained. However, two patterns were the most common: the group 2 pattern, characterized by a major 35-kilodalton polypeptide band, and the group 5 pattern, identified by principal bands of 37 and 56 kilodaltons. The group 2 pattern was characteristic of strains isolated during hospital outbreaks and from sporadic cases of pseudomembranous colitis and antibiotic-associated diarrhea. The group 5 pattern was obtained only from isolates from healthy neonates and children. A correlation between electrophoretic characteristics and virulence can be hypothesized, namely that group 2 strains are more prone to induce diseases and cause outbreaks. It is noteworthy that strains isolated from children with diarrhea of unknown etiology, not related to antibiotic use, belong to the "virulent" group 2; strains from leukemic patients showed a variety of different patterns, and only two belong to group 2. This characterization can be used to aid studies on the virulence and clinical significance of C. difficile.
Clostridium difficile is the major cause of nosocomial antibiotic-associated diarrhoea with the potential risk of progressing to severe clinical outcomes including death. It is not unusual for Clostridium difficile infection to progress to complications of toxic megacolon, bowel perforation and even Gram-negative sepsis following pathological changes in the intestinal mucosa. These complications are however less commonly seen in community-acquired Clostridium difficile infection than in hospital-acquired Clostridium difficile infection. To the best of our knowledge, this was the first case of community-acquired Clostridium difficile infection of its type seen in Jamaica.
We report a case of a 22-year-old female university student who was admitted to the University Hospital of the West Indies, Jamaica with a presumptive diagnosis of pseudomembranous colitis PMC. She presented with a 5-day history of diarrhoea following clindamycin treatment for coverage of a tooth extraction due to a dental abscess. Her clinical condition deteriorated and progressed from diarrhoea to toxic megacolon, bowel perforation and Gram-negative sepsis. Clostridium difficile NAP12/ribotype 087 was isolated from her stool while blood cultures grew Klebsiella pneumoniae. Despite initial treatment intervention with empiric therapy of metronidazole and antibiotic clearance of Klebsiella pneumoniae from the blood, the patient died within 10 days of hospital admission.
We believe that clindamycin used for coverage of a dental abscess was an independent risk factor that initiated the disruption of the bowel micro-flora, resulting in overgrowth of Clostridium difficile NAP12/ribotype 087. This uncommon strain, which is the same ribotype (087) as ATCC 43255, was apparently responsible for the increased severity of the infection and death following toxic megacolon, bowel perforation and pseudomembranous colitis involving the entire large bowel. K. pneumoniae sepsis, resolved by antibiotic therapy was secondary to Clostridium difficile infection. The case registers community-acquired Clostridium difficile infection as producing serious complications similar to hospital-acquired Clostridium difficile infection and should be treated with the requisite importance.
Clostridium difficile; Klebsiella pneumoniae; Community-Acquired Infection; Diarrhoea; Clindamycin; Pseudomembranous Colitis; Toxic Megacolon
Clostridium difficile is the bacterial pathogen identified as the cause of pseudomembranous colitis and is principally responsible for nosocomial antibiotic-associated diarrhea and colitis. The pathologic findings associated with this infection are believed to be caused by two large (∼300-kDa) exotoxins, toxins A and B. Because of the mucosal nature of this infection, vaccination strategies aimed at providing prophylactic or therapeutic immune protection have included immunization by mucosal routes. Using the hamster model of C. difficile infection, we examined the protective efficacy of inactivated toxin (toxoid) vaccine formulations prepared as either culture filtrate or partially purified toxoid. We compared combination parenteral and mucosal vaccination regimens involving intranasal, intragastric, or rectal routes of immunization and found that rectal immunization in conjunction with intramuscular (i.m.) vaccination provided full protection of hamsters from death and diarrhea while the other mucosal routes did not. Protection was associated with high levels of toxin-neutralizing antibodies in serum. The requirement for adjuvants for protection was assessed by using sequential i.m. and rectal or i.m. vaccination regimens. Unexpectedly, i.m. immunization without adjuvant conferred the highest protection from death and diarrhea; this regimen elicited the highest serum anti-toxin B titers as well as toxin B neutralizing titers. Passive transfer of mouse antitoxin antibodies protected hamsters in a dose-dependent manner, demonstrating the principal role of circulating antitoxin antibodies in immunity from this toxin-mediated mucosal disease. These results suggest that prophylactic parenteral vaccination or intravenous immunotherapy could provide protection from C. difficile disease in humans.
Although antimicrobial agent-associated colitis has been recognized as a clinicopathologic entity for years, the cause of this disease has been determined only recently. Virtually all cases of pseudomembranous colitis and some cases of antimicrobial agent-associated nonspecific colitis or diarrhea have been shown to be caused by a toxin of Clostridium difficile. Methods for cultivating C difficile from feces and for detecting the toxin have been developed. Oral administration of vancomycin has proved to be effective for the treatment of C difficile-induced colitis, although isolated instances of relapse after treatment have been documented.
The discovery of C difficile as a human intestinal pathogen has provided an explanation for some, but not all cases of antimicrobial agent-associated diarrhea. The epidemiology, pathogenesis and means of prevention of C difficile toxin-induced diarrhea remain to be determined.
Background: Clostridium difficile infection (CDI) is a recent epidemic in the United States, particularly in the hospital setting. Oral metronidazole is standard therapy for C. difficile infection, but resistance to metronidazole is becoming a clinical challenge.
Methods: We evaluated the efficacy of the nonsystemic oral antibiotic rifaximin for the treatment of metronidazole-resistant C. difficile infection. Twenty-five patients with C. difficile infection were enrolled in the study. All had mild-to-moderate C. difficile infection (5–10 bowel movements a day without sepsis) unresponsive to metronidazole (i.e. stools positive for toxins A and B after oral metronidazole 500 mg three times daily [t.i.d.] for 5 days). After discontinuation of metronidazole, rifaximin 400 mg t.i.d. for 14 days was prescribed. Patients were followed for 56 days and stool was tested for C. difficile using polymerase chain reaction (PCR) to assess the effect of treatment. A negative PCR test result was interpreted as a favorable response to rifaximin.
Results: Sixteen of 22 patients (73%) were eligible for study inclusion and completed rifaximin therapy experienced eradication of infection (stool negative for C. difficile) immediately after rifaximin therapy and 56 days post-treatment. Three patients (12%) discontinued therapy because of abdominal distention. Rifaximin was generally well tolerated.
Conclusions: In conclusion, rifaximin may be considered for treatment of mild-to-moderate C. difficile infection that is resistant to metronidazole. Larger randomized trials are needed to confirm these positive findings.
Clostridium difficile infection; metronidazole; resistant; rifaximin
Faecal metronidazole and hydroxymetronidazole concentrations measured by high pressure liquid chromatography are reported during 10 episodes of Clostridium difficile colitis in nine patients. Bactericidal faecal concentrations were present in all patients with acute disease receiving oral or intravenous metronidazole, and all responded to therapy. Metronidazole and hydroxymetronidazole concentrations fell as the diarrhoea improved and neither substance was detectable in the faeces of five patients after recovery. This demonstration of intracolonic therapeutic concentrations of metronidazole supports the clinical experience of oral metronidazole being effective in the treatment of antibiotic associated diarrhoea caused by C difficile and also suggests a potential role for intravenous metronidazole in this disease.
Clostridium difficile (C. difficile) is now the leading cause of nosocomial diarrhea in the USA, accounting for 30% of patients with antibiotic-associated diarrhea, 70% of those with antibiotic-associated colitis, and most cases of pseudomembranous colitis. The organism has evolved over the last 8 years to become more virulent and resistant to antimicrobials (NAP1/027 strain) causing a more severe form of the disease that has increased mortality and healthcare costs. While it is generally accepted that the problem results from the overuse of antibiotics, and in particular second and third generation cephalosporins, fluoroquinolones and macrolides, recent studies suggest that acid suppression with proton pump inhibitors (PPIs) may be equally culpable. A further common, but less recognized, etiological factor is the prolonged use of elemental diets. Such diets are totally absorbed within the small intestine and therefore deprive the colonic microbiota of their source of nutrition, namely dietary fiber, fructose oligosaccharides, and resistant starch. The resultant suppression of colonic fermentation leads to suppression of the “good” bacteria, such as butyrate-producers (butyrate being essential for colonic mucosal health), and bifidobacteria and the creation of a “permissive” environment for C. difficile colonization and subsequent infection. Based on this analysis, the best chance of suppressing the emerging C. difficile epidemic is to adopt a 3-pronged attack consisting of (1) avoidance of the use of prophylactic antibiotics, (2) the avoidance of prophylactic PPIs, and (3) the conversion of elemental diet feeding to a diet containing adequate indigestible carbohydrate after the first week of critical illness. In this review, we highlight the rising worldwide incidence of C. difficile associated diarrhea and the role played by non-residue diets in destabilizing the colonic microbiota.
Clostridium difficile; Elemental diets; Enteral nutrition; Microbiota