Clostridium difficile Infection Caused by the Epidemic BI/NAP1/027 Strain
2009; Elsevier BV; Volume: 136; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2009.02.073
ISSN1528-0012
AutoresJennifer R. O’Connor, Stuart Johnson, Dale N. Gerding,
Tópico(s)Microscopic Colitis
ResumoRates and severity of Clostridium difficile infection (CDI) in hospitals in North America and Europe have increased since 2000 and correlate with dissemination of an epidemic strain characterized by higher than usual toxin A and B production, the presence of a third toxin, binary toxin, and high-level resistance to fluoroquinolone antibiotics. The strain, which is restriction endonuclease analysis group BI, pulse-field gel electrophoresis type NAP1, and polymerase chain reaction ribotype 027, is designated BI/NAP1/027. How this strain has become so widely distributed geographically and produces such severe CDI is the subject of active investigation. The deletion at position 117 of the tcdC gene, a repressor of toxin A and B production, is one possible contributor to increased levels of the toxins. The role of binary toxin is unknown. Recent isolates of BI/NAP1/027 were found to be resistant to fluoroquinolones, which is likely to contribute to the dissemination of this strain. Other virulence factors such as increased sporulation and surface layer protein adherence are also under investigation. Infections caused by this organism are particularly frequent among elderly hospitalized patients, in whom the attributable 30-day mortality is greater than 5%. Major risk factors for BI/NAP1/027 infection include advanced age, hospitalization, and exposure to specific antimicrobials, especially fluoroquinolones and cephalosporins. When CDI is severe, vancomycin treatment is more effective than metronidazole; for mild disease either agent can be used. Control of hospital outbreaks caused by BI/NAP1/027 is difficult but possible through a combination of barrier precautions, environmental cleaning, and antimicrobial stewardship. Rates and severity of Clostridium difficile infection (CDI) in hospitals in North America and Europe have increased since 2000 and correlate with dissemination of an epidemic strain characterized by higher than usual toxin A and B production, the presence of a third toxin, binary toxin, and high-level resistance to fluoroquinolone antibiotics. The strain, which is restriction endonuclease analysis group BI, pulse-field gel electrophoresis type NAP1, and polymerase chain reaction ribotype 027, is designated BI/NAP1/027. How this strain has become so widely distributed geographically and produces such severe CDI is the subject of active investigation. The deletion at position 117 of the tcdC gene, a repressor of toxin A and B production, is one possible contributor to increased levels of the toxins. The role of binary toxin is unknown. Recent isolates of BI/NAP1/027 were found to be resistant to fluoroquinolones, which is likely to contribute to the dissemination of this strain. Other virulence factors such as increased sporulation and surface layer protein adherence are also under investigation. Infections caused by this organism are particularly frequent among elderly hospitalized patients, in whom the attributable 30-day mortality is greater than 5%. Major risk factors for BI/NAP1/027 infection include advanced age, hospitalization, and exposure to specific antimicrobials, especially fluoroquinolones and cephalosporins. When CDI is severe, vancomycin treatment is more effective than metronidazole; for mild disease either agent can be used. Control of hospital outbreaks caused by BI/NAP1/027 is difficult but possible through a combination of barrier precautions, environmental cleaning, and antimicrobial stewardship. Stuart JohnsonView Large Image Figure ViewerDownload Hi-res image Download (PPT)Dale N. GerdingView Large Image Figure ViewerDownload Hi-res image Download (PPT)In 2000, the University of Pittsburgh Medical Center began to observe an increase in severe Clostridium difficile infections (CDIs) that required colectomy (17 patients in a single year).1Dallal R.M. Harbrecht B.G. Boujoukas A.J. et al.Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications.Ann Surg. 2002; 235: 363-372Crossref PubMed Scopus (535) Google Scholar Although the cause of this outbreak was not recognized for 3–4 years, this was the beginning of an increasing rate of CDI in US hospitals that continued, unabated, through 2006. Other hospitals in widely scattered US cities, and especially Montreal, Canada, soon began reporting a similar increase in severe CDI, with high rates of mortality.2Loo V.G. Poirier L. Miller M.A. et al.A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.N Engl J Med. 2005; 353: 2442-2449Crossref PubMed Scopus (1712) Google Scholar Hospitals throughout the United States reported increased numbers of severe cases of CDI from 2001 to 2003; they submitted isolates for typing to the Centers for Disease Control and Prevention and the Hines VA Hospital C difficile research laboratories. Their analysis showed a common strain of C difficile that infected patients in Maine, Pennsylvania, New Jersey, Georgia, Illinois, and Oregon hospitals. The pathogen was characterized as toxinotype III, restriction endonuclease analysis (REA) group BI, by pulsed-field gel electrophoresis as North American pulsed-field type NAP1, and subsequently by polymerase chain reaction (PCR) ribotyping as type 027, or BI/NAP1/027.3McDonald L.C. Killgore G.E. Thompson A. et al.An epidemic, toxin gene-variant strain of Clostridium difficile.N Engl J Med. 2005; 353: 2433-2441Crossref PubMed Scopus (1788) Google Scholar, 4Killgore G. Thompson A. Johnson S. et al.Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing.J Clin Microbiol. 2008; 46: 431-437Crossref PubMed Scopus (278) Google Scholar This was followed by perhaps the most devastating single hospital CDI outbreaks, which occurred in 2003–2004 and then in 2004–2005, at Stoke Mandeville Hospital in the United Kingdom and resulted in a total of 334 CDI cases and 38 deaths.5Investigation into outbreaks of Clostridium difficile at Stoke Mandeville Hospital, Buckinghamshire Hospitals NHS Trust: Commission for Healthcare Audit and Inspection, 2006.Google Scholar Subsequently, cases caused by BI/NAP1/027 have been documented in 40 states in the United States, all provinces of Canada, and numerous European countries (Figure 1). Overall, the rates of CDI have continued to increase in US hospitals, although not documented as caused by BI/NAP1/027, and mortality attributed to CDI has increased in parallel with rates, especially among the elderly (Figure 2).Figure 1Extent of BI/NAP1/027 distribution. (A) States in the United States that have had ≥1 hospital that has reported CDI caused by the BI/NAP1/027 epidemic strain as of October 2008 (red).116Division of Healthcare Quality Promotion (DHQP)States with BI/NAP1/027 strain of C difficile. Centers for Disease Control and Prevention, Atlanta, GA2008Google Scholar (B) Percentage of C difficile isolates in Canadian provinces (no data are available for territories) with the BI/NAP1/027 strain in the 2005 Canadian Nosocomial Infection Surveillance Program (CNISP) survey.97Miller M. Canadian experience with Clostridium difficile; tracking a pathogen.2008Google Scholar (C) Hospitals in Europe reporting outbreaks (stars) and sporadic cases (circles) of CDI caused by the BI/NAP1/027 strain.89Kuijper E.J. Barbut F. Brazier J.S. et al.Update of Clostridium difficile infection due to PCR ribotype 027 in Europe, 2008.Euro Surveill. 2008; (13.pii:18942)Google ScholarView Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Frequency and mortality of CDI. (A) National estimates of US short-stay hospital discharges with C difficile infection as any listed diagnosis or the primary hospitalization diagnosis, based on the national inpatient sample117Elixhauser A. Jhung M. Clostridium difficile-associated disease in US Hospitals, 1993-2005. Agency for Healthcare Research and Quality, Rockville, MD2008Google Scholarand unpublished data, 2008). (B) C difficile–related mortality based on listings on US death certificates from 1999 to 2004, with age-adjusted mortality rates per million people for the same years (shown in box).118Redelings M.D. Sorvillo F. Mascola L. Increase in Clostridium difficile-related mortality rates, United States, 1999-2004.Emerg Infect Dis. 2007; 13: 1417-1419PubMed Google ScholarView Large Image Figure ViewerDownload Hi-res image Download (PPT) The C difficile strain that caused these epidemics is not new. Fourteen group BI isolates, dating from 1984 to 1992, were found in the Hines VA Hospital C difficile collection, but they had caused only sporadic cases of CDI during a 10-year period. Analysis of historic BI/NAP1/027 isolates has shown them to be virtually identical to the recent outbreak strains, with the exception that the older isolates were not resistant to the newer fluoroquinolone antibiotics, gatifloxacin, and moxifloxacin (current epidemic strains are resistant to these antibiotics). Most CDI that has been attributed to the BI/NAP1/027 strain has been found in hospitals and is commonly associated with outbreaks or epidemics. This certainly was the case in Pittsburgh, Quebec, Stoke Mandeville, and numerous other North American hospitals.3McDonald L.C. Killgore G.E. Thompson A. et al.An epidemic, toxin gene-variant strain of Clostridium difficile.N Engl J Med. 2005; 353: 2433-2441Crossref PubMed Scopus (1788) Google Scholar, 5Investigation into outbreaks of Clostridium difficile at Stoke Mandeville Hospital, Buckinghamshire Hospitals NHS Trust: Commission for Healthcare Audit and Inspection, 2006.Google Scholar, 6Muto C.A. Pokrywka M. Shutt K. et al.A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use.Infect Control Hosp Epidemiol. 2005; 26: 273-280Crossref PubMed Scopus (557) Google Scholar, 7Hubert B. Loo V.G. Bourgault A.M. et al.A portrait of the geographic dissemination of the Clostridium difficile North American pulsed-field type 1 strain and the epidemiology of C difficile-associated disease in QuebeC.Clin Infect Dis. 2007; 44: 238-244Crossref PubMed Scopus (169) Google Scholar, 8Pepin J. Valiquette L. Alary M.E. et al.Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity.CMAJ. 2004; 171: 466-472Crossref PubMed Scopus (963) Google Scholar In the hospital setting, CDI occurs most frequently in elderly patients; it has been shown that risk and mortality of CDI from the BI/NAP1/027 strain increases with patient age (Table 1).2Loo V.G. Poirier L. Miller M.A. et al.A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.N Engl J Med. 2005; 353: 2442-2449Crossref PubMed Scopus (1712) Google Scholar, 7Hubert B. Loo V.G. Bourgault A.M. et al.A portrait of the geographic dissemination of the Clostridium difficile North American pulsed-field type 1 strain and the epidemiology of C difficile-associated disease in QuebeC.Clin Infect Dis. 2007; 44: 238-244Crossref PubMed Scopus (169) Google Scholar The BI/NAP1/027 strain was not observed to cause disease in the community until recently. In a typing study of isolates from hospital-onset and health care facility–associated CDI, 54% of these isolates and 50% of community-associated C difficile isolates were NAP1 (the predominant type).9Thompson A. Nicholson A. Wiggs L. et al.Strain diversity among Clostridium difficile isolates from healthcare- and community-associated.2008Google Scholar In a Foodborne Diseases Active Surveillance Network surveillance study, 22% of 60 community-associated–C difficile isolates from 5 states were NAP1, suggesting that this epidemic strain can be isolated from patients in the community as well as in the hospital.10Angulo F, Sharapov U, Long C, et al. An epidemic hospital strain as a cause of community-associated Clostridium difficile-associated disease: FoodNet pilot study, 2006 [abstract P5]. Presented at the second international Clostridium difficile Symposium, Maribor, Slovenia, June 7, 2007.Google Scholar Nonetheless, in the community, CDI caused by any strain is relatively rare compared with the rate in hospitals.Table 1CDI Rates and Mortality Increase With Patient AgeAgeCDI rate per 1000 admissionsAttributable 30-day mortality rate, % 90 y74.414.0Adapted from Loo et al.2Loo V.G. Poirier L. Miller M.A. et al.A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.N Engl J Med. 2005; 353: 2442-2449Crossref PubMed Scopus (1712) Google Scholar Open table in a new tab Adapted from Loo et al.2Loo V.G. Poirier L. Miller M.A. et al.A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.N Engl J Med. 2005; 353: 2442-2449Crossref PubMed Scopus (1712) Google Scholar Potential community sources of CDI include soil; salt, fresh, and tap water; pet animals and food animals; and meats and vegetables.11al Saif N. Brazier J.S. The distribution of Clostridium difficile in the environment of South Wales.J Med Microbiol. 1996; 45: 133-137Crossref PubMed Scopus (213) Google Scholar The BI/NAP1/027 strain was identified in meat in Canada and in Arizona.12Rodriguez-Palacios A. Staempfli H.R. Duffield T. et al.Clostridium difficile in retail ground meat, Canada.Emerg Infect Dis. 2007; 13: 485-487Crossref PubMed Scopus (231) Google Scholar, 13Songer J, Trinh H, Killgore G, et al. Is Clostridium difficile in the food chain? [abstract XIV-F1]. Presented at the ninth biennial congress of the Anaerobe Society of Americas, Long Beach, CA, June 26, 2008.Google Scholar However, the most common C difficile strains found in animals and meat are toxinotype V, PCR ribotype 078, and REA type BK.12Rodriguez-Palacios A. Staempfli H.R. Duffield T. et al.Clostridium difficile in retail ground meat, Canada.Emerg Infect Dis. 2007; 13: 485-487Crossref PubMed Scopus (231) Google Scholar, 13Songer J, Trinh H, Killgore G, et al. Is Clostridium difficile in the food chain? [abstract XIV-F1]. Presented at the ninth biennial congress of the Anaerobe Society of Americas, Long Beach, CA, June 26, 2008.Google Scholar, 14Jhung M.A. Thompson A.D. Killgore G.E. et al.Toxinotype V Clostridium difficile in humans and food animals.Emerg Infect Dis. 2008; 14: 1039-1045Crossref PubMed Scopus (178) Google Scholar These strains share with type BI/NAP1/027 the presence of binary toxin and tcdC gene deletions. To date there has been no conclusive evidence that consuming food contaminated with C difficile has led to clinical CDI in human beings. The means by which the current epidemic BI/NAP1/027 strain has become so widely distributed in multiple countries so quickly has not been determined, but a common vehicle such as food remains an enticing avenue for further research. The greatest risk factors for CDI caused by the BI/NAP1/027 strain are advanced patient age, hospitalization, and exposure to specific antimicrobials, especially fluoroquinolones and cephalosporins.2Loo V.G. Poirier L. Miller M.A. et al.A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.N Engl J Med. 2005; 353: 2442-2449Crossref PubMed Scopus (1712) Google Scholar, 6Muto C.A. Pokrywka M. Shutt K. et al.A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use.Infect Control Hosp Epidemiol. 2005; 26: 273-280Crossref PubMed Scopus (557) Google Scholar, 15Gaynes R. Rimland D. Killum E. et al.Outbreak of Clostridium difficile infection in a long-term care facility: association with gatifloxacin use.Clin Infect Dis. 2004; 38: 640-645Crossref PubMed Scopus (231) Google Scholar, 16Pepin J. Saheb N. Coulombe M.A. et al.Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in QuebeC.Clin Infect Dis. 2005; 41: 1254-1260Crossref PubMed Scopus (852) Google Scholar The specific fluoroquinolones that have been identified as risk factors include levofloxacin, moxifloxacin, gatifloxacin, and ciprofloxacin, presumably as a result of the fluoroquinolone-resistance present in the epidemic strain. Cephalosporin antibiotics, to which virtually all C difficile strains are resistant, have also been implicated as a risk in hospitals in which the epidemic strain is present, including use for surgical prophylaxis.2Loo V.G. Poirier L. Miller M.A. et al.A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.N Engl J Med. 2005; 353: 2442-2449Crossref PubMed Scopus (1712) Google Scholar, 17Labbe A.C. Poirier L. MacCannell D. et al.Clostridium difficile infections in a Canadian tertiary care hospital before and during a regional epidemic associated with the BI/NAP1/027 strain.Antimicrob Agents Chemother. 2008; 52: 3180-3187Crossref PubMed Scopus (131) Google Scholar Exposures to stomach acid–reducing agents such as histamine type 2 blockers and proton pump inhibitors have been identified inconsistently as risk factors for CDI in hospitals in which the epidemic strain is present.2Loo V.G. Poirier L. Miller M.A. et al.A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.N Engl J Med. 2005; 353: 2442-2449Crossref PubMed Scopus (1712) Google Scholar, 16Pepin J. Saheb N. Coulombe M.A. et al.Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in QuebeC.Clin Infect Dis. 2005; 41: 1254-1260Crossref PubMed Scopus (852) Google Scholar, 18Dial S. Alrasadi K. Manoukian C. et al.Risk of Clostridium difficile diarrhea among hospital inpatients prescribed proton pump inhibitors: cohort and case-control studies.CMAJ. 2004; 171: 33-38Crossref PubMed Scopus (488) Google Scholar Other than the new fluoroquinolone resistance, the organism-specific factors that have enabled the BI/NAP1/027 strains to be so successful recently in their dissemination and ability to cause severe CDI remain largely speculative but are the subject of intensive research efforts. All virulent C difficile strains carry a 19.6-kb pathogenicity locus (PaLoc), which contains 5 genes (Figure 3):tcdR, tcdB, tcdE, tcdA, and tcdC The tcdA and tcdB genes encode toxins A and B, respectively; these toxins are monoglucosyltransferases that modify rho proteins in host cells leading to collapse of the actin cytoskeleton and cell death.19Voth D.E. Ballard J.D. Clostridium difficile toxins: mechanism of action and role in disease.Clin Microbiol Rev. 2005; 18: 247-263Crossref PubMed Scopus (911) Google Scholar The tcdE gene encodes a holin-like protein that is proposed to facilitate release of toxins A and B from the bacterial cell, because these toxins do not possess signal peptides.20Tan K.S. Wee B.Y. Song K.P. Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile.J Med Microbiol. 2001; 50: 613-619PubMed Google Scholar The regulation of toxin production in C difficile is not completely characterized, but current data show that toxin expression is controlled by the positive regulator, TcdR, its antagonist, TcdC, and the global regulator, CodY (Figure 3).21Dineen S.S. Villapakkam A.C. Nordman J.T. et al.Repression of Clostridium difficile toxin gene expression by CodY.Mol Microbiol. 2007; 66: 206-219Crossref PubMed Scopus (179) Google Scholar, 22Mani N. Dupuy B. Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor.Proc Natl Acad Sci U S A. 2001; 98: 5844-5849Crossref PubMed Scopus (206) Google Scholar, 23Mani N. Lyras D. Barroso L. et al.Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression.J Bacteriol. 2002; 184: 5971-5978Crossref PubMed Scopus (109) Google Scholar, 24Matamouros S. England P. Dupuy B. Clostridium difficile toxin expression is inhibited by the novel regulator TcdC.Mol Microbiol. 2007; 64: 1274-1288Crossref PubMed Scopus (180) Google Scholar During logarithmic-phase growth, tcdR, tcdB, tcdE, and tcdA are expressed (at a low level) from a single transcript that originates from a putative promoter upstream of tcdR, at a rate proportional to cell density, whereas the expression of tcdC is maximal (Figure 3A).25Hundsberger T. Braun V. Weidmann M. et al.Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile.Eur J Biochem. 1997; 244: 735-742Crossref PubMed Scopus (161) Google Scholar In stationary phase, the expression of tcdC decreases, whereas tcdR, tcdA, and tcdB expression increases. During this time, tcdR, tcdA, and tcdB are transcribed from individual promoters in a TcdR-dependent manner (Figure 3B).22Mani N. Dupuy B. Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor.Proc Natl Acad Sci U S A. 2001; 98: 5844-5849Crossref PubMed Scopus (206) Google Scholar, 23Mani N. Lyras D. Barroso L. et al.Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression.J Bacteriol. 2002; 184: 5971-5978Crossref PubMed Scopus (109) Google Scholar, 25Hundsberger T. Braun V. Weidmann M. et al.Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile.Eur J Biochem. 1997; 244: 735-742Crossref PubMed Scopus (161) Google Scholar, 26Dupuy B. Sonenshein A.L. Regulated transcription of Clostridium difficile toxin genes.Mol Microbiol. 1998; 27: 107-120Crossref PubMed Scopus (225) Google Scholar TcdR is a 22-kDa protein that is part of group 5 of the sigma 70 factor family, which has similarities to extracytoplasmic function–like sigma factors.27Dupuy B. Matamouros S. Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase sigma-factors.Res Microbiol. 2006; 157: 201-205Crossref PubMed Scopus (39) Google Scholar, 28Helmann J.D. The extracytoplasmic function (ECF) sigma factors.Adv Microb Physiol. 2002; 46: 47-110Crossref PubMed Scopus (571) Google Scholar This protein binds to the RNA polymerase holoenzyme22Mani N. Dupuy B. Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor.Proc Natl Acad Sci U S A. 2001; 98: 5844-5849Crossref PubMed Scopus (206) Google Scholar to facilitate expression of tcdR, tcdA, tcdB, and probably tcdE. TcdC, a 26-kDa protein, is likely to be an antisigma factor that acts as a negative regulator of toxin production.24Matamouros S. England P. Dupuy B. Clostridium difficile toxin expression is inhibited by the novel regulator TcdC.Mol Microbiol. 2007; 64: 1274-1288Crossref PubMed Scopus (180) Google Scholar, 29Braun V. Hundsberger T. Leukel P. et al.Definition of the single integration site of the pathogenicity locus in Clostridium difficile.Gene. 1996; 181: 29-38Crossref PubMed Scopus (258) Google Scholar The tcdC structural gene is located downstream of the other 4 PaLoc genes and is encoded on the opposite strand (Figure 3).25Hundsberger T. Braun V. Weidmann M. et al.Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile.Eur J Biochem. 1997; 244: 735-742Crossref PubMed Scopus (161) Google Scholar TcdC is a membrane-associated protein that most likely interacts with and sequesters TcdR to inhibit transcription of the toxin genes.24Matamouros S. England P. Dupuy B. Clostridium difficile toxin expression is inhibited by the novel regulator TcdC.Mol Microbiol. 2007; 64: 1274-1288Crossref PubMed Scopus (180) Google Scholar, 30Govind R. Vediyappan G. Rolfe R.D. Fralick J.A. Evidence that Clostridium difficile TcdC is a membrane-associated protein.J Bacteriol. 2006; 188: 3716-3720Crossref PubMed Scopus (21) Google Scholar Previous analysis of tcdC polymorphisms among C difficile clinical isolates found that most strains that carried nonsense mutations or deletions in tcdC also had variant tcdA and tcdB genes.31Spigaglia P. Mastrantonio P. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates.J Clin Microbiol. 2002; 40: 3470-3475Crossref PubMed Scopus (251) Google Scholar The investigators observed that these strains were persistent in the hospital population during a 2-year period, were responsible for outbreaks of CDI, and conferred higher levels of in vitro cytotoxicity than some other clinical isolates. Thus, it was proposed that the variant tcdC genes in these toxin-variant strains may have been responsible for their higher levels of cytotoxicity.31Spigaglia P. Mastrantonio P. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates.J Clin Microbiol. 2002; 40: 3470-3475Crossref PubMed Scopus (251) Google Scholar Recently, a clonal group of epidemic C difficile isolates (BI/NAP1/027) were characterized that produced approximately 16-fold more toxin A and 23-fold more toxin B than some other clinical isolates.32Warny M. Pepin J. Fang A. et al.Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe.Lancet. 2005; 366: 1079-1084Abstract Full Text Full Text PDF PubMed Scopus (1224) Google Scholar This study and others identified an 18-bp deletion in the tcdC gene of the BI/NAP1/027 C difficile strains. It was suggested that this deletion alone may account for a loss of TcdC function, which could be associated with increased expression of toxins A and B.2Loo V.G. Poirier L. Miller M.A. et al.A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.N Engl J Med. 2005; 353: 2442-2449Crossref PubMed Scopus (1712) Google Scholar, 32Warny M. Pepin J. Fang A. et al.Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe.Lancet. 2005; 366: 1079-1084Abstract Full Text Full Text PDF PubMed Scopus (1224) Google Scholar However, further studies showed that this 18-bp deletion alone did not impair the negative regulatory function of the TcdC protein, either in vitro or in vivo.24Matamouros S. England P. Dupuy B. Clostridium difficile toxin expression is inhibited by the novel regulator TcdC.Mol Microbiol. 2007; 64: 1274-1288Crossref PubMed Scopus (180) Google Scholar In addition to the 18-bp deletion in TcdC, the BI/NAP1/027 epidemic strains have subsequently been found to carry a 1-bp deletion in tcdC, located 117 bp into the open reading frame. This deletion is predicted to result in a truncated TcdC protein that is unlikely to be functional,24Matamouros S. England P. Dupuy B. Clostridium difficile toxin expression is inhibited by the novel regulator TcdC.Mol Microbiol. 2007; 64: 1274-1288Crossref PubMed Scopus (180) Google Scholar, 33Curry S.R. Marsh J.W. Muto C.A. et al.tcdC genotypes associated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difficile.J Clin Microbiol. 2007; 45: 215-221Crossref PubMed Scopus (173) Google Scholar, 34MacCannell D.R. Louie T.J. Gregson D.B. et al.Molecular analysis of Clostridium difficile PCR ribotype 027 isolates from Eastern and Western Canada.J Clin Microbiol. 2006; 44: 2147-2152Crossref PubMed Scopus (149) Google Scholar which could explain the observation that recent epidemic strains that carry this tcdC deletion produce higher in vitro levels of toxins A and B than some strains that do not carry this tcdC polymorphism. This hypothesis could in turn account, at least in part, for the increased virulence of the BI/NAP1/027 strains. To confirm the hypothesis that increased virulence of BI/NAP1/027 strains is due to nonfunctional TcdC, it will be necessary to construct a set of BI/NAP1/027-derived isogenic strains with intact or inactivated tcdC, then test these strains for their virulence in an animal model. Targeted gene disruption in certain C difficile strains is now possible,21Dineen S.S. Villapakkam A.C. Nordman J.T. et al.Repression of Clostridium difficile toxin gene expression by CodY.Mol Microbiol. 2007; 66: 206-219Crossref PubMed Scopus (179) Google Scholar, 35Heap J.T. Pennington O.J. Cartman S.T. et al.The ClosTron: a universal gene knock-out system for the genus
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