Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Genomic organization and molecular characterization of SM1, a temperate bacteriophage of Streptococcus mitis

The direct binding of Streptococcus mitis to human platelets is mediated in part by two proteins (PblA and PblB) encoded by a lysogenic bacteriophage (SM1). Since SM1 is the first prophage of S. mitis that has been identified and because of the possible role of these phage-encoded proteins in virulence, we sought to characterize SM1 in greater detail. Sequencing of the SM1 genome revealed that it consisted of 34,692 bp, with an overall G+C content of 39 mol%. Fifty-six genes encoding proteins of 40 or more amino acids were identified. The genes of SM1 appear to be arranged in a modular, life cycle-specific organization. BLAST analysis also revealed that the proteins of SM1 have homologies to proteins from a wide variety of lambdoid phages. Bioinformatic analyses, in addition to N-terminal sequencing of the proteins, led to the assignment of possible functions to a number of proteins, including the integrase, the terminase, and two major structural proteins. Examination of the phage structural components indicates that the phage head may assemble using stable multimers of the major capsid protein, in a process similar to that of phage r1t. These findings indicate that SM1 may be part of a discrete subfamily of the Siphoviridae that includes at least phages r1t of Lactococcus lactis and SF370.3 of Streptococcus pyogenes.     

Molecular characterization of a Clostridium difficile bacteriophage and its cloned biologically active endolysin

Clostridium difficile infection is increasing in both frequency and severity, with the emergence of new highly virulent strains highlighting the need for more rapid and effective methods of control. Here, we show that bacteriophage endolysin can be used to inhibit and kill C. difficile. The genome sequence of a novel bacteriophage that is active against C. difficile was determined, and the bacteriophage endolysin gene was subcloned and expressed in Escherichia coli. The partially purified endolysin was active against 30 diverse strains of C. difficile, and importantly, this group included strains of the major epidemic ribotype 027 (B1/NAP1). In contrast, a range of commensal species that inhabit the gastrointestinal tract, including several representatives of the clostridium-like Firmicutes, were insensitive to the endolysin. This endolysin provides a platform for the generation of both therapeutic and detection systems to combat the C. difficile problem. To investigate a method for the protected delivery and production of the lysin in the gastrointestinal tract, we demonstrated the expression of active CD27L endolysin in the lactic acid bacterium Lactococcus lactis MG1363.     

Structural insights into the molecular organization of the S-layer from Clostridium difficile

Clostridium difficile expresses a surface layer (S-layer) which coats the surface of the bacterium and acts as an adhesin facilitating interaction of the bacterium with host enteric cells. The S-layer contains a high-molecular-weight S-layer protein (HMW SLP) and its low-molecular-weight partner protein (LMW SLP). We show that these proteins form a tightly associated non-covalent complex, the H/L complex, and we identify the regions of both proteins responsible for complex formation. The 2.4 Å X-ray crystal structure of a truncated derivative of the LMW SLP reveals two domains. Domain 1 has a two-layer sandwich architecture while domain 2, predicted to orientate towards the external environment, contains a novel fold. Small-angle X-ray scattering analysis of the H/L complex shows an elongated molecule, with the two SLPs arranged 'end-to-end' interacting with each other through a small contact area. Alignment of LMW SLPs, which exhibit high sequence diversity, reveals a core of conserved residues that could reflect functional conservation, while allowing for immune evasion through sequence variation. These structures are the first described for the S-layer of a bacterial pathogen, and provide insights into the assembly and biogenesis of the S-layer.     

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

ABSTRACT: BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status. RESULTS: Toxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, beta2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.     

Laboratory diagnosis of Clostridium difficile associated diarrhoea and molecular characterization of clinical isolates

We evaluated a three-step algorithm for laboratory diagnosis of Clostridium difficile-associated diarrhoea (CDAD). First, stool specimens were screened using an EIA test for glutamate dehydrogenase detection. Screen-positive specimens were tested by a rapid cytotoxintoxin A/B assay and subjected to stool culture. All cultures positive for C. difficile underwent toxigenic culture. The results showed that toxigenic culture allowed us to recover 37/156 (24.4%) stool samples harbouring toxigenic C. difficile that would have been missed by using faecal cytotoxin assay alone. This determined an increase in infection prevalence of 4.2% (from 11.4% to 15.6 %). Furthermore, to characterize the clinical Clostridium difficile isolates and the distribution of PCR ribotypes circulating in the San Carlo Borromeo hospital, molecular typing using semi-automated repetitive-sequence-based PCR (rep- PCR) and PCR ribotyping, and an evaluation of the antibiotic resistance were also performed. Among them, 71 indistinguishable strains were detected by rep-PCR and 83 by PCR-ribotyping revealing C. difficile outbreaks in our hospital. A total of 6 different ribotypes were obtained by PCR ribotyping. The most frequent ribotype was 018 (88.2%) that also showed resistance to moxifloxacin. In one case, uncommon PCR ribotype 186 was also identified.     

Genomic organization and molecular analysis of virulent bacteriophage 2972 infecting an exopolysaccharide-producing Streptococcus thermophilus strain

The Streptococcus thermophilus virulent pac-type phage 2972 was isolated from a yogurt made in France in 1999. It is a representative of several phages that have emerged with the industrial use of the exopolysaccharide-producing S. thermophilus strain RD534. The genome of phage 2972 has 34,704 bp with an overall G+C content of 40.15%, making it the shortest S. thermophilus phage genome analyzed so far. Forty-four open reading frames (ORFs) encoding putative proteins of 40 or more amino acids were identified, and bioinformatic analyses led to the assignment of putative functions to 23 ORFs. Comparative genomic analysis of phage 2972 with the six other sequenced S. thermophilus phage genomes confirmed that the replication module is conserved and that cos- and pac-type phages have distinct structural and packaging genes. Two group I introns were identified in the genome of 2972. They interrupted the genes coding for the putative endolysin and the terminase large subunit. Phage mRNA splicing was demonstrated for both introns, and the secondary structures were predicted. Eight structural proteins were also identified by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Detailed analysis of the putative minor tail proteins ORF19 and ORF21 as well as the putative receptor-binding protein ORF20 showed the following interesting features: (i) ORF19 is a hybrid protein, because it displays significant identity with both pac- and cos-type phages; (ii) ORF20 is unique; and (iii) a protein similar to ORF21 of 2972 was also found in the structure of the cos-type phage DT1, indicating that this structural protein is present in both S. thermophilus phage groups. The implications of these findings for phage classification are discussed.     

Genomic Relatedness of Clostridium difficile strains from different toxinotypes and serogroups

Clostridium difficile strains can be divided into sixteen toxinotypes (0 and I to XV) according to changes in their toxin genes. To determine the genomic similarity between toxinotypes, two molecular typing techniques were used, AP-PCR and PFGE. Strains were selected from five serogroups (A1, A15, E, F, X) and represented non-toxinogenic isolates, strains with toxin genes identical to the reference C. difficile strain, VPI 10463 (toxinotype 0), and strains with variant toxin genes from toxinotypes III, IV, V, VI, VII, VIII, IX, and XI. The strains studied formed three main clusters, which correlated well with serogroups: in the first were strains from serogroup A15 and E; in the second, serogroup A1 strains; and in the third, strains from serogroups F and X. Within these three clusters strains of a single toxinotype were grouped together. Toxinotypes III, IV and VIII were more similar to strains with ordinary toxin genes or non-toxinogenic isolates within the same serogroup than to other toxinotypes. Toxinotypes V, VI, VII, and XI, which exhibit similar changes in their toxin genes, seem to be more closely related one to another than to other toxinotypes. It can be concluded that variant Clostridium difficile strains do not have a common ancestor and that groups of different toxinotypes arose independently from strains with ordinary toxin genes.     

Proteomic and Genomic Characterization of Highly Infectious Clostridium difficile 630 Spores

Clostridium difficile, a major cause of antibiotic-associated diarrhea, produces highly resistant spores that contaminate hospital environments and facilitate efficient disease transmission. We purified C. difficile spores using a novel method and show that they exhibit significant resistance to harsh physical or chemical treatments and are also highly infectious, with <7 environmental spores per cm² reproducibly establishing a persistent infection in exposed mice. Mass spectrometric analysis identified ∼336 spore-associated polypeptides, with a significant proportion linked to translation, sporulation/germination, and protein stabilization/degradation. In addition, proteins from several distinct metabolic pathways associated with energy production were identified. Comparison of the C. difficile spore proteome to those of other clostridial species defined 88 proteins as the clostridial spore “core” and 29 proteins as C. difficile spore specific, including proteins that could contribute to spore-host interactions. Thus, our results provide the first molecular definition of C. difficile spores, opening up new opportunities for the development of diagnostic and therapeutic approaches.Clostridium difficile is a gram-positive, spore-forming, anaerobic bacterium that can asymptomatically colonize the intestinal tracts of humans and other mammals (3, 30, 39). Antibiotic treatment can result in C. difficile overgrowth and can lead to clinical disease, ranging from diarrhea to life-threatening pseudomembranous colitis, particularly in immunocompromised hosts (2, 4, 7). In recent years, C. difficile has emerged as the major cause of nosocomial antibiotic-induced diarrhea, and it is frequently associated with outbreaks (21, 22). A contributing factor is that C. difficile can be highly infectious and difficult to contain, especially when susceptible patients are present in the same hospital setting (13).Person-to-person transmission of C. difficile is associated with the excretion of highly resistant spores in the feces of infected patients, creating an environmental reservoir that can confound many infection control measures (29, 44). Bacterial spores, which are metabolically dormant cells that are formed following asymmetric cell division, normally have thick concentric external layers, the spore coat and cortex, that protect the internal cytoplasm (15, 42). Upon germination, spores lose their protective external layers and resume vegetative growth (24, 27, 36). Bacillus spores and the spores of most Clostridium species germinate in response to amino acids, carbohydrates, or potassium ions (24, 36). In contrast, C. difficile spores show an increased level of germination in response to cholate derivatives found in bile (40, 41). Thus, spores are well adapted for survival and dispersal under a wide range of environmental conditions but will germinate in the presence of specific molecular signals (24, 36).While the spores of a number of Bacillus species, such as Bacillus subtilis and Bacillus anthracis, and those of other Clostridium species, such as Clostridium perfringens (15, 20), have been well characterized, research on C. difficile spores has been relatively limited. A greater understanding of C. difficile spore biology could be exploited to rationalize disinfection regimes, molecular diagnostics, and the development of targeted treatments such as vaccines. Here we describe a novel method to isolate highly purified C. difficile spores that maintain their resistance and infectious characteristics, thus providing a unique opportunity to study C. difficile spores in the absence of vegetative cells. A thorough proteomic and genomic analysis of the spore provides novel insight into the unique composition and predictive biological properties of C. difficile spores that should underpin future research into this high-profile but poorly understood pathogen.     

Isolation and characterization of temperate bacteriophages of Clostridium difficile

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages phiC2, phiC5, phiC6, and phiC8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of phiC2 showed nucleotide homology to the sequenced C. difficile strain CD630.     

Detection and characterization of Clostridium difficile in retail chicken

Aims: This study was designed to evaluate the prevalence of Clostridium difficile contamination of retail chicken. Methods and Results: Chicken legs, thighs and wings were purchased using a standardized method from retail outlets across Ontario, Canada. Selective culture was used for qualitative and quantitative detection of C. difficile. Clostridium difficile was isolated from 26/203 (12·8%) chicken samples; 10/111 (9·0%) thighs, 13/72 (18%) wings and 3/20 (15%) legs (P = 0·19). All isolates were ribotype 078, a strain that has been associated with food animals and potentially community‐associated disease in humans. All positive samples were positive only on enrichment culture. Conclusions: Clostridium difficile could be found relatively commonly in retail chicken meat, albeit at low levels. Significance and Impact of the Study: This is the first study to report C. difficile in chicken meat. Contamination of meat with C. difficile strains implicated in human infections raises concerns about food as a source of C. difficile infection. The relevance of food contamination is completely unclear at this point but food should be investigated as a source of infection.     

Molecular characterization and antimicrobial susceptibilities of extra-intestinal Clostridium difficile isolates

Amongst 25 extra-intestinal clinical isolates of Clostridium difficile, A(+)B(+) (72%) and A(-)B(+) (4%) toxigenic phenotypes, as well as the non-toxigenic phenotype (A(-)B(-)) (24%), were identified. The A(-)B(-) isolates did not express toxin, yet carried part of the tcdA and tcdB gene and are of a previously unreported toxinotype. Six A(+)B(+) isolates also carried binary toxin genes. Resistance to erythromycin (20%), clindamycin (48%), tetracycline (16%), moxifloxacin (16%) and imipenem (11%) occurred but with no apparent correlation to phenotype. None of the strains was resistant to vancomycin or metronidazole. Imipenem-resistance decreased by EDTA, but susceptibility to meropenem suggests the presence of an imipenem specific metalloenzyme.     

Antibiotics and Clostridium difficile

Clostridium difficile is now established as a major nosocomial pathogen. C. difficile infection is seen almost exclusively as a complication of antibiotic therapy, and is particularly associated with clindamycin and third-generation cephalosporins. Depletion of the indigenous gut microflora by antibiotic therapy has long been established as a major factor in the disease. However, the direct influence of antimicrobials upon virulence mechanisms such as toxin production and adhesion in the bowel, and the exact mechanisms by which the organism causes disease remain to be elucidated.     

Genomic organization of plant terpene synthases and molecular evolutionary implications

Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.     

Genomic diversity and organization of virulence genes in the pathogenic anaerobe Clostridium perfringens

Pulsed-field gel electrophoresis has been used to assess genomic diversity and to identify virulence regions in 10 strains, representing all five serotypes, of the anaerobic pathogen Clostridium perfringens. Detailed physical and gene maps of the approximately 3.6 Mb circular chromosomes have been established in eight cases and used to deduce a consensus map. With one exception the chromosomal arrangement was relatively constant and map comparison allowed three hypervariable regions to be identified. One of these was associated with the enterotoxin gene, cpe, which is an important cause of human diarrhoea following the ingestion of food contaminated with C. perfringens. Another variable region spanning the major virulence gene plc, which encodes the cytolytic toxin, alpha, was located near oriC in all cases whereas the gene for another lethal typing toxin, epsilon, was borne by an episome. It now seems likely that the serological variations, and the changes in the pathogenic spectrum which constitute the C. perfringens typing system, may be due entirely to the loss, or acquisition, of extrachromosomal genetic elements.     

Purification and characterization of S layer proteins from Clostridium difficile GAI 0714

The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.