A genomic survey of Clostridioides difficile isolates from hospitalized patients in Melbourne, Australia.

IMPORTANCE: There has been a decrease in healthcare-associated Clostridioides difficile infection in Australia, but an increase in the genetic diversity of infecting strains, and an increase in community-associated cases. Here, we studied the genetic relatedness of C. difficile isolated from patients at a major hospital in Melbourne, Australia. Diverse ribotypes were detected, including those associated with community and environmental sources. Some types of isolates were more likely to carry antimicrobial resistance determinants, and many of these were associated with mobile genetic elements. These results correlate with those of other recent investigations, supporting the observed increase in genetic diversity and prevalence of community-associated C. difficile, and consequently the importance of sources of transmission other than symptomatic patients. Thus, they reinforce the importance of surveillance for in both hospital and community settings, including asymptomatic carriage, food, animals, and other environmental sources to identify and circumvent important sources of C. difficile transmission.

In silico Identification of Novel Toxin Homologs and Associated Mobile Genetic Elements in Clostridium perfringens.

Clostridium perfringens causes a wide range of diseases in a variety of hosts, due to the production of a diverse set of toxins and extracellular enzymes. The C. perfringens toxins play an important role in pathogenesis, such that the presence and absence of the toxins is used as a typing scheme for the species. In recent years, several new toxins have been discovered that have been shown to be essential or highly correlated to diseases; these include binary enterotoxin (BecAB), NetB and NetF. In the current study, genome sequence analysis of C. perfringens isolates from diverse sources revealed several putative novel toxin homologs, some of which appeared to be associated with potential mobile genetic elements, including transposons and plasmids. Four novel toxin homologs encoding proteins related to the pore-forming Leukocidin/Hemolysin family were found in type A and G isolates. Two novel toxin homologs encoding proteins related to the epsilon aerolysin-like toxin family were identified in Type A and F isolates from humans, contaminated food and turkeys. A novel set of proteins related to clostridial binary toxins was also identified. While phenotypic characterisation is required before any of these homologs can be established as functional toxins, the in silico identification of these novel homologs on mobile genetic elements suggests the potential toxin reservoir of C. perfringens may be much larger than previously thought.

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens.

BACKGROUND: Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome. RESULTS: Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis. CONCLUSIONS: The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.

Conjugation-Mediated Horizontal Gene Transfer of Clostridium perfringens Plasmids in the Chicken Gastrointestinal Tract Results in the Formation of New Virulent Strains.

Clostridium perfringens is a gastrointestinal pathogen capable of causing disease in a variety of hosts. Necrotic enteritis in chickens is caused by C. perfringens strains that produce the pore-forming toxin NetB, the major virulence factor for this disease. Like many other C. perfringens toxins and antibiotic resistance genes, NetB is encoded on a conjugative plasmid. Conjugative transfer of the netB-containing plasmid pJIR3535 has been demonstrated in vitro with a netB-null mutant. This study has investigated the effect of plasmid transfer on disease pathogenesis, with two genetically distinct transconjugants constructed under in vitro conditions, within the intestinal tract of chickens. This study also demonstrates that plasmid transfer can occur naturally in the host gut environment without the need for antibiotic selective pressure to be applied. The demonstration of plasmid transfer within the chicken host may have implications for the progression and pathogenesis of C. perfringens-mediated disease. Such horizontal gene transfer events are likely to be common in the clostridia and may be a key factor in strain evolution, both within animals and in the wider environment.IMPORTANCEClostridium perfringens is a major gastrointestinal pathogen of poultry. C. perfringens strains that express the NetB pore-forming toxin, which is encoded on a conjugative plasmid, cause necrotic enteritis. This study demonstrated that the conjugative transfer of the netB-containing plasmid to two different nonpathogenic strains converted them into disease-causing strains with disease-causing capability similar to that of the donor strain. Plasmid transfer of netB and antibiotic resistance was also demonstrated to occur within the gastrointestinal tract of chickens, with approximately 14% of the isolates recovered comprising three distinct, in vivo-derived, transconjugant types. The demonstration of in vivo plasmid transfer indicates the potential importance of strain plasticity and the contribution of plasmids to strain virulence.

Evidence that compatibility of closely related replicons in Clostridium perfringens depends on linkage to parMRC-like partitioning systems of different subfamilies.

Clostridium perfringens produces an extensive repertoire of toxins and extracellular enzymes, many of which are intimately involved in the progression of disease and are encoded by genes on conjugative plasmids. In addition, many C. perfringens strains can carry up to five of these conjugative toxin or antimicrobial resistance plasmids, each of which has a similar 35kb backbone. This conserved backbone includes the tcp conjugation locus and the central control region (CCR), which encodes genes involved in plasmid regulation, replication and partitioning, including a parMRC partitioning locus. Most conjugative plasmids in C. perfringens have a conserved replication protein, raising questions as to how multiple, closely related plasmids are maintained within a single strain. Bioinformatics analysis has highlighted the presence of at least 10 different parMRC partitioning system families (parMRCA-J) in these plasmids, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. No two plasmids that encode genes belonging to the same partitioning family have been observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility. In an attempt to validate the proposed parMRC incompatibility groups, genetically marked C. perfringens plasmids encoding identical parMRCC or parMRCD homologues or different combinations of parMRCA, parMRCC and parMRCD family homologues were introduced into a single strain via conjugation. The stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored over the course of two days in the absence of direct selection. The results showed that plasmids with identical parMRCC or parMRCD homologues were incompatible and could not coexist in the absence of external selection. By contrast, plasmids that encoded different parMRC homologues were compatible and could coexist in the same cell in the absence of selection, with the exception of strains housing parMRCC and parMRCD combinations, which showed a minor incompatibility phenotype. In conclusion, we have provided the first direct evidence of plasmid incompatibility in Clostridium spp. and have shown experimentally that the compatibility of conjugative C. perfringens plasmids correlates with the presence of parMRC-like partitioning systems of different phylogenetic subfamilies.

Genomic diversity of necrotic enteritis-associated strains of Clostridium perfringens: a review.

The investigation of genomic variation between Clostridium perfringens isolates from poultry has been an important tool to enhance our understanding of the genetic basis of strain pathogenicity and the epidemiology of virulent and avirulent strains within the context of necrotic enteritis (NE). The earliest studies used whole genome profiling techniques such as pulsed-field gel electrophoresis to differentiate isolates and determine their relative levels of relatedness. DNA sequencing has been used to investigate genetic variation in (a) individual genes, such as those encoding the alpha and NetB toxins; (b) panels of housekeeping genes for multi-locus sequence typing and (c) most recently whole genome sequencing to build a more complete picture of genomic differences between isolates. Conclusions drawn from these studies include: differential carriage of large conjugative plasmids accounts for a large proportion of inter-strain differences; plasmid-encoded genes are more highly conserved than chromosomal genes, perhaps indicating a relatively recent origin for the plasmids; isolates from NE-affected birds fall into three distinct sequence-based clades while non-pathogenic isolates from healthy birds tend to be more genomically diverse. Overall, the NE causing strains are closely related to C. perfringens isolates from other birds and other diseases whereas the non-pathogenic poultry strains are generally more remotely related to either the pathogenic strains or the strains from other birds. Genomic analysis has indicated that genes in addition to netB are associated with NE pathogenic isolates. Collectively, this work has resulted in a deeper understanding of the pathogenesis of this important poultry disease.

Disruption of the Gut Microbiome: Clostridium difficile Infection and the Threat of Antibiotic Resistance.

Clostridium difficile is well recognized as the leading cause of antibiotic-associated diarrhea, having a significant impact in both health-care and community settings. Central to predisposition to C. difficile infection is disruption of the gut microbiome by antibiotics. Being a Gram-positive anaerobe, C. difficile is intrinsically resistant to a number of antibiotics. Mobile elements encoding antibiotic resistance determinants have also been characterized in this pathogen. While resistance to antibiotics currently used to treat C. difficile infection has not yet been detected, it may be only a matter of time before this occurs, as has been seen with other bacterial pathogens. This review will discuss C. difficile disease pathogenesis, the impact of antibiotic use on inducing disease susceptibility, and the role of antibiotic resistance and mobile elements in C. difficile epidemiology.

Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen.

The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions.

Rho activation regulates CXCL12 chemokine stimulated actin rearrangement and restitution in model intestinal epithelia.

Chemokines are critical regulatory factors that direct migration, proliferation and maturation of receptor expressing target cells within gut mucosa. The aim of the present study was to define the cellular mechanisms whereby engagement of the essential chemokine CXCL12 to CXCR4 regulates restitutive epithelial cell migration. Non-transformed IEC-6 cells or polarized T84 epithelial monolayers were wounded and F-actin accumulation assessed using fluorescence microscopy and flow cytometry. Immunoblot analysis, pull-down assays, fluorescence microscopy and wound healing assays defined activation of Rho, Rho-kinase (ROCK), and myosin light chain (MLC) and the role for those Rho effectors in CXCL12-regulated epithelial restitution. CXCL12 increased RhoGTP and F-actin localization to the leading edge of wounded IEC-6 and T84 monolayers. CXCL12 congruently stimulated an increase in active MLC that was inhibited by blockade of ROCK and myosin light chain kinase and regulated epithelial migration. Our data in model intestinal epithelia suggest CXCR4 and CXCL12 may function as an autocrine and paracrine mucosal signaling network regulating the competency of the epithelial barrier to withstand injury and mediate repair following damage.

Flagellin-independent regulation of chemokine host defense in Campylobacter jejuni-infected intestinal epithelium.

Campylobacter jejuni is a leading cause of bacterial food-borne diarrheal disease throughout the world and the most frequent antecedent of autoimmune neuropathy Guillain-Barré syndrome. While infection is associated with immune memory, little is known regarding the role of the epithelium in targeting dendritic cells (DC) for initiating the appropriate adaptive immune response to C. jejuni. The objective of this study was to define the role for the intestinal epithelium in the induction of the adaptive immune response in C. jejuni infection by assessing the production of DC and T-cell chemoattractants. Human T84 epithelial cells were used as model intestinal epithelia. Infection of T84 cells with C. jejuni dose- and time-dependently up-regulated DC and T-cell chemokine gene transcription and secretion. Induction required live bacteria and was in the physiologically relevant direction for attraction of mucosal immunocytes. C. jejuni-activated NF-kappaB signaling was shown to be essential for proinflammatory chemokine secretion. Notably, C. jejuni secretion occurred independently of flagellin identification by Toll-like receptor 5. Secretion of a DC chemoattractant by differing clinical C. jejuni isolates suggested adherence/invasion were key virulence determinants of epithelial chemokine secretion. The regulated epithelial expression of DC and T-cell chemoattractants suggests a mechanism for the directed trafficking of immune cells required for the initiation of adaptive immunity in campylobacteriosis. Chemokine secretion occurs despite Campylobacter evasion of the flagellin pattern recognition receptor, suggesting that alternate host defense strategies limit disease pathogenesis.

CXCL12 activation of CXCR4 regulates mucosal host defense through stimulation of epithelial cell migration and promotion of intestinal barrier integrity.

Intestinal epithelial cell migration plays a key role in gastrointestinal mucosal barrier formation, enterocyte development, differentiation, turnover, wound healing, and adenocarcinoma metastasis. Chemokines, through engagement of their corresponding receptors, are potent mediators of directed cell migration and are critical in the establishment and regulation of innate and adaptive immune responses. The aim of this study was to define the role for the chemokine CXCL12 and its sole cognate receptor CXCR4 in regulating intestinal epithelial cell migration and to determine its impact on barrier integrity. CXCL12 stimulated the dose-dependent chemotactic migration of human T84 colonic epithelial cells. Epithelial cell migration was inhibited by CXCR4 neutralizing antibody, pertussis toxin, LY-294002, and PD-98059, thereby implicating Galpha(i), phosphatidylinositol 3-kinase (PI3-kinase), and the ERK1/2 MAP kinase pathways in CXCR4-specific signaling. CXCL12 was also shown to increase barrier integrity, as defined by transepithelial resistance and paracellular flux across differentiating T84 monolayers. To determine whether CXCL12 regulated epithelial restitution, we used the normal nontransformed intestinal epithelial cell-6 (IEC-6) wound healing model. By using RT-PCR, immunoblot analysis, and immunofluorescence microscopy, we first showed expression of both CXCR4 and its ligand by IEC-6 cells. We then demonstrated that CXCL12 activated comparable signaling mechanisms to stimulate epithelial migration in the absence of proliferation in wounded IEC-6 monolayers. Taken together, these data indicate that CXCL12 signaling via CXCR4 directs intestinal epithelial cell migration, barrier maturation, and restitution, consistent with an important mechanistic role for these molecules in mucosal barrier integrity and innate host defense.

The Clostridium perfringens TetA(P) efflux protein contains a functional variant of the Motif A region found in major facilitator superfamily transport proteins.

The Clostridium perfringens tetracycline resistance protein, TetA(P), is an inner-membrane protein that mediates the active efflux of tetracycline from the bacterial cell. This protein comprises 420 aa and is predicted to have 12 transmembrane domains (TMDs). Comparison of the TetA(P) amino acid sequence to that of several members of the major facilitator superfamily (MFS) identified a variant copy of the conserved Motif A. This region consists of the sequence E59xPxxxxxDxxxRK72 and is located within the putative loop joining TMDs 2 and 3 in the predicted structural model of the TetA(P) protein. To study the functional importance of the conserved residues, site-directed mutagenesis was used to construct 17 point mutations that were then analysed for their effect on tetracycline resistance and their ability to produce an immunoreactive TetA(P) protein. Changes to the conserved Phe-58 residue were tolerated, whereas three independent substitutions of Pro-61 abolished tetracycline resistance. Examination of the basic residues showed that Arg-71 is required for function, whereas tetracycline resistance was retained when Lys-72 was substituted with arginine. These results confirm that the region encoding this motif is important for tetracycline resistance and represents a distant version of the Motif A region found in other efflux proteins and members of the MFS family. In addition, it was shown that Glu-117 of the TetA(P) protein, which is predicted to be located in TMD4, is important for resistance although a derivative with an aspartate residue at this position is also functional.

Comparison of Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis and the CW459tet(M) element from Clostridium perfringens shows that they have similar conjugation regions but different insertion and excision modules.

Comparative analysis of the conjugative transposons Tn5397 from Clostridium difficile and Tn916 from Enterococcus faecalis, and the CW459tet(M) element from Clostridium perfringens, has revealed that these tetracycline-resistance elements are closely related. All three elements contain the tet(M) resistance gene and have sequence similarity throughout their central region. However, they have very different integration/excision modules. Instead of the int and xis genes that are found in Tn916, Tn5397 has a large resolvase gene, tndX. The C. perfringens element encodes the putative Int459 protein, which is a member of the integrase family of site-specific recombinases but is not closely related to Int from Tn916. Based on these studies it is concluded that the clostridial elements have a modular genetic organization and were derived independently from distinct mobile genetic elements.