In silico, in vitro and in vivo analysis of putative virulence factors identified in large clostridial toxin-negative, binary toxin- producing C. difficile strains.

The relevance of large clostridial toxin-negative, binary toxin-producing (A-B-CDT+) Clostridium difficile strains in human infection is still controversial. In this study, we investigated putative virulence traits that may contribute to the role of A-B-CDT+C. difficile strains in idiopathic diarrhea. Phenotypic assays were conducted on 148 strains of C. difficile comprising 10 different A-B-CDT+C. difficile ribotypes (RTs): 033, 238, 239, 288, 585, 586, QX143, QX444, QX521 and QX629. A subset of these isolates (n = 53) was whole-genome sequenced to identify genetic loci associated with virulence and survival. Motility studies showed that with the exception of RT 239 all RTs tested were non-motile. C. difficile RTs 033 and 288 had deletions in the F2 and F3 regions of their flagella operon while the F2 region was absent from strains of RTs 238, 585, 586, QX143, QX444, QX521 and QX629. The flagellin and flagella cap genes, fliC and fliD, respectively, involved in adherence and host colonization, were conserved in all strains, including reference strains. All A-B-CDT+C. difficile strains produced at least three extracellular enzymes (deoxyribonuclease, esterase and mucinase) indicating that these are important extracellular proteins. The toxicity of A-B-CDT+C. difficile strains in Vero cells was confirmed, however, pathogenicity was not demonstrated in a mouse model of disease. Despite successful colonization by most strains, there was no evidence of disease in mice. This study provides the first in-depth analysis of A-B-CDT+C. difficile strains and contributes to the current limited knowledge of these strains as a cause of C. difficile infection.

Epsilon toxin is essential for the virulence of Clostridium perfringens type D infection in sheep, goats, and mice.

Clostridium perfringens type D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX to C. perfringens type D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulent C. perfringens type D wild-type strain (WT), an isogenic ETX null mutant (etx mutant), and a strain where the etx mutation has been reversed (etx complemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenic etx mutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation of etx knockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis.

Enterotoxic Clostridia: Clostridioides difficile Infections.

Clostridioides difficile is a Gram-positive, anaerobic, spore forming pathogen of both humans and animals and is the most common identifiable infectious agent of nosocomial antibiotic-associated diarrhea. Infection can occur following the ingestion and germination of spores, often concurrently with a disruption to the gastrointestinal microbiota, with the resulting disease presenting as a spectrum, ranging from mild and self-limiting diarrhea to severe diarrhea that may progress to life-threating syndromes that include toxic megacolon and pseudomembranous colitis. Disease is induced through the activity of the C. difficile toxins TcdA and TcdB, both of which disrupt the Rho family of GTPases in host cells, causing cell rounding and death and leading to fluid loss and diarrhea. These toxins, despite their functional and structural similarity, do not contribute to disease equally. C. difficile infection (CDI) is made more complex by a high level of strain diversity and the emergence of epidemic strains, including ribotype 027-strains which induce more severe disease in patients. With the changing epidemiology of CDI, our understanding of C. difficile disease, diagnosis, and pathogenesis continues to evolve. This article provides an overview of the current diagnostic tests available for CDI, strain typing, the major toxins C. difficile produces and their mode of action, the host immune response to each toxin and during infection, animal models of disease, and the current treatment and prevention strategies for CDI.

Status of vaccine research and development for Clostridium difficile.

Clostridium difficile associated disease is fundamentally associated with dysbiosis of the gut microbiome as a consequence of antibiotic use. This is because this sporulating, obligate anaerobe germinates and proliferates rapidly in the dysbiotic gut, which is an indirect consequence of their use. During its growth, C. difficile produces two toxins, toxin A (TcdA) and toxin B (TcdB), which are responsible for the majority of clinical symptoms associated with the disease. Three parenterally delivered vaccines, based on detoxified or recombinant forms of these toxins, have undergone or are undergoing clinical trials. Each offers the opportunity to generate high titres of toxin neutralising antibodies. Whilst these data suggest these vaccines may reduce primary symptomatic disease, they do not in their current form reduce the capacity of the organism to persist and shed from the vaccinated host. The current progress of vaccine development is considered with advantages and limitations of each highlighted. In addition, several alternative approaches are described that seek to limit C. difficile germination, colonisation and persistence. It may yet prove that the most effective treatments to limit infection, disease and spread of the organism will require a combination of therapeutic approaches. The potential use and efficacy of these vaccines in low and middle income countries will be depend on the development of a cost effective vaccine and greater understanding of the distribution and extent of disease in these countries.

Clostridial Genetics: Genetic Manipulation of the Pathogenic Clostridia.

The past 10 years have been revolutionary for clostridial genetics. The rise of next-generation sequencing led to the availability of annotated whole-genome sequences of the important pathogenic clostridia: Clostridium perfringens, Clostridioides (Clostridium) difficile, and Clostridium botulinum, but also Paeniclostridium (Clostridium) sordellii and Clostridium tetani. These sequences were a prerequisite for the development of functional, sophisticated genetic tools for the pathogenic clostridia. A breakthrough came in the early 2000s with the development of TargeTron-based technologies specific for the clostridia, such as ClosTron, an insertional gene inactivation tool. The following years saw a plethora of new technologies being developed, mostly for C. difficile, but also for other members of the genus, including C. perfringens. A range of tools is now available, allowing researchers to precisely delete genes, change single nucleotides in the genome, complement deletions, integrate novel DNA into genomes, or overexpress genes. There are tools for forward genetics, including an inducible transposon mutagenesis system for C. difficile. As the latest addition to the tool kit, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies have also been adopted for the construction of single and multiple gene deletions in C. difficile. This article summarizes the key genetic technologies available to manipulate, study, and understand the pathogenic clostridia.

The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia.

Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.

Effect of an inactivated Parapoxvirus based immunomodulator (Baypamun) on post weaning diarrhoea syndrome and wasting pig syndrome of piglets.

Post weaning diarrhoea syndrome (PWDS) and wasting pig syndrome (WPS) are two entities of the pig industry induced by stress factors occurring at the early weaning and further complicated by opportunistic pathogens. In the present paper we examine--through one pilot and one final trial--whether prevention of these two syndromes can be achieved in the field by immunomodulation of the pig e.g., by improving its non-specific immunological response. In both trials two groups of early weaned piglets were submitted to the treatments. In the pilot trial the first group received no treatment and served as controls and the animals of the second group were injected twice with an inactivated Parapoxvirus based immunomodulator (Baypamun, Bayer, Germany). In the final trial the first group of piglets received no treatment and served as a control group. The animals of the second group were injected twice with a freeze-dried immunomodulator (Baypamun). Piglets were compared with regard to the severity of PWS, mortality, growth performance parameters, and for the presence of pathogens in their faeces. The results have shown that Baypamun treated piglets performed significantly better than negative control piglets in all the parameters examined. It was concluded that stimulation of the non-specific immunity of piglets may be helpful in preventing the appearance of PWDS and WPS in the field.

Experimental study of tendon healing early phase: is IGF-1 expression influenced by platelet rich plasma gel?

BACKGROUND: It is well established that growth factors play a critical role in the healing process of connective tissues. To our knowledge, there are no studies in literature concerning the influence of PRP on growth factors expression. HYPOTHESIS: The aim of this study was to assess the effect of a single application of platelet rich plasma (PRP) gel in a patellar tendon defect on the spatial and temporal expression of Insulin-like Growth Factor 1 (IGF-1) during tendon healing. MATERIALS AND METHODS: Twenty-four animals were randomized to receive PRP (PRPFast, Bioteck) in a gel form (PRP group) and 24 to serve as untreated controls (Control group). A defect of 3 mm x 10 mm was surgically created on the tendon under general anaesthetic and in the PRP group, PRP gel was applied to fill the tendon defect whereas no treatment was applied in the control group. Six animals (12 limbs) from each treatment-group were sacrificed after one, two, three and four weeks following treatment. Histological and immunohistochemical staining were performed. RESULTS: Histology revealed a faster healing process in the tendons of PRP group in comparison with the controls. In the first 2 weeks of healing, IGF-1 was found intracellularly in various type cells, whereas in the last 2 weeks of healing, IGF-1 was detected mainly in tenocytes. Both cytoplasmic and nuclear expressions were present, whereas the larger amounts of immunoexpression were localized in both epitenon and endotenon. A superior expression of IGF-1 was seen in PRP group compared with controls (p<0.0001) in both the epitenon and endotenon at each time point except at 4th week of healing where a superior expression of IGF-1 was shown in the endotenon of control group, compared to the PRP group (p<0.0001). CONCLUSION: PRP may improve tendon defect healing by overexpression of IGF-1. LEVEL OF EVIDENCE: Laboratory control animal study.

The use of autologous PRP gel as an aid in the management of acute trauma wounds.

Autologous platelet-rich plasma (PRP) gel is increasingly used in the treatment of a variety of soft and bony tissue defects, such as accelerating bone formation and in the management of chronic non-healing wounds. We performed this study to assess the benefits of using autologous PRP gel in the treatment of acute limb soft tissue wounds. 59 patients with acute wounds (open fractures, closed fractures with skin necrosis and friction burns) were randomised into two groups. Group A (32 patients) were treated with conventional dressings and Group B (27 patients) were managed with local application of PRP gel. Gustillo grade IIIb or IIIc open fractures were not included in this study, as these injuries required coverage with flap. The clinical endpoints were the healing rate and/or the time required to bring about adequate tissue regeneration in order to undergo reconstructive plastic surgery. The rate of wound healing rate was significantly faster in Group B at week 1, 2 and 3 (p=0.003, p<0.001 and p<0.001, respectively). The mean time to plastic reconstruction in Group B was 21.26 days, S.D.=1.35 vs 40.6 days in Group A, S.D.=5.27 (p<0.001). This study has shown that PRP gel treatment can be a valuable and effective aid in the management of acute trauma wounds.

Internal fixation of proximal humerus fractures using the Polarus intramedullary nail.

INTRODUCTION: It has been stated that proximal humeral fractures are the last unsolved fractures in orthopedics. In this study, we evaluate the results of the Polarus nail, in order to constitute to the controversial problem of the treatment of proximal humerus fractures. PATIENTS AND METHODS: Between 2003 and 2005, 27 patients with severely displaced proximal humeral fractures were treated with the Polarus intramedullary system. The fractures were classified according to the Neer classification system. There were 16 2-part fractures and 11 3-part fractures. At the 12-months follow up we graded the clinical outcome of the patients according to the Neer scoring system. RESULTS: The duration from time of injury to operation ranged from 1 to 6 days. The mean operative time was 55 min and no need of blood transfusion was recorded. Bony union was obtained in 27 patients. There were no cases of nonunion and the period from operation to the appearance of sufficient bridging callus on radiographs was 5-11 weeks (mean 6 weeks). In one patient, a backed-out proximal screw was recorded. Stiffness of the shoulder was seen in one patient and avascular necrosis of the head of humerus in another. Related to the clinical outcome, six patients had an excellent result, 15 patients had a satisfactory result, four patients had an unsatisfactory result, and two patients had a poor result. The 77.78% of the patients had an excellent or satisfactory clinical outcome. There was significant difference in the functional outcome between patients younger than 65 years (91.55) and those older than 65 years (80.22) (P < 0.05). No significant difference was recorded in the Neer score between the patients with a 2-part fracture and the patients with a 3-part fracture (80.18) (P > 0.05). CONCLUSION: The Polarus nail is designed to provide stable fixation with a straightforward insertion and targeting procedure. The fact that 77.78% of the patients had an excellent or satisfactory clinical outcome, suggest that Polarus humeral rod system can be a worthy alternative to traditional operative methods for displaced proximal fractures of the humerus.

Transposition of Tn4451 and Tn4453 involves a circular intermediate that forms a promoter for the large resolvase, TnpX.

Tn4451 is the paradigm element of a family of mobilizable chloramphenicol resistance transposons from Clostridium perfringens and Clostridium difficile. The unique feature of these 6.3 kb elements is that their excision to form a circular molecule is mediated by TnpX, a member of the large resolvase family of site-specific recombinases. By optimizing the transposition assay system in Escherichia coli, we showed that Tn4453a from C. difficile transposed at a higher frequency than the C. perfringens element, Tn4451, and that transposition of both Tn4451 and Tn4453a was significantly enhanced by the provision of a multicopy tnpX gene in trans. The complete nucleotide sequence of Tn4453a was determined, but its comparison with Tn4451 did not reveal why it transposed at a higher frequency. Using experiments involving a chromosomal derivative of Tn4453a, we have confirmed that the circular form is the transposition intermediate. As the tnpX gene is located very close to one end of these elements, primer extension analysis was used to determine the transcription start point. The results showed that the formation of the circular intermediate creates a strong tnpX promoter, which consists of a -10 box originally located at the left end of the transposon and a -35 box originally located at the right end. The data provide strong evidence that transcription of tnpX is likely to occur from the non-replicating circular intermediate, which would facilitate the subsequent insertion of the transient circular molecule. It is postulated that, when the transposon is in an integrated state, transcription of tnpX would depend on the presence of an appropriately spaced -35 sequence in the DNA flanking the insertion site or the presence of an alternative upstream promoter.

Genetic organization and distribution of tetracycline resistance determinants in Clostridium perfringens.

The Tet P determinant from the conjugative Clostridium perfringens R plasmid pCW3 two functional overlapping tetracycline resistance genes, tetA(P) and tetB(P). The tetA(P) gene encodes a putative 46-kDa transmembrane protein which mediates active efflux of tetracycline from the cell, while tetB(P) encodes a putative 72.6-kDa protein which has significant similarity to Tet M-like tetracycline resistance proteins (J. Sloan, L.M. McMurry, D. Lyras, S. B. Levy, and J. I. Rood, Mol. Microbiol. 11:403-415, 1994). In the present study, hybridization and PCR analysis of 81 tetracycline-resistant isolates of C. perfringens showed that they all carried the tetA(P) gene. Most of these isolates (93%) carried a second tetracycline resistance gene, with 53% carrying tetB(P) and 40% carrying a tet(M)-like gene. Despite the wide distribution of the tetB(P) and tet(M) genes, no isolate which carried both of these determinants was detected. In isolates that carried both tetA(P) and tetB(P) these genes overlapped, as in pCW3. Isolates carrying this combination of genes originated from diverse geographical locations and environmental sources. The single Clostridium paraputrificum isolate examined carried tetA(P), indicating that this gene is not confined to C.perfringens. However, neither tetA(P) nor tetB(P) was detected in the nine Clostridium difficile isolates tested. Nucleotide sequence analysis of isolates lacking tetB(P) revealed that they contained the tetA408(P) gene, which lacked the codons for the 12 carboxy-terminal amino acids of the TetA(P) protein.

Conjugative transfer of RP4-oriT shuttle vectors from Escherichia coli to Clostridium perfringens.

A chromosomally integrated copy of the IncP plasmid RP4 was shown to mediate the conjugative transfer of shuttle plasmids containing an oriT site from Escherichia coli to the anaerobic pathogen Clostridium perfringens. Two versatile shuttle plasmids, pJIR1456 and pJIR1457, which will be invaluable for the introduction of cloned genes into C. perfringens, were constructed.

The occurence of haemangioblastoma in three brothers of one family.

The occurence of haemangioblastomas in three brothers is reported. From a review of the literature it becomes evident that only a few of such cases have been confirmed histologically. The interesting features of our three haemangioblastoma cases, which have all been confirmed histologically, are discussed.

Induction of pCW3-encoded tetracycline resistance in Clostridium perfringens involves a host-encoded factor.

The tetracycline resistance determinant Tet P, which is encoded by the conjugative plasmid pCW3 from Clostridium perfringens, is induced by subinhibitory concentrations of tetracycline. In this study we have shown that the inducible phenotype is strain dependent. When pCW3 is present in derivatives of the wild-type strains CW234 and CW362 resistance is inducible. However, transfer to derivatives of strain 13 leads to a constitutive phenotype that is only observed in this strain background. Based on these results it is proposed that induction of the pCW3-encoded tet(P) genes in C. perfringens requires a host-encoded factor that is either absent or nonfunctional in strain 13 derivatives.

Characterization of a Tra 2 function of RP1 that affects growth of Pseudomonas aeruginosa PAO and surface exclusion in Escherichia coli K12.

pVS438, a clone of part of the Tra 2 region of RP1 in RSF1010, confers two unusual phenotypes: poor growth (Slo+) in Pseudomonas aeruginosa PAO and surface exclusion (Sfx+) in Escherichia coli K12. Both of these phenotypes were found to be encoded by a 1.8-kb fragment of RP1 (from 25.9-27.7 kb) that spans the traB gene. However, whether both phenotypes, neither, or only Slo+ is expressed by this fragment depends on its location and orientation in RSF1010. In pVS438, where this fragment occurs in the SmR locus of RSF1010, expression of the Sfx+ phenotype is due to augmented transcription from the two promoters that cotranscribe the SuRSmR genes. When augmentation is abolished by insertion of Tn5 between these promoters and the cloned fragment, or by insertion of the fragment elsewhere in RSF1010, a Slo+Sfx- phenotype results. DNA that confers only the Slo+ phenotype was mapped to the 26.2-26.8 kb region of RP1 between traE and traB and the designation, traS, given to the gene responsible. Despite the recognition of a traS+ (Slo+) component of DNA within that encoding the Slo+ and Sfx+ phenotypes, this gene seems nevertheless to be responsible for the Sfx+ phenotype since hydroxylamine-induced Slo- mutants of pVS438 are usually also Sfx-. These apparently conflicting observations and the precise interplay between the Slo+, Sfx+, and TraB+ phenotypes were not resolved. Finally, traS is not essential for plasmid transfer since pVS438 and a Slo-Sfx- derivative of it can both equally complement an RP1tra-deletion mutant of part of the Tra 2 region.

The macrolide-lincosamide-streptogramin B resistance determinant from Clostridium difficile 630 contains two erm(B) genes.

The ErmB macrolide-lincosamide-streptogramin B (MLS) resistance determinant from Clostridium difficile 630 contains two copies of an erm(B) gene, separated by a 1.34-kb direct repeat also found in an Erm(B) determinant from Clostridium perfringens. In addition, both erm(B) genes are flanked by variants of the direct repeat sequence. This genetic arrangement is novel for an ErmB MLS resistance determinant.

The surface exclusion system of RP1: investigation of the roles of trbJ and trbK in the surface exclusion, transfer, and slow-growth phenotypes.

The contiguous trbJ and trbK genes of RP1 were cloned individually to study their effects. Surface exclusion was conferred only by trbK and only when gene dosage was high or when trbJ was also present in cis or in trans. This suggests that in the low-copy-number RP1, surface exclusion is due to a two-gene interaction in which trbK is the dominant partner. Among surface exclusion genes, trbJ is novel in yielding a periplasmic product that is also essential for conjugal transfer. This cellular location and the disturbed membrane function that accompanies TrbJ-processing probably accounts for the retarded growth caused by trbJ+ clones in Pseudomonas aeruginosa strain PAO.

Transcriptional analysis of the tet(P) operon from Clostridium perfringens.

The Clostridium perfringens tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes, tetA(P) and tetB(P), which mediate resistance by different mechanisms. Detailed transcriptional analysis has shown that the inducible tetA(P) and tetB(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the tetA(P) start codon. Deletion of P3 or alteration of the spacing between the -35 and -10 regions significantly reduced the level of transcription in a reporter construct. Induction was shown to be mediated at the level of transcription. Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of the tetA(P) gene. Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct. It is postulated that the T1 terminator is an intrinsic control element of the tet(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline.

The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline-resistance determinants.

The complete nucleotide sequence and mechanism of action of the tetracycline-resistance determinant, Tet P, from Clostridium perfringens has been determined. Analysis of the 4.4 kb of sequence data revealed the presence of two open reading frames, designated as tetA(P) and tetB(P). The tetA(P) gene appears to encode a 420 amino acid protein (molecular weight 46,079) with twelve transmembrane domains. This gene was shown to be responsible for the active efflux of tetracycline from resistant cells. Although there was some amino acid sequence similarity between the putative TetA(P) protein and other tetracycline efflux proteins, analysis suggested that TetA(P) represented a different type of efflux protein. The tetB(P) gene would encode a putative 652 amino acid protein (molecular weight 72,639) with significant sequence similarity to Tet(M)-like cytoplasmic proteins that specify a ribosomal-protection tetracycline-resistance mechanism. In both C. perfringens and Escherichia coli, tetB(P) encoded low-level resistance to tetracycline and minocycline whereas tetA(P) only conferred tetracycline resistance. The tetA(P) and tetB(P) genes appeared to be linked in an operon, which represented a novel genetic arrangement for tetracycline-resistance determinants. It is proposed that tetB(P) evolved from the conjugative transfer into C. perfringens of a tet(M)-like gene from another bacterium.