A molecular characterization of<i>Clostridium difficile</i>isolates from humans, animals and their environments

O'Neill, G.L.; Adams, J. E.; Bowman, R.A.; Riley, Thomas V.

doi:10.1017/s095026880005696x

Cited by 45 publications

(31 citation statements)

References 27 publications

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“…It has been speculated that domestic animals could constitute a reservoir of C. difficile isolates and a potential source for human acquisition (O'Neill et al, 1993). Since the present multilocus analysis of virulenceassociated genes, MLST analysis of housekeeping genes (Lemée et al, 2004a) and PCR ribotyping (Arroyo et al, 2005) have not characterized any host specificity, we can also presume that animal isolates could constitute a source for human community infections.…”

Section: Multilocus Sequence Data Analysis Of Virulenceassociated Genesmentioning

confidence: 88%

Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile

et al. 2005

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A multilocus sequence analysis of ten virulence-associated genes was performed to study the genetic relationships between 29 Clostridium difficile isolates of various origins, hosts and clinical presentations, and selected from the main lineages previously defined by multilocus sequence typing (MLST) of housekeeping genes. Colonization-factor-encoding genes (cwp66, cwp84, fbp68, fliC, fliD, groEL and slpA), toxin A and B genes (tcdA and tcdB), and the toxin A and B positive regulator gene (tcdD) were investigated. Binary toxin genes (cdtA and cdtB) were also detected, and internal fragments were sequenced for positive isolates. Virulence-associated genes exhibited a moderate polymorphism, comparable to the polymorphism of housekeeping genes, whereas cwp66 and slpA genes appeared highly polymorphic. Isolates recovered from human pseudomembranous colitis cases did not define a specific lineage. The presence of binary toxin genes, detected in five of the 29 isolates (17 %), was also not linked to clinical presentation. Conversely, toxigenic A"B+ isolates defined a very homogeneous lineage, which is distantly related to other isolates. By clustering analysis, animal isolates were intermixed with human isolates. Multilocus sequence analysis of virulence-associated genes is consistent with a clonal population structure for C. difficile and with the lack of host specificity. The data suggest a co-evolution of several of the virulence-associated genes studied (including toxins A and B and the binary toxin genes) with housekeeping genes, reflecting the genetic background of C. difficile, whereas flagellin, cwp66 and slpA genes may undergo recombination events and/or environmental selective pressure. INTRODUCTIONSince its recognition as the main cause of pseudomembranous colitis (PMC) in 1978, Clostridium difficile has emerged as an important enteropathogen (Bartlett et al., 1978;Johnson & Gerding, 1998;Larson et al., 1978). C. difficile is currently responsible for virtually all cases of PMC and for 10-25 % of antibiotic-associated diarrhoea (AAD) (Bartlett, 1994;Kelly et al., 1994). It is also the leading cause of nosocomial diarrhoea, and many hospital outbreaks have been described throughout the world (Barbut et al., 1994; Cartmill et al., 1994; McEllistrem et al., 2005). The two main virulence factors are exotoxins, toxins A (enterotoxin) and B (cytotoxin), both of which damage the human colonic mucosa and are potent tissue-damaging enzymes (Bongaerts & Lyerly, 1994;Borriello, 1998). Until recently, it was presumed that a strain must produce both toxins to be considered fully pathogenic. However, it is still unclear (i) why strains that do not produce toxin A (A2B+ variant strains) are yet responsible for diarrhoea and even PMC (Alfa et al., 2000;Toyokawa et al., 2003), and (ii) why some patients infected by toxinogenic strains will present a mild diarrhoea, whereas others will present a fulminant PMC.Abbreviations: AAD, antibiotic-associated diarrhoea; MLST, multilocus sequence typing; PaLoc, pathogenicity locus; P...

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Section: Multilocus Sequence Data Analysis Of Virulenceassociated Genesmentioning

confidence: 88%

Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile

et al. 2005

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“…Although C. diffi cile is recognized as a cause of disease in several animal species (19)(20)(21)(22), little investigation has been conducted on the potential for interspecies transmission of C. diffi cile to humans. Previous studies have suggested the possibility of C. diffi cile transmission between humans and domestic pets (23,24), but no interspecies transmission has been documented, and few studies have examined the possible link between CDAD in food animals and humans. Identifi cation of the same variant toxinotype strain as responsible for both human and animal disease in our study suggests at least 3 possible causes for human toxinotype V CDAD: 1) exposure of humans and animals to a common environmental source of C. diffi cile, 2) human disease caused by transmission by means of direct or indirect (e.g., through contaminated produce, water, or the environment) contact with infected live animals, and 3) human disease linked to consumption of products from food-producing animals.…”

Section: Discussionmentioning

confidence: 99%

Toxinotype VClostridium difficilein Humans and Food Animals

Jhung

Thompson

Killgore

et al. 2008

Emerg. Infect. Dis.

197

155

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Clostridium diffi cile is a recognized pathogen in neonatal pigs and may contribute to enteritis in calves. Toxinotype V strains have been rare causes of human C. diffi cileassociated disease (CDAD). We examined toxinotype V in human disease, the genetic relationship of animal and human toxinotype V strains, and in vitro toxin production of these strains. From 2001 through 2006, 8 (1.3%) of 620 patient isolates were identifi ed as toxinotype V; before 2001, 7 (<0.02%) of ≈6,000 isolates were identifi ed as toxinotype V. Six (46.2%) of 13 case-patients for whom information was available had community-associated CDAD. Molecular characterization showed a high degree of similarity between human and animal toxinotype V isolates; all contained a 39-bp tcdC deletion and most produced binary toxin. Further study is needed to understand the epidemiology of CDAD caused by toxinotype V C. diffi cile, including the potential of foodborne transmission to humans. R ecent evidence suggests that the epidemiology of Clostridium diffi cile-associated disease (CDAD) is increasing in incidence and severity (1-3). These changes are due, at least in part, to the emergence of a more virulent C. diffi cile strain, designated NAP1 (based on its pulsed-fi eld gel electrophoresis [PFGE] pattern), BI (by restriction endonuclease analysis [REA]), toxinotype III (by PCR characterization of the pathogenicity locus), and 027 (by PCR ribotyping) (4). However, the emergence of BI/NAP1/027 may not be solely responsible for changes in CDAD epidemiology, and the origin of this and other virulent strains is still largely unknown. C. diffi cile has also recently emerged as a pathogen or commensal in food animals such as neonatal pigs and beef and dairy calves (5-7); most of these animal isolates are toxigenic. Although several ribotypes have been identifi ed in calves, the predominant ribotype in both calves and pigs is a toxinotype V strain (8,9). Moreover, recent reports suggest that C. diffi cile strains recognized as causes of human disease may contaminate retail meats (10).To better understand whether food sources could be a source of infection for humans, we investigated recent and past human CDAD caused by toxinotype V C. diffi cile and compared isolates from these cases with those recovered from neonatal pigs and calves. We documented apparent changes in the frequency with which these toxinotype V strains cause human CDAD and compared the molecular characterization and toxin production of these strains with those of recent epidemic (i.e., BI/NAP1/027) and nonepidemic isolates. Materials and Methods Human Case Finding and Defi nitionsCase fi nding was performed by reviewing recent and past human isolates of interest from 2 sources. Cases were defi ned as patients with clinical isolates identifi ed as toxinotype V by analysis of restriction fragment length polymorphisms (RFLPs) of toxin-encoding genes. First, we reviewed 620 C. diffi cile human isolates sent to the Centers for Disease Control and Prevention (CDC) from healthcare facilities and ...

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“…A correlation between a primary antibiotic treatment and the animal disease was demonstrated, as for the human disease (6,40). However, few studies were interested in genetic relationships of human and animal C. difficile isolates (37,50). Restriction endonuclease analysis found no correlation between C. difficile isolates recovered from pets and human isolates (37), but the isolates studied were collected from different locations.…”

Section: Discussionmentioning

confidence: 99%

Multilocus Sequence Typing Analysis of Human and Animal Clostridium difficile Isolates of Various Toxigenic Types

et al. 2004

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A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 72 Clostridium difficile isolates from various hosts, geographic sources, PCR ribotypes, and toxigenic types (determined by PCR targeting tcdA and tcdB genes). MLST was performed by DNA sequence analysis of seven housekeeping genes (aroE, ddl, dutA, tpi, recA, gmk, and sodA). The number of alleles ranged from five (dutA and ddl) to eleven (recA). Allelic profiles allowed the definition of 34 different sequence types (STs). These STs lacked correlation with geographic source but were well correlated to toxigenic type. The dendrogram generated from a matrix of pairwise genetic distances showed that animal isolates did not constitute a distinct lineage from human isolates and that there was no hypervirulent lineage within the population of toxigenic human isolates (isolates recovered from pseudomembranous colitis and antibioticassociated diarrhea did not cluster in distinct lineages). However, A ؊ B ؉ variant isolates shared the same ST that appeared as a divergent lineage in the population studied, indicating a single evolutionary origin. The population structure was further examined by analysis of allelic polymorphism. The dendrogram generated from composite sequence-based analysis revealed a homogeneous population associated with three divergent lineages, one of which was restricted to A ؊ B ؉ variant isolates. C. difficile exhibited a clonal population structure, as revealed by the estimation of linkage disequilibrium (Ia) between loci. The analysis of alleles within clonal complexes estimated that point mutation generated new alleles at a frequency eightfold higher than recombinational exchange, and the congruence of the dendrograms generated from separate housekeeping loci confirmed the mutational evolution of this species.

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A molecular characterization ofClostridium difficileisolates from humans, animals and their environments

Cited by 45 publications

References 27 publications

Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile

Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile

Toxinotype VClostridium difficilein Humans and Food Animals

Multilocus Sequence Typing Analysis of Human and Animal Clostridium difficile Isolates of Various Toxigenic Types

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