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Toxin A-negative, toxin B-positive Clostridium difficile
International Journal of Infectious Diseases (2007) 11, 5—10
http://intl.elsevierhealth.com/journals/ijid
PERSPECTIVE
Toxin A-negative, toxin B-positive Clostridium difficile ´amus Fanning a, Lorraine Kyne b Denise Drudy a,*, Se a
Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland b Department of Medicine for the Older Person, Mater Misericordiae University Hospital, Dublin 7, Ireland Received 30 November 2005; received in revised form 31 March 2006; accepted 17 April 2006 Corresponding Editor: Timothy Barkham, Singapore
KEYWORDS +
A B Clostridium difficile; Infectious diarrhea; Bacterial genotyping
Summary Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Many pathogenic strains of Clostridium difficile produce two toxins TcdA and TcdB, both of which are pro-inflammatory and enterotoxic in human intestine. Clinically relevant toxin A-negative, toxin B-positive (AB+) strains of Clostridium difficile that cause diarrhea and colitis in humans have been isolated with increasing frequency worldwide. This perspective describes these important toxin variant strains and highlights the need to use Clostridium difficile diagnostic methods that can detect both TcdA and TcdB. # 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Introduction Clostridium difficile is a common nosocomial pathogen and a major cause of infectious diarrhea in hospitalized patients.1,2 Colonization with C. difficile is associated with a wide spectrum of clinical presentations ranging from asymptomatic carriage to fulminant pseudomembranous colitis.3 In recent years the incidence of C. difficile and the number of cases associated with severe outcomes have increased worldwide.4—8 This may be related to several factors, including changing demographics of patients admitted to hospital, changing infection control policies, or the emergence of more virulent strains of C. difficile.7,9,10 Two structurally similar toxins denoted as TcdA (308 kDa) and TcdB (270 kDa) are the main virulence determinants
* Corresponding author. Tel.: +353 1 716 6096; fax: +353 1 716 6091. E-mail address: [email protected] (D. Drudy).
associated with Clostridium difficile-associated disease (CDAD) and most pathogenic strains of C. difficile produce both toxins (A+B+). Both of these large toxins are glucosyltransferases that catalyze the monoglucosylation of threonine 35/37 of small GTP-binding proteins Rho, Rac, and Cdc42 within target cells, and thus modulate several physiological cellular events resulting in cell death. Structurally these toxins have three domains: the enzymatic activity is located in the N terminus of the protein, the middle domain is the putative translocation region, and the C terminus is involved in receptor binding. The N termini of TcdA and TcdB demonstrate 74% homology that accounts for their similar substrate specificity. The carboxy-terminal domain of both toxins carry a number of short homologous regions termed combined repetitive oligopeptides (CROPs). The role of these toxins in the pathogenesis of CDAD has been well described.11 Both toxin A and B are proinflammatory, cytotoxic, and enterotoxic in the human colon.12,13 These toxins are encoded by two genes tcdA and tcdB,
1201-9712/$32.00 # 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2006.04.003
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D. Drudy et al.
Figure 1 Schematic representation of the Clostridium difficile pathogenicity locus of Clostridium difficile 10463 and the alterations found in the four AB+ strain types described to date. Filled bars represent the repetitive sequences of both toxin genes. All four types differ in the length of the deletion (indicated by the triangle and open bar) in tcdA and the size of the deletion where known is indicated. The presence of the 30 end of tcdA in strain XVII has not been confirmed and is therefore indicated with an asterisk.
mapping to a 19.6-kb pathogenicity locus (PaLoc) containing additional regulatory genes.14 C. difficile isolates with varying genetic modifications within the PaLoc have been investigated and 28 different toxinotypes have been described.15,16 These include variant C. difficile that have deletions, insertions, or polymorphic restriction sites in one or more of the genes on the PaLoc but which can still produce functional toxin proteins TcdA and TcdB and toxin-variant strains (AB+) which fail to produce detectable toxin A15,17 (Figure 1). Toxin variant strains that produce either toxin A or toxin B are the most commonly isolated variant C. difficile with modifications in their PaLoc. Until recently, it was thought that all toxigenic strains associated with disease produced both TcdA and TcdB. Early studies in animal models indicated that TcdA and TcdB acted synergistically but that TcdA was required to produce the initial damage in the colon.18 Consequently much of the research on the pathogenesis of CDAD has focused on the inflammation modulated by toxin A. However many recent reports demonstrate the clinical importance of A-negative toxin B positive (AB+) isolates.19—22 Outbreaks of C. difficile diarrhea, sporadic cases of infection and cases of pseudomembranous colitis (PMC) caused by AB+ C. difficile have been previously documented.20,23—25
AB+ Clostridium difficile To date four different AB+ Clostridium difficile strains have been described and characterized using Rupnik’s toxinotyping (PCR-RFLP) scheme.15 In the early 1990s two AB+ strain types were identified that produced TcdB but no detectable TcdA. Strain 8864, the first toxin variant strain to be characterized, caused hemorrhage, fluid accumulation, and tissue damage in ligated ileal rabbit loops.26,27 Molecular
analysis identified a large deletion of 5.9-kb in the PaLoc mapping to the 30 end of tcdA and tcdC and which prevented production of TcdA at a transcriptional level. In addition there is a 1.1-kb insertion between tcdA and tcdE.28 Toxinotyping designates this strain type as toxinotype X15 (Figure 1). Early studies indicated that this strain type produced a modified form of TcdB29 and subsequent analyses determined that TcdB-8864 induced a different cytopathic effect (CPE) compared to TcdB-10463 (TcdB-8864 glucosylates the Rho proteins Rac1, Rap 1/2, and Ral)28 (Figure 2). Only one organism of strain type 8864 has ever been isolated (from an asymptomatic adult) and although the clinical significance of this strain in humans is questionable its virulence in animal models confirms its pathogenic potential. The second category of AB+ strain types identified were serogroup F strains (type strain 1470) that were considered non-pathogenic due to their frequent isolation from asymptomatic infants and their lack of pathogenicity in animal models.30,31 Like strain 8864 these are also truncated in the 30 region of the repetitive domain of tcdA. They contain a 1.8-kb deletion in tcdA, together with a mutation that introduces a stop codon corresponding to amino acid position 47, leading to the truncation of TcdA-147032 (Figure 1). Toxinotyping classified these organisms as type VIII. Analysis of TcdB-1470 indicated that this toxin also has polymorphisms in its tcdB gene resulting in altered glucosylation and differential cytopathic effects.33 Sequence analysis of TcdB from strains 8864 and 1470 showed a high degree of sequence conservation in the enzymatic domain with substitutions at only 8 of 560 amino acid positions.28 These novel cytotoxins are a hybrid between TcdB from reference strain VPI 10463 and Clostridium sordellii lethal toxin (TclS).33 While the receptor binding domain shares 100% homology with TcdB10463 the enzymatic domain is only 79% homologous (Figure 2). These hybrid toxins suggest that genetic exchange
Toxin AB+ Clostridium difficile
7
Figure 2 Receptor binding domain of variant Clostridium difficile toxin B and toxin B from VPI 10463 demonstrate 99% homology. The enzymatic domain of variant Clostridium difficile toxin B is only 79% homologous to toxin B from VPI 10463. Variant toxin B glucosylates different Rho proteins than toxin B from VPI 10463. This is further demonstrated by the differential cytopathic effect on fibroblast cells.
and recombination may have occurred between large clostridial toxin (LCT) producing strains of clostridia. Most recently two additional AB+ strains, toxinotypes XVI and XVII, have been described.17 Interestingly these newer toxinotypes contain the genes that code the Clostridium difficile binary toxin. The molecular mechanism responsible for the lack of toxin A production in these newer toxinotypes has not yet been described although these strains also have deletions in the CROP region of tcdA17 (Figure 1). Although four AB+ strain types have been described, toxinotype VIII appears to be the most clinically significant toxin variant strain type. These have been isolated from four documented outbreaks, sporadic cases of diarrhea, and cases of pseudomembranous colitis. In contrast, only one strain of toxinotype XVI has been associated with clinical disease and the other two toxinotypes X and XVII have been isolated from asymptomatic patients.
outbreak was 13% while there were seven episodes of severe diarrhea and one death. Clonality of the 16 available outbreak isolates was determined using 16—23S rRNA ribotyping. This outbreak subsided following a change in the surgical prophylactic antibiotic policy such that clindamycin was withdrawn, along with the introduction of strategic infection control measures. In 2001, 10 patients with identical AB+ C. difficile strains were identified in a cancer care hospital in Japan.34 Our group recently described an AB+ C. difficile outbreak in one Dublin hospital that affected 73 patients (Drudy et al., submitted for publication). Molecular typing of 90 recovered isolates determined that 95% of the isolates were AB+ (PCR ribotype 017, toxinotype VIII).
Outbreaks of AB+ Clostridium difficile
AB+ Clostridium difficile causes the same spectrum of disease that is associated with A+B+ strains ranging from asymptomatic colonization through to the more severe fulminant colitis. The rate of C. difficile recurrence observed in our study (36%) and other outbreaks due to toxin-variant strains, is similar to recurrence rates reported by us, and others, for toxin A+B+ strains. However, outbreaks described by us and other authors have documented increased disease severity associated with these AB+ isolates.35 The mechanism(s) responsible for this observed increase in disease severity are not known. We have previously demonstrated that the immune response to toxin A was important in determining clinical presentation and course of CDAD.35,36 Recent in vitro studies have indicated that like TcdA, TcdB is also a potent enterotoxin capable of inducing 10-fold more toxicity in human colonic explants than toxin A.13,37 Warny
To date there have been four documented outbreaks associated with AB+ Clostridium difficile. The first outbreak reported in 1998 by Alfa et al. occurred in a Canadian hospital over a three month period.19,23 There were 16 cases of nosocomial C. difficile diarrhea on four hospital wards; eight of the cases spent time on one hospital ward. This outbreak was associated with a 19% recurrence rate of CDAD and two deaths. Following the outbreak, the hospital infection control policy was changed and the laboratory testing for C. difficile was changed from a toxin A ELISA, which failed to detect these AB+ strains, to the cell culture cytotoxicity assay. A second AB+ outbreak occurred in The Netherlands between 1997 and 1998.20 In this outbreak 24 patients with AB+ C. difficile were identified. The recurrence rate in this
Disease spectrum and transmissibility of AB+ Clostridium difficile
8 and colleagues have demonstrated that emerging epidemic A+B+ clones produce 23 times more TcdB and 16 times more TcdA compared to toxinotype 0 strains.10 Although the kinetics of toxin B production in AB+ isolates have not yet been measured these strains do not produce toxin A and therefore the pathology observed in cases of severe colitis are likely to result from host inflammatory events modulated by TcdB. These findings add to an increasing number of investigations that highlight the importance of toxin B in the pathogenesis of CDAD. The mechanisms of nosocomial acquisition, persistence, and transmission of Clostridium difficile are complex and not fully understood for all strain types. Other investigators have determined that greater sporulation and spore germination capacity along with increased antimicrobial resistance may contribute to the persistence of A+B+ clonal isolates in the nosocomial setting.10,38—40 The mechanisms by which AB+ strains persist have not been widely studied. However, the increased resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics and fluoroquinolones seen in our outbreak isolates is likely to contribute to persistence in both the hospital environment and the human gut. Ongoing surveillance at our hospital indicates the persistence of this strain even though the incidence rate remains low. Surveillance following the outbreak in The Netherlands demonstrated that their AB+ isolates did not persist. These AB+ strains are no longer found in patients at that institution (E. Kuiper, personal communication).
Antibiotic resistance in AB+ Clostridium difficile An early investigation by Delme ´e and Avesani showed that serogroup F strains (AB+) were usually susceptible to clindamycin and erythromycin.41 Since that time the emergence of resistance to MLS antibiotics has been documented. Sixteen AB+ isolates from the outbreak in The Netherlands were investigated for antibiotic resistance and all were resistant to clindamycin, erythromycin, and tetracycline and contained the ermB gene that encoded MLS resistance.20 The isolates from the Canadian and Japanese outbreaks were not tested for antibiotic susceptibility. The AB+ isolates from our hospital were also resistant to MLS antibiotics and contained the ermB gene. In addition our AB+ strains were resistant to a number of newer classes of fluoroquinolones including ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, and gatifloxacin. To our knowledge this is the first description of fluoroquinolone resistance in this AB+ strain type. Pituch et al. described the emergence of a clindamycin resistant clone of AB+ C. difficile isolates in Polish hospitals.42 As C. difficile is not routinely cultured in many hospital laboratories the true incidence of antimicrobial resistance among C. difficile is not known. A current prospective study undertaken by the European C. difficile study group is investigating antimicrobial resistance in C. difficile and will generate data with regard to antibiotic resistance in AB+ and other C. difficile strain types.
Prevalence of AB+ Clostridium difficile These toxin variant strains have been isolated from several countries22,43,44 with varying prevalence rates.34,45—47 For
D. Drudy et al. example, rates of 0.2% have been reported from a multicenter study in the USA where 2 of 102 Clostridium difficile isolates from six clinical settings were AB+.47 In the UK prevalence rates of 3% have been described representing 43 isolates from 9 of 35 hospitals that submitted strains for typing to the Anaerobic Reference Laboratory in Cardiff.48 In France, rates of 3% from 25 different hospitals in Paris were reported.45 In contrast, AB+ prevalence rates as high as 39% have been described in one Japanese study.46 Furthermore, a recent study in Israel documented AB+ C. difficile rates of 56%.49 A recent study in Argentina indicated that AB+ strains have completely replaced A+B+ strains over a four-year time period wherein AB+ isolates increased from 12.5% in 2000 to 97.9% in 2003.22
Laboratory diagnosis of AB+ Clostridium difficile Most laboratory diagnostic tests for Clostridium difficile are based on either faecal culture or direct toxin detection in faecal samples. The cell culture cytotoxicity assay is considered the gold standard for toxin detection and can detect picogram quantities of toxin after 48 hours. In addition cell culture can identify these AB+ strains due to the different CPE induced by these strains as a result of their different glucosyltransferase substrate specificities. Enzyme immunoassays (EIAs) which can detect either TcdA and/or TcdB are the most commonly used diagnostic approach due to their ease of use and quick turn-around time although these methods are less sensitive compared to the cytotoxicity assay. Toxin A-specific EIAs use antibodies targeted against the CROP region of tcdA, which is deleted in AB+ strain types, which therefore fail to react in these assays. A recent survey carried out by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) study group on C. difficile determined that among European laboratories performing C. difficile toxin detection, 58% used ELISAs that can only detect toxin A and therefore toxin AB+ strains would fail to be detected in these clinical settings.50 Failure to use standardized C. difficile diagnostic methods that include both toxin A and B could lead to significant under-reporting of C. difficile. C. difficile culture lacks specificity due to the presence of non-toxigenic isolates and takes 48 hours to complete. However, culture is the only way to type strains and to test for antimicrobial susceptibility and therefore the only way in which the emergence and spread of more virulent or resistant strains can be identified and monitored.
Molecular analysis of AB+ Clostridium difficile Several different molecular typing methods have been applied to AB+ variants including restriction endonuclease analysis (REA),51 16—23S rRNA ribotyping,52 amplified fragment length polymorphism (AFLP),53 multi-locus sequence typing (MLST),54 and toxinotyping (PCR-RFLP).15 A recent study by Lemee et al. applied MLST to 72 C. difficile isolates from various hosts, geographic locations, PCR ribotypes, and toxigenic types. The study isolates included eight AB+ isolates from the USA, Japan, and France cultured from human adults and children. All eight isolates belonged to the same sequence type and clustered in a very homogenous MLST
Toxin AB+ Clostridium difficile phylogenetic lineage despite their origins from unrelated patients. Furthermore, a null allele for one of the seven housekeeping genes investigated further supported the low genetic diversity of these AB+ types.54 van den Berg et al. used AFLP and 16—23S rRNA ribotyping to analyze 39 AB+ strains from seven different countries. AFLP recognized two genotypes among the 39 isolates while two different ribotyping methods could differentiate two and three PCR ribotypes, respectively. All three methods exhibited similar discriminatory power when typing AB+ strain types.53 One international study has compared the relatedness of 23 AB+ strains using three typing methods: serogrouping, 16—23S rRNA PCR ribotyping, and REA.43 This study documented a high degree of similarity between AB+ strains from the USA and Europe. Twenty-one of the 23 isolates had a 1.8kb deletion in the 30 end of tcdA corresponding to toxinotype VIII and 20 of the 21 examined were PCR ribotype 017. REA was the most discriminatory typing method distinguishing 11 REA types among the 23 isolates. This method could further subtype the 20 PCR 017/serogroup F strains into six different REA types. PCR ribotyping and serogrouping identified four and three distinct types respectively. The remaining two AB+ strains investigated in this international study were strain 8864, which was unique using all three typing methods. In addition a PCR ribotype 110/serogroup X/REA DA1 strain had the same toxin genotype as VPI 10463 but did not produce any toxin A in vitro.
Concluding remarks The incidence of AB+ Clostridium difficile strains appears to be increasing worldwide. These strain types now represent a substantial number of C. difficile isolates. These isolates may be under-reported due to the widespread use of diagnostics that detect TcdA only. Although further studies are required to determine whether the pathogenesis of toxin variant CDAD (AB+) differs from that seen with toxin A+B+ strains, clinicians should be aware of the risk of severe disease associated with these variant strain types. TcdB is likely to play a more prominent role in the pathogenesis of C. difficile than previously considered, and future work should focus on the expression and regulation of this toxin as well as modulation of the host immune response. However the lack of suitable genetic tools to manipulate C. difficile and lack of isogenic mutants may hinder progress. New therapeutic approaches for CDAD such as toxin binders, passive immunotherapy or active immunization through vaccination will now need to target both TcdA and TcdB.
Acknowledgements The authors acknowledge the financial support provided by the National Institute on Aging, The Health Research Board, Ireland, The Mater Foundation and The Newman Scholarship Programme, University College Dublin. Conflict of interest: No conflict of interest to declare.
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http://intl.elsevierhealth.com/journals/ijid
PERSPECTIVE
Toxin A-negative, toxin B-positive Clostridium difficile ´amus Fanning a, Lorraine Kyne b Denise Drudy a,*, Se a
Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland b Department of Medicine for the Older Person, Mater Misericordiae University Hospital, Dublin 7, Ireland Received 30 November 2005; received in revised form 31 March 2006; accepted 17 April 2006 Corresponding Editor: Timothy Barkham, Singapore
KEYWORDS +
A B Clostridium difficile; Infectious diarrhea; Bacterial genotyping
Summary Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Many pathogenic strains of Clostridium difficile produce two toxins TcdA and TcdB, both of which are pro-inflammatory and enterotoxic in human intestine. Clinically relevant toxin A-negative, toxin B-positive (AB+) strains of Clostridium difficile that cause diarrhea and colitis in humans have been isolated with increasing frequency worldwide. This perspective describes these important toxin variant strains and highlights the need to use Clostridium difficile diagnostic methods that can detect both TcdA and TcdB. # 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Introduction Clostridium difficile is a common nosocomial pathogen and a major cause of infectious diarrhea in hospitalized patients.1,2 Colonization with C. difficile is associated with a wide spectrum of clinical presentations ranging from asymptomatic carriage to fulminant pseudomembranous colitis.3 In recent years the incidence of C. difficile and the number of cases associated with severe outcomes have increased worldwide.4—8 This may be related to several factors, including changing demographics of patients admitted to hospital, changing infection control policies, or the emergence of more virulent strains of C. difficile.7,9,10 Two structurally similar toxins denoted as TcdA (308 kDa) and TcdB (270 kDa) are the main virulence determinants
* Corresponding author. Tel.: +353 1 716 6096; fax: +353 1 716 6091. E-mail address: [email protected] (D. Drudy).
associated with Clostridium difficile-associated disease (CDAD) and most pathogenic strains of C. difficile produce both toxins (A+B+). Both of these large toxins are glucosyltransferases that catalyze the monoglucosylation of threonine 35/37 of small GTP-binding proteins Rho, Rac, and Cdc42 within target cells, and thus modulate several physiological cellular events resulting in cell death. Structurally these toxins have three domains: the enzymatic activity is located in the N terminus of the protein, the middle domain is the putative translocation region, and the C terminus is involved in receptor binding. The N termini of TcdA and TcdB demonstrate 74% homology that accounts for their similar substrate specificity. The carboxy-terminal domain of both toxins carry a number of short homologous regions termed combined repetitive oligopeptides (CROPs). The role of these toxins in the pathogenesis of CDAD has been well described.11 Both toxin A and B are proinflammatory, cytotoxic, and enterotoxic in the human colon.12,13 These toxins are encoded by two genes tcdA and tcdB,
1201-9712/$32.00 # 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2006.04.003
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Figure 1 Schematic representation of the Clostridium difficile pathogenicity locus of Clostridium difficile 10463 and the alterations found in the four AB+ strain types described to date. Filled bars represent the repetitive sequences of both toxin genes. All four types differ in the length of the deletion (indicated by the triangle and open bar) in tcdA and the size of the deletion where known is indicated. The presence of the 30 end of tcdA in strain XVII has not been confirmed and is therefore indicated with an asterisk.
mapping to a 19.6-kb pathogenicity locus (PaLoc) containing additional regulatory genes.14 C. difficile isolates with varying genetic modifications within the PaLoc have been investigated and 28 different toxinotypes have been described.15,16 These include variant C. difficile that have deletions, insertions, or polymorphic restriction sites in one or more of the genes on the PaLoc but which can still produce functional toxin proteins TcdA and TcdB and toxin-variant strains (AB+) which fail to produce detectable toxin A15,17 (Figure 1). Toxin variant strains that produce either toxin A or toxin B are the most commonly isolated variant C. difficile with modifications in their PaLoc. Until recently, it was thought that all toxigenic strains associated with disease produced both TcdA and TcdB. Early studies in animal models indicated that TcdA and TcdB acted synergistically but that TcdA was required to produce the initial damage in the colon.18 Consequently much of the research on the pathogenesis of CDAD has focused on the inflammation modulated by toxin A. However many recent reports demonstrate the clinical importance of A-negative toxin B positive (AB+) isolates.19—22 Outbreaks of C. difficile diarrhea, sporadic cases of infection and cases of pseudomembranous colitis (PMC) caused by AB+ C. difficile have been previously documented.20,23—25
AB+ Clostridium difficile To date four different AB+ Clostridium difficile strains have been described and characterized using Rupnik’s toxinotyping (PCR-RFLP) scheme.15 In the early 1990s two AB+ strain types were identified that produced TcdB but no detectable TcdA. Strain 8864, the first toxin variant strain to be characterized, caused hemorrhage, fluid accumulation, and tissue damage in ligated ileal rabbit loops.26,27 Molecular
analysis identified a large deletion of 5.9-kb in the PaLoc mapping to the 30 end of tcdA and tcdC and which prevented production of TcdA at a transcriptional level. In addition there is a 1.1-kb insertion between tcdA and tcdE.28 Toxinotyping designates this strain type as toxinotype X15 (Figure 1). Early studies indicated that this strain type produced a modified form of TcdB29 and subsequent analyses determined that TcdB-8864 induced a different cytopathic effect (CPE) compared to TcdB-10463 (TcdB-8864 glucosylates the Rho proteins Rac1, Rap 1/2, and Ral)28 (Figure 2). Only one organism of strain type 8864 has ever been isolated (from an asymptomatic adult) and although the clinical significance of this strain in humans is questionable its virulence in animal models confirms its pathogenic potential. The second category of AB+ strain types identified were serogroup F strains (type strain 1470) that were considered non-pathogenic due to their frequent isolation from asymptomatic infants and their lack of pathogenicity in animal models.30,31 Like strain 8864 these are also truncated in the 30 region of the repetitive domain of tcdA. They contain a 1.8-kb deletion in tcdA, together with a mutation that introduces a stop codon corresponding to amino acid position 47, leading to the truncation of TcdA-147032 (Figure 1). Toxinotyping classified these organisms as type VIII. Analysis of TcdB-1470 indicated that this toxin also has polymorphisms in its tcdB gene resulting in altered glucosylation and differential cytopathic effects.33 Sequence analysis of TcdB from strains 8864 and 1470 showed a high degree of sequence conservation in the enzymatic domain with substitutions at only 8 of 560 amino acid positions.28 These novel cytotoxins are a hybrid between TcdB from reference strain VPI 10463 and Clostridium sordellii lethal toxin (TclS).33 While the receptor binding domain shares 100% homology with TcdB10463 the enzymatic domain is only 79% homologous (Figure 2). These hybrid toxins suggest that genetic exchange
Toxin AB+ Clostridium difficile
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Figure 2 Receptor binding domain of variant Clostridium difficile toxin B and toxin B from VPI 10463 demonstrate 99% homology. The enzymatic domain of variant Clostridium difficile toxin B is only 79% homologous to toxin B from VPI 10463. Variant toxin B glucosylates different Rho proteins than toxin B from VPI 10463. This is further demonstrated by the differential cytopathic effect on fibroblast cells.
and recombination may have occurred between large clostridial toxin (LCT) producing strains of clostridia. Most recently two additional AB+ strains, toxinotypes XVI and XVII, have been described.17 Interestingly these newer toxinotypes contain the genes that code the Clostridium difficile binary toxin. The molecular mechanism responsible for the lack of toxin A production in these newer toxinotypes has not yet been described although these strains also have deletions in the CROP region of tcdA17 (Figure 1). Although four AB+ strain types have been described, toxinotype VIII appears to be the most clinically significant toxin variant strain type. These have been isolated from four documented outbreaks, sporadic cases of diarrhea, and cases of pseudomembranous colitis. In contrast, only one strain of toxinotype XVI has been associated with clinical disease and the other two toxinotypes X and XVII have been isolated from asymptomatic patients.
outbreak was 13% while there were seven episodes of severe diarrhea and one death. Clonality of the 16 available outbreak isolates was determined using 16—23S rRNA ribotyping. This outbreak subsided following a change in the surgical prophylactic antibiotic policy such that clindamycin was withdrawn, along with the introduction of strategic infection control measures. In 2001, 10 patients with identical AB+ C. difficile strains were identified in a cancer care hospital in Japan.34 Our group recently described an AB+ C. difficile outbreak in one Dublin hospital that affected 73 patients (Drudy et al., submitted for publication). Molecular typing of 90 recovered isolates determined that 95% of the isolates were AB+ (PCR ribotype 017, toxinotype VIII).
Outbreaks of AB+ Clostridium difficile
AB+ Clostridium difficile causes the same spectrum of disease that is associated with A+B+ strains ranging from asymptomatic colonization through to the more severe fulminant colitis. The rate of C. difficile recurrence observed in our study (36%) and other outbreaks due to toxin-variant strains, is similar to recurrence rates reported by us, and others, for toxin A+B+ strains. However, outbreaks described by us and other authors have documented increased disease severity associated with these AB+ isolates.35 The mechanism(s) responsible for this observed increase in disease severity are not known. We have previously demonstrated that the immune response to toxin A was important in determining clinical presentation and course of CDAD.35,36 Recent in vitro studies have indicated that like TcdA, TcdB is also a potent enterotoxin capable of inducing 10-fold more toxicity in human colonic explants than toxin A.13,37 Warny
To date there have been four documented outbreaks associated with AB+ Clostridium difficile. The first outbreak reported in 1998 by Alfa et al. occurred in a Canadian hospital over a three month period.19,23 There were 16 cases of nosocomial C. difficile diarrhea on four hospital wards; eight of the cases spent time on one hospital ward. This outbreak was associated with a 19% recurrence rate of CDAD and two deaths. Following the outbreak, the hospital infection control policy was changed and the laboratory testing for C. difficile was changed from a toxin A ELISA, which failed to detect these AB+ strains, to the cell culture cytotoxicity assay. A second AB+ outbreak occurred in The Netherlands between 1997 and 1998.20 In this outbreak 24 patients with AB+ C. difficile were identified. The recurrence rate in this
Disease spectrum and transmissibility of AB+ Clostridium difficile
8 and colleagues have demonstrated that emerging epidemic A+B+ clones produce 23 times more TcdB and 16 times more TcdA compared to toxinotype 0 strains.10 Although the kinetics of toxin B production in AB+ isolates have not yet been measured these strains do not produce toxin A and therefore the pathology observed in cases of severe colitis are likely to result from host inflammatory events modulated by TcdB. These findings add to an increasing number of investigations that highlight the importance of toxin B in the pathogenesis of CDAD. The mechanisms of nosocomial acquisition, persistence, and transmission of Clostridium difficile are complex and not fully understood for all strain types. Other investigators have determined that greater sporulation and spore germination capacity along with increased antimicrobial resistance may contribute to the persistence of A+B+ clonal isolates in the nosocomial setting.10,38—40 The mechanisms by which AB+ strains persist have not been widely studied. However, the increased resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics and fluoroquinolones seen in our outbreak isolates is likely to contribute to persistence in both the hospital environment and the human gut. Ongoing surveillance at our hospital indicates the persistence of this strain even though the incidence rate remains low. Surveillance following the outbreak in The Netherlands demonstrated that their AB+ isolates did not persist. These AB+ strains are no longer found in patients at that institution (E. Kuiper, personal communication).
Antibiotic resistance in AB+ Clostridium difficile An early investigation by Delme ´e and Avesani showed that serogroup F strains (AB+) were usually susceptible to clindamycin and erythromycin.41 Since that time the emergence of resistance to MLS antibiotics has been documented. Sixteen AB+ isolates from the outbreak in The Netherlands were investigated for antibiotic resistance and all were resistant to clindamycin, erythromycin, and tetracycline and contained the ermB gene that encoded MLS resistance.20 The isolates from the Canadian and Japanese outbreaks were not tested for antibiotic susceptibility. The AB+ isolates from our hospital were also resistant to MLS antibiotics and contained the ermB gene. In addition our AB+ strains were resistant to a number of newer classes of fluoroquinolones including ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, and gatifloxacin. To our knowledge this is the first description of fluoroquinolone resistance in this AB+ strain type. Pituch et al. described the emergence of a clindamycin resistant clone of AB+ C. difficile isolates in Polish hospitals.42 As C. difficile is not routinely cultured in many hospital laboratories the true incidence of antimicrobial resistance among C. difficile is not known. A current prospective study undertaken by the European C. difficile study group is investigating antimicrobial resistance in C. difficile and will generate data with regard to antibiotic resistance in AB+ and other C. difficile strain types.
Prevalence of AB+ Clostridium difficile These toxin variant strains have been isolated from several countries22,43,44 with varying prevalence rates.34,45—47 For
D. Drudy et al. example, rates of 0.2% have been reported from a multicenter study in the USA where 2 of 102 Clostridium difficile isolates from six clinical settings were AB+.47 In the UK prevalence rates of 3% have been described representing 43 isolates from 9 of 35 hospitals that submitted strains for typing to the Anaerobic Reference Laboratory in Cardiff.48 In France, rates of 3% from 25 different hospitals in Paris were reported.45 In contrast, AB+ prevalence rates as high as 39% have been described in one Japanese study.46 Furthermore, a recent study in Israel documented AB+ C. difficile rates of 56%.49 A recent study in Argentina indicated that AB+ strains have completely replaced A+B+ strains over a four-year time period wherein AB+ isolates increased from 12.5% in 2000 to 97.9% in 2003.22
Laboratory diagnosis of AB+ Clostridium difficile Most laboratory diagnostic tests for Clostridium difficile are based on either faecal culture or direct toxin detection in faecal samples. The cell culture cytotoxicity assay is considered the gold standard for toxin detection and can detect picogram quantities of toxin after 48 hours. In addition cell culture can identify these AB+ strains due to the different CPE induced by these strains as a result of their different glucosyltransferase substrate specificities. Enzyme immunoassays (EIAs) which can detect either TcdA and/or TcdB are the most commonly used diagnostic approach due to their ease of use and quick turn-around time although these methods are less sensitive compared to the cytotoxicity assay. Toxin A-specific EIAs use antibodies targeted against the CROP region of tcdA, which is deleted in AB+ strain types, which therefore fail to react in these assays. A recent survey carried out by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) study group on C. difficile determined that among European laboratories performing C. difficile toxin detection, 58% used ELISAs that can only detect toxin A and therefore toxin AB+ strains would fail to be detected in these clinical settings.50 Failure to use standardized C. difficile diagnostic methods that include both toxin A and B could lead to significant under-reporting of C. difficile. C. difficile culture lacks specificity due to the presence of non-toxigenic isolates and takes 48 hours to complete. However, culture is the only way to type strains and to test for antimicrobial susceptibility and therefore the only way in which the emergence and spread of more virulent or resistant strains can be identified and monitored.
Molecular analysis of AB+ Clostridium difficile Several different molecular typing methods have been applied to AB+ variants including restriction endonuclease analysis (REA),51 16—23S rRNA ribotyping,52 amplified fragment length polymorphism (AFLP),53 multi-locus sequence typing (MLST),54 and toxinotyping (PCR-RFLP).15 A recent study by Lemee et al. applied MLST to 72 C. difficile isolates from various hosts, geographic locations, PCR ribotypes, and toxigenic types. The study isolates included eight AB+ isolates from the USA, Japan, and France cultured from human adults and children. All eight isolates belonged to the same sequence type and clustered in a very homogenous MLST
Toxin AB+ Clostridium difficile phylogenetic lineage despite their origins from unrelated patients. Furthermore, a null allele for one of the seven housekeeping genes investigated further supported the low genetic diversity of these AB+ types.54 van den Berg et al. used AFLP and 16—23S rRNA ribotyping to analyze 39 AB+ strains from seven different countries. AFLP recognized two genotypes among the 39 isolates while two different ribotyping methods could differentiate two and three PCR ribotypes, respectively. All three methods exhibited similar discriminatory power when typing AB+ strain types.53 One international study has compared the relatedness of 23 AB+ strains using three typing methods: serogrouping, 16—23S rRNA PCR ribotyping, and REA.43 This study documented a high degree of similarity between AB+ strains from the USA and Europe. Twenty-one of the 23 isolates had a 1.8kb deletion in the 30 end of tcdA corresponding to toxinotype VIII and 20 of the 21 examined were PCR ribotype 017. REA was the most discriminatory typing method distinguishing 11 REA types among the 23 isolates. This method could further subtype the 20 PCR 017/serogroup F strains into six different REA types. PCR ribotyping and serogrouping identified four and three distinct types respectively. The remaining two AB+ strains investigated in this international study were strain 8864, which was unique using all three typing methods. In addition a PCR ribotype 110/serogroup X/REA DA1 strain had the same toxin genotype as VPI 10463 but did not produce any toxin A in vitro.
Concluding remarks The incidence of AB+ Clostridium difficile strains appears to be increasing worldwide. These strain types now represent a substantial number of C. difficile isolates. These isolates may be under-reported due to the widespread use of diagnostics that detect TcdA only. Although further studies are required to determine whether the pathogenesis of toxin variant CDAD (AB+) differs from that seen with toxin A+B+ strains, clinicians should be aware of the risk of severe disease associated with these variant strain types. TcdB is likely to play a more prominent role in the pathogenesis of C. difficile than previously considered, and future work should focus on the expression and regulation of this toxin as well as modulation of the host immune response. However the lack of suitable genetic tools to manipulate C. difficile and lack of isogenic mutants may hinder progress. New therapeutic approaches for CDAD such as toxin binders, passive immunotherapy or active immunization through vaccination will now need to target both TcdA and TcdB.
Acknowledgements The authors acknowledge the financial support provided by the National Institute on Aging, The Health Research Board, Ireland, The Mater Foundation and The Newman Scholarship Programme, University College Dublin. Conflict of interest: No conflict of interest to declare.
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