The Hypervirulent Strain of Clostridium Difficile: NAP1/B1/027 - A Brief Overview.
ABSTRACT: Clostridium difficile is a gram-positive bacterium notorious for causing epidemic diarrhea globally with a significant health burden. The pathogen is clinically challenging with increasing antibiotic resistance and recurrence rate. We provide here an in-depth review of one particular strain/ribotype 027, commonly known as NAP1/B1/027 or North American pulsed-field gel electrophoresis type 1, restriction endonuclease analysis type B1, polymerase chain reaction ribotype 027, which has shown a much higher recurrence rate than other strains.
Project description:Clostridium difficile is the most common cause of antibiotic-associated diarrhoea worldwide. Over the past 10 years, the incidence and severity of disease have increased in North America and Europe due to the emergence of a hypervirulent clone designated PCR ribotype 027. In this study, we sought to identify phenotypic differences among a collection of 26 presumed PCR ribotype 027 strains from the US and the UK isolated between 1988 and 2008 and also re-evaluated the PCR ribotype. We demonstrated that some of the strains typed as BI by restriction endonuclease analysis, and presumed to be PCR ribotype 027, were in fact other PCR ribotypes such as 176, 198 and 244 due to slight variation in banding pattern compared to the 027 strains. The reassigned 176, 198 and 244 ribotype strains were isolated in the US between 2001 and 2004 and appeared to have evolved recently from the 027 lineage. In addition, the UK strains were more motile and more resistant to most of the antibiotics compared to the US counterparts. We conclude that there should be a heightened awareness of newly identified PCR ribotypes such as 176, 198 and 244, and that they may be as problematic as the notorious 027 strains.
Project description:Clostridium difficile (C. difficile) strains belonging to PCR ribotype 027, PFGE type NAP1, REA type B1 and toxinotype III, termed NAP1/027, have been implicated in the increased frequency of outbreaks of Clostridium difficile-associated diarrhoea (CDAD) in North America and Europe. The NAP1/027 strains appears to be more virulent with an increased mortality and frequency of relapse. Current European C. difficile microarrays are designed to the first sequenced and annotated C. difficile complete genome - strain 630 (ribotype 12). A high density oligonucleotide microarray was designed to C. difficile 630 (CD630) sequence and extra probes corresponding to two PCR ribotypes O27 strains C. difficile R20291 and QCD-32g58 were also included. Comparative genomic hybridisation was used to identify markers of ribotype 027 strains and markers to identify CD630. Strains hybridised to the array included the most prevalent ribotypes found in the UK and Europe (106 and 001) as well as the emerging hypervirulent ribotype 078.
Project description:Clostridium difficile PCR ribotype 027 comprised 0.2% of a collection of Swedish isolates in 1997-2001 (3 of 1,325 isolates). These isolates had lower moxifloxacin MICs than the epidemic type 027 isolates, but they had the same tcdC sequence and toxin yield. Type 027 produced 3- to 13-fold more toxin than did major Swedish types. One epidemic strain (027/NAP1a) sporulated more than did other type 027 isolates, a feature that should contribute to its survival and spread.
Project description:Allogeneic hematopoietic stem cell transplantation (HSCT) recipients are at high risk for developing Clostridium difficile infection (CDI). We studied the incidence, risk factors, NAP1/027 prevalence, and clinical outcomes, including acute lower gastrointestinal graft-versus-host disease (GI GVHD), associated with early CDI in this population. A retrospective review was conducted of patients who underwent allogeneic HSCT at Memorial Sloan Kettering Cancer Center from January 1, 2005 to September 30, 2010. Early CDI was defined as infection occurring from day -10 to day +40 from stem cell infusion. Among 793 patients who received allogeneic HSCTs, early CDI occurred in 11.9%; 56% cases were between day -5 and day +5. Overall incidence was 25.2 cases/10,000 at-risk days. There was a high prevalence of NAP1/027 strains during peak incidence (61% in 2008). NAP1/027 was the most common strain in both adult and pediatric cases (24% and 23%, respectively). CDI was clinically mild, including those due to NAP1/027. Metronidazole was the primary treatment for 91 of 94 patients, 7 of 8 cases refractory to metronidazole had no response to vancomycin, and none was due to NAP1/027. Relapse of CDI was common (31%). The cumulative incidence of GI GVHD in patients with and without early CDI was 6.8% and 8%, respectively (P = .5). Most cases of CDI occurred during conditioning or immediately after transplant. Despite high prevalence of NAP1/027, we found only mild disease. Most patients were treated successfully with metronidazole, irrespective of NAP1/027 status. There was no significant association between early CDI and subsequent development of GI GVHD. This study demonstrates the high incidence of CDI early after allogeneic HSCT with wide diversity among infecting strains. Despite the high prevalence of NAP1/027, the disease is mild but relapses are common. No association was found between CDI and subsequent development of GI GVHD.
Project description:Comparative analysis of the Clostridium difficile BI/NAP1/027 strain R20291 and ClosTron-derived ermB mutants in the hamster infection model are compromised by the clindamycin susceptibility of the parent. Mutants can appear more virulent. We have rectified this anomaly by genome engineering. The variant created (CRG20291) represents an ideal control strain for virulence assays of ClosTron mutants.
Project description:The Clostridium difficile exotoxin, TcdB, which is a major virulence factor, varies between strains of this pathogen. Herein, we show that TcdB from the epidemic BI/NAP1/027 strain of C. difficile is more lethal, causes more extensive brain hemorrhage, and is antigenically variable from TcdB produced by previously studied strains of this pathogen (TcdB003). In mouse intoxication assays, TcdB from a ribotype 027 strain (TcdB027) was at least four fold more lethal than TcdB003. TcdB027 caused a previously undescribed brain hemorrhage in mice and this correlated with a heightened sensitivity of brain microvascular endothelial cells to the toxin. TcdB003 and TcdB027 also differed in their antigenic profiles and did not share cross-neutralizing epitopes in a major immunogenic region of the protein. Solid phase humoral mapping of epitopes in the carboxy-terminal domains (CTD) of TcdB027 and TcdB003 identified 11 reactive epitopes that varied between the two forms of TcdB, and 13 epitopes that were shared or overlapping. Despite the epitope differences and absence of neutralizing epitopes in the CTD of TcdB027, a toxoid form of this toxin primed a strong protective response. These findings indicate TcdB027 is a more potent toxin than TcdB003 as measured by lethality assays and pathology, moreover the sequence differences between the two forms of TcdB alter antigenic epitopes and reduce cross-neutralization by antibodies targeting the CTD.
Project description:Clostridium difficile infection (CDI) is a leading cause of hospital-acquired diarrhea. In recent decades, the emergence of the "hypervirulent" BI/NAP1/027 strains of C. difficile significantly increased the morbidity and mortality of CDI. The pathogenesis of CDI is primarily mediated by the action of two toxins, TcdA and TcdB, with TcdB being the major virulent factor in humans. In this report, we describe the engineering of a panel of designed ankyrin repeat proteins (DARPins) that potently neutralize TcdB from the BI/NAP1/027 strains (e.g., TcdBUK1). The most effective DARPin, D16, inhibits TcdBUK1 with a 50% effective concentration (EC50) of 0.5 nM, which is >66-fold lower than that of the FDA-approved anti-TcdB antibody bezlotoxumab (EC50, ∼33 nM). Competitive enzyme-linked immunosorbent assays (ELISAs) showed that D16 blocks interactions between TcdB and its receptor, chondroitin sulfate proteoglycan 4 (CSPG4). The dimeric DARPin U3D16, which pairs D16 with DARPin U3, a disrupter of the interaction of TcdB with Frizzled 1/2/7 receptor, exhibits 10-fold-to-20-fold-enhanced neutralization potency against TcdB from C. difficile strains VPI 10463 (laboratory strain) and M68 (CF/NAP9/017) but identical activity against TcdBUK1 relative to D16. Subsequent ELISAs revealed that TcdBUK1 did not significantly interact with Frizzled 1/2/7. Computation modeling revealed 4 key differences at the Frizzled 1/2/7 binding interface which are likely responsible for the significantly reduced binding affinity.IMPORTANCE We report the engineering and characterization of designed ankyrin proteins as potent neutralizers of TcdB toxin secreted by a hypervirulent ribotype 027 strain of Clostridium difficile We further show that although TcdB toxins from both ribotype 027 and VPI 10461 interact efficiently with TcdB receptors CSPG4 and Pvrl3, TcdB027 lacks significant ability to bind the only known physiologically relevant TcdB receptor, Frizzled 1/2/7.
Project description:TcdA and TcdB exotoxins are the main virulence factors of Clostridium difficile, one of the most deadly nosocomial pathogens. Recent data suggest that prophages can influence the regulation of toxin expression. Here we present the characterization of ?CD38-2, a pac-type temperate Siphoviridae phage that stimulates toxin expression when introduced as a prophage into C. difficile. Host range analysis showed that ?CD38-2 was able to infect 99/207 isolates of C. difficile representing 11 different PCR ribotypes. Of 89 isolates corresponding to the NAP1/027 hypervirulent strain, which recently caused several outbreaks in North America and Europe, 79 (89%) were sensitive to ?CD38-2. The complete double-stranded DNA (dsDNA) genome was determined, and a putative function could be assigned to 24 of the 55 open reading frames. No toxins or virulence factors could be identified based on bioinformatics analyses. Our data also suggest that ?CD38-2 replicates as a circular plasmid in C. difficile lysogens. Upon introduction of ?CD38-2 into a NAP1/027 representative isolate, up to 1.6- and 2.1-fold more TcdA and TcdB, respectively, were detected by immunodot blotting in culture supernatants of the lysogen than in the wild-type strain. In addition, real-time quantitative reverse transcriptase PCR (qRT-PCR) analyses showed that the mRNA levels of all five pathogenicity locus (PaLoc) genes were higher in the CD274 lysogen. Our study provides the first genomic sequence of a new pac-type Siphoviridae phage family member infecting C. difficile and brings further evidence supporting the role of prophages in toxin production in this important nosocomial pathogen.
Project description:The continued rise of Clostridium difficile infections worldwide has been accompanied by the rapid emergence of a highly virulent clone designated PCR-ribotype 027. To understand more about the evolution of this virulent clone, we made a three-way genomic and phenotypic comparison of an 'historic' non-epidemic 027 C. difficile (CD196), a recent epidemic and hypervirulent 027 (R20291) and a previously sequenced PCR-ribotype 012 strain (630).Although the genomes are highly conserved, the 027 genomes have 234 additional genes compared to 630, which may contribute to the distinct phenotypic differences we observe between these strains relating to motility, antibiotic resistance and toxicity. The epidemic 027 strain has five unique genetic regions, absent from both the non-epidemic 027 and strain 630, which include a novel phage island, a two component regulatory system and transcriptional regulators.A comparison of a series of 027 isolates showed that some of these genes appeared to have been gained by 027 strains over the past two decades. This study provides genetic markers for the identification of 027 strains and offers a unique opportunity to explain the recent emergence of a hypervirulent bacterium.
Project description:Clostridium difficile is one of the leading causes of antibiotic-associated diarrhea in health care facilities worldwide. Here, we report the genome sequence of C. difficile strain G46, ribotype 027, isolated from an outbreak in Glamorgan, Wales, in 2006.