Progressive ulcerative keratitis in dogs in the United Kingdom: Microbial isolates, antimicrobial sensitivity, and resistance patterns.

OBJECTIVES: The objective of the study was to identify bacterial pathogens and their antimicrobial sensitivity profile associated with cases of canine progressive ulcerative keratitis. MATERIALS AND METHODS: Analysis of microbial culture and sensitivity results from dogs with progressive ulcerative keratitis presenting to a UK referral practice between December 2018 and August 2020. RESULTS: Positive bacterial cultures were obtained from 80/148 (54%) of the canine ulcers sampled with 99 bacterial isolates cultured. Streptococcus canis (n = 29), Pseudomonas aeruginosa (n = 19), and Staphylococcus pseudintermedius (n = 16) were the most common isolates. Pseudomonas aeruginosa was more likely to be isolated whether the ulcer was clinically malacic at the time of sampling (OR = 10.1, p < .001). Ulcers treated prior to culture with fusidic acid were 7.6 times more likely to be positive than those treated with any other antimicrobial(s). Bacterial isolates demonstrated resistance against neomycin (85%), fusidic acid (78%), and tetracycline (68%). Conversely, isolates were most likely to be sensitive to gentamicin (88%), ofloxacin (77%), ciprofloxacin (73%), and chloramphenicol (64%). Antimicrobial combinations of chloramphenicol or gentamicin with a fluoroquinolone (ofloxacin or ciprofloxacin) or chloramphenicol combined with gentamicin were the most effective on in vitro analysis (over 90% susceptibility of all isolates). CONCLUSION: The most common bacterial species associated with canine progressive ulcerative keratitis in a UK referral population were S. canis, P. aeruginosa, and S. pseudintermedius. Combination antimicrobial therapy is recommended pending culture and sensitivity results given the varied antimicrobial susceptibility profiles and significant bacterial in vitro resistance to antimicrobial monotherapy.

An assessment of bacterial contamination of indirect ophthalmoscopes and condensing lenses used in clinical practice: A multi-center study.

PURPOSE: To investigate bacterial contamination of indirect ophthalmoscopes and condensing lenses used in three UK veterinary referral centers, and the impact of an implemented cleaning protocol. METHODS: Bacteriology samples from 10 indirect ophthalmoscopes and 10 condensing lenses were taken at each center (n = 30 T0), before initiating one of three cleaning frequencies (every 2 weeks/once weekly/daily) for 28 days. The most contaminated indirect ophthalmoscope and condensing lens from each center were re-sampled 30 min prior to (T1; n = 9) and 30 min after (T2; n = 9) the final clean. Sensitivity testing was completed using MIC. RESULTS: Seventy-three isolates representing 15 different bacterial populations (genus/species) were cultured from 36 of 48 (75%) swabs tested. The most frequently cultured isolates were Staphylococcus spp. 30%, Micrococcus 22%, and Bacillus 14%. Pseudomonas aeruginosa, Pantoea, and Staphylococcus pseudintermedius demonstrated resistance to >50% of antibiotics against which they were tested. Eighty-three percent of T0 samples (54 isolates across 11 species, median 2 isolates/swab), all T1 samples (15 isolates across 8 species, median 2 isolates/swab), and 22% of T2 samples (4 isolates across 4 species, median 0 isolates/swab) were contaminated. Head contact points were most contaminated irrespective of time point. A T1 sample was 57 times more likely (95% CI: 2.4-1376) to have a positive culture than a T2 sample (p = .01). CONCLUSIONS: Baseline contamination was high, representing a potential source of nosocomial infection in ophthalmic patients and handlers of diagnostic equipment. No center implemented a cleaning protocol prior to this study. Routine cleaning reduces bacterial contamination.

Experimental acute Clostridium perfringens type D enterotoxemia in sheep is not characterized by specific renal lesions.

Type D enterotoxemia, caused by Clostridium perfringens epsilon toxin (ETX), is one of the most economically important clostridial diseases of sheep. Acute type D enterotoxemia is characterized by well-documented lesions in the nervous, cardiocirculatory, and pulmonary systems. However, discrepancies and confusion exist as to whether renal lesions are part of the spectrum of lesions of this condition, which is controversial considering that for many decades it has been colloquially referred to as "pulpy kidney disease." Here, the authors assess renal changes in an experimental model of acute type D enterotoxemia in sheep and evaluate the possible role of ETX in their genesis. Four groups of 6 sheep each were intraduodenally inoculated with either a wild-type virulent C. perfringens type D strain, an etx knockout mutant unable to produce ETX, the etx mutant strain complemented with the wild-type etx gene that regains the ETX toxin production, or sterile culture medium (control group). All sheep were autopsied less than 24 hours after inoculation; none of them developed gross lesions in the kidneys. Ten predefined histologic renal changes were scored in each sheep. The proportion of sheep with microscopic changes and their severity scores did not differ significantly between groups. Mild intratubular medullary hemorrhage was observed in only 2 of the 12 sheep inoculated with the wild-type or etx-complemented bacterial strains, but not in the 12 sheep of the other 2 groups. The authors conclude that no specific gross or histologic renal lesions are observed in sheep with experimental acute type D enterotoxemia.

Extensive genome analysis identifies novel plasmid families in Clostridium perfringens.

Globally, the anaerobic bacterium Clostridium perfringens causes severe disease in a wide array of hosts; however, C. perfringens strains are also carried asymptomatically. Accessory genes are responsible for much of the observed phenotypic variation and virulence within this species, with toxins frequently encoded on conjugative plasmids and many isolates carrying up to 10 plasmids. Despite this unusual biology, current genomic analyses have largely excluded isolates from healthy hosts or environmental sources. Accessory genomes, including plasmids, also have often been excluded from broader scale phylogenetic investigations. Here we interrogate a comprehensive collection of 464 C. perfringens genomes and identify the first putative non-conjugative enterotoxin (CPE)-encoding plasmids and a putative novel conjugative locus (Bcp) with sequence similarity to a locus reported from Clostridium botulinum. We sequenced and archived 102 new C. perfringens genomes, including those from rarely sequenced toxinotype B, C, D and E isolates. Long-read sequencing of 11 C. perfringens strains representing all toxinotypes (A-G) identified 55 plasmids from nine distinct plasmid groups. Interrogation of the 464 genomes in this collection identified 1045 plasmid-like contigs from the nine plasmid families, with a wide distribution across the C. perfringens isolates. Plasmids and plasmid diversity play an essential role in C. perfringens pathogenicity and broader biology. We have expanded the C. perfringens genome collection to include temporal, spatial and phenotypically diverse isolates including those carried asymptomatically in the gastrointestinal microbiome. This analysis has resulted in the identification of novel C. perfringens plasmids whilst providing a comprehensive understanding of species diversity.

TcpA from the Clostridium perfringens plasmid pCW3 is more closely related to the DNA translocase FtsK than to coupling proteins.

Conjugative DNA transfer is a major factor in the dissemination of antibiotic resistance and virulence genes. In the Gram-positive pathogen Clostridium perfringens, the majority of conjugative plasmids share the conserved tcp locus that governs the assembly of the transfer system. Here, we describe multiple structures of the coupling protein TcpA, an essential ATPase that is suggested to provide the mechanical force to propel the DNA through the transfer apparatus. The structures of TcpA in the presence and absence of nucleotides revealed conformational rearrangements and highlight a crucial role for the unstructured C terminus. Our findings reveal that TcpA shares most structural similarity with the FtsK DNA translocase, a central component of the bacterial cell division machinery. Our structural data suggest that conjugation in C. perfringens may have evolved from the bacterial chromosome segregation system and, accordingly, suggest the possibility that double-stranded DNA is transferred through the Tcp conjugation apparatus.

Ocular pathology in aquarium fish with a focus on the Syngnathidae and Apogonidae families.

This study catalogued ocular pathology in fish histopathology submissions to a specialist diagnostic service and investigated associations with species and systemic disease, with a focus on species of conservation interest. Cross-tabulations and Fisher's exact tests were used to identify associations among the variables and results are reported as prevalence ratios (PRs) with 95% confidence intervals (CI). Of 12,488 reports reviewed, ocular histology examination was available for 4,572 submissions, in which histopathological ocular lesions were identified in 18% (813/4572). Most diagnoses (701/813; 87%) were in marine fish. Inflammatory conditions were most common (608/813; 75%), with identification of a bacterial aetiology in 42% (255/608) and a parasitic aetiology in 30% (183/608). Most bacterial infections were due to mycobacteriosis (153/255; 60%) and most parasitic infections were due to scuticociliatosis (114/184; 62%). The Syngnathidae, Centriscidae and Cichlidae families were each more likely than all other families combined to be diagnosed with ocular manifestations of mycobacteriosis (PRs = 2.6, 4.4 and 2.9, respectively, P <0.0001 for each). The Syngnathidae were also more likely to be diagnosed with ocular scuticociliatosis (PR = 1.9, P <0.0001). Fifty-four percent (39/72) of ocular mycobacteriosis and 38% (9/24) of gas bubble disease cases affected threatened or near threatened Syngnathidae species. The Apogonidae were more likely than any other family to have ocular iridovirus (PR = 10.3, 95% CI = 5.5-19.4, P <0.0001) and neoplasia (PR = 8.2, 95% CI = 4.2-16.3, P <0.0001). The endangered Banggai cardinalfish (Pterapogon kauderni) accounted for 13/15 ocular iridovirus and 16/18 mycobacteriosis cases in this family. All cases of neoplasia in the Apogonidae occurred in pajama cardinalfish (Sphaeramia nematoptera). These results should inform clinical diagnosis of ocular disease in aquarium fish and influence training for aquarists, highlighting ocular pathology as a potential early warning of systemic disease. The findings also have direct/indirect consequences for the welfare and conservation of some of these popular flagship fish species.

Outcome of Surgical Extraction of Linguoverted Deciduous Mandibular Canine Teeth, Performed as an Interceptive Orthodontic Procedure in Puppies, on Permanent Occlusion.

Linguoversion of deciduous mandibular canine teeth can be a painful condition, interferes with the development and growth of the jaws, and potentially leads to further malocclusions affecting permanent dentition. Extraction of linguoverted deciduous mandibular canines is considered an interceptive orthodontic procedure that would allow unimpeded development of the jaws and permanent teeth. This study assessed clinical records of 124 dogs that had linguoverted deciduous mandibular canine teeth surgically extracted between October 2010 and September 2019 in a veterinary dental referral clinic. Seventy-seven cases fulfilled the study criteria. Fifty-one percent of these patients required further orthodontic treatment of the permanent occlusion and forty-nine percent demonstrated atraumatic permanent occlusion. The study found no correlation of the outcome with age at the time of surgery. The class of malocclusion (class 1 or class 2) at the time of surgery was also not associated with the outcome.

The Specificity of ParR Binding Determines the Incompatibility of Conjugative Plasmids in Clostridium perfringens.

Plasmids that encode the same replication machinery are generally unable to coexist in the same bacterial cell. However, Clostridium perfringens strains often carry multiple conjugative toxin or antibiotic resistance plasmids that are closely related and encode similar Rep proteins. In many bacteria, plasmid partitioning upon cell division involves a ParMRC system; in C. perfringens plasmids, there are approximately 10 different ParMRC families, with significant differences in amino acid sequences between each ParM family (15% to 54% identity). Since plasmids carrying genes belonging to the same ParMRC family are not observed in the same strain, these families appear to represent the basis for plasmid compatibility in C. perfringens. To understand this process, we examined the key recognition steps between ParR DNA-binding proteins and their parC binding sites. The ParR proteins bound to sequences within a parC site from the same ParMRC family but could not interact with a parC site from a different ParMRC family. These data provide evidence that compatibility of the conjugative toxin plasmids of C. perfringens is mediated by their parMRC-like partitioning systems. This process provides a selective advantage by enabling the host bacterium to maintain separate plasmids that encode toxins that are specific for different host targets. IMPORTANCE Toxins produced by the Gram-positive pathogen Clostridium perfringens are primarily encoded by genes found on different conjugative plasmids. These plasmids encode highly similar replication proteins and therefore should be incompatible, but they are often found to coexist within the same isolate. In this study, we showed that a series of phylogenetically related ParMRC plasmid partitioning systems, structures that are normally responsible for ensuring that plasmids segregate correctly at cell division, dictate which toxin plasmid combinations can coexist within the same bacterial cell. We dissected the recognition steps between the DNA-binding ParMRC component, ParR, and the plasmid-derived centromere, parC. Our data suggested a mechanism by which plasmids encoding ParMRC systems from the same family are incompatible, whereas plasmids encoding ParMRC systems from distinct families are compatible. This work provides insight into how these cells can maintain multiple highly similar toxin plasmids, which is a critical first step in understanding how to limit the disease-causing potential of C. perfringens.

Results of a Clinical Trial Showing Changes to the Faecal Microbiome in Racing Thoroughbreds after Feeding a Nutritional Supplement.

Next-generation sequencing (NGS) has been used to evaluate the effect of various interventions on the equine microbiome. The aim of this randomised blinded clinical trial was to determine if a prebiotic nutritional supplement would result in a change from baseline in the faecal microbiome composition of racing Thoroughbred horses in training being fed a high concentrate/grain-based diet to be more similar to that found in forage fed/pasture grazed horses. Thirty-two horses on one training yard were randomised to either receive the supplement or not. Faecal samples were collected at baseline, 6 and 12 weeks for NGS of the 16S ribosomal subunit gene. Twenty-two horses completed the trial, met the inclusion criteria and were included in the intention to treat analysis; 20 horses were included in the per protocol analysis. The mean and median percent decreases in Bacteroidetes, increases in Firmicutes and the Firmicutes:Bacteroidetes ratio were significantly greater than zero for the treated horses only. Supplemented horses (8/10) were more likely than control horses (2/10) to show an increase in Firmicutes of a ≥9% with ≥24% increase in Clostridia, ≥5% decrease in Bacteroidetes, ≥16% increase in the F:B ratio and ≥2% increase in Actinobacteria (RR = 4, 95% CI: 1.1-14.4, p = 0.01). This provides useful information for further investigations on long-term effects on the microbiome and on health and racing-related outcomes.

Low Number of Owner-Reported Suspected Transmission of Foodborne Pathogens From Raw Meat-Based Diets Fed to Dogs and/or Cats.

The aim of this worldwide survey was to determine owner-reported frequency of pathogen transmission to humans living in or in contact with households feeding their pets raw, minimally processed (MP) diets. A total of 5,611 responses were gathered from 62 countries with 77.1% of households feeding only MP diets to dog and/or cat(s) with no confirmed cases of pathogen transmission or infection by laboratory testing. Eleven households (0.20%; 95% CI, 0.10-0.36) were classified as having experienced "probable" transmission, and 20 households (0.36%; 95% CI, 0.22-0.56) were classified as having experienced "possible" transmission to result in a total of 31 households (0.55%; 95% CI, 0.38-0.79) being identified as potential cases of transmission. The remainder of households (n = 5,580 = 99.45%; 95% CI, 99.21-99.62) were not considered to have experienced potential transmission of foodborne pathogens based on their responses to the survey. The most frequently reported pathogens were Salmonella (n = 11, 0.2%), Campylobacter (n = 6, 0.1%), and Escherichia coli (n = 4, 0.1%), with the most common age group being adults age 18-65 (n = 29, 78.4% of cases). Beef and chicken were the most common proteins reported as being fed in case households, although this was not associated with pathogen transmission. Households feeding a greater number of different protein sources, including pork, turkey, duck, rabbit, and salmon, were associated with decreased risk of pathogen transmission. Additional risk factors associated with pathogen transmission included preparing either MP diets in a separate location, with different utensils than human food, mixing MP diets with dry (kibble) diets and feeding a limited variety of protein sources. Based on the results of this survey, confirmed pathogen transmission from MP diets to humans appears to be rare. We conclude that potential or probable cases of pathogen transmission is likely dependent upon hygiene and food safety measures, and more education surrounding food safety should reduce risk.

Cardiopulmonary Lesions in Sheep Produced by Experimental Acute Clostridium Perfringens Type D Enterotoxemia.

Enterotoxemia caused by Clostridium perfringens type D is one of the most prevalent clostridial diseases of sheep. The lesions of the acute form of this disease, particularly the cerebral lesions, are well characterized; however, detailed descriptions of the cardiac and pulmonary lesions are lacking. Here we describe cardiopulmonary lesions in experimental acute type D enterotoxemia in sheep and determine the role of epsilon toxin (ETX) in the development of these lesions. Four groups of 6 sheep were intraduodenally inoculated with either a wild-type C. perfringens type D strain; its etx knockout mutant, which is unable to produce ETX; the etx mutant complemented with the wild-type etx gene, which regains the ETX toxigenic ability; or sterile culture medium as a control. All sheep were subjected to postmortem examination within 24 hours of inoculation. Lesion scores were compared between groups for pulmonary edema; hydrothorax; ascites; hydropericardium; endocardial, myocardial and epicardial hemorrhages; microscopic lesions of acute myocardial degeneration and necrosis; and myocardial, endocardial, and epicardial edema, hemorrhage, and inflammation. Only sheep inoculated with the wild-type and complemented ETX-toxigenic bacterial strains developed cardiopulmonary lesions, which were present in varying degrees of severity and proportions. These lesions were not present in sheep inoculated with the etx mutant or in the negative control. We conclude that severe acute cardiopulmonary lesions in sheep with experimental enterotoxemia are associated with the capacity of the strains to produce ETX. These changes are likely contributors to the clinical signs and even death of affected animals.

The ever-expanding tcp conjugation locus of pCW3 from Clostridium perfringens.

The spore-forming, anaerobic Gram positive pathogen Clostridium perfringens encodes many of its disease-causing toxins on closely related conjugative plasmids. Studies of the tetracycline resistance plasmid pCW3 have identified many of the genes involved in conjugative transfer, which are located in the tcp conjugation locus. Upstream of this locus is an uncharacterised region (the cnaC region) that is highly conserved. This study examined the importance in pCW3 conjugation of several highly conserved proteins encoded in the cnaC region. Conjugative mating studies suggested that the SrtD, TcpN and Dam proteins were required for efficient pCW3 transfer between C. perfringens cells from the same strain background. The requirement of these proteins for conjugation was amplified in matings between C. perfringens cells of different strain backgrounds. Additionally, the putative collagen adhesin protein, CnaC, was only required for the optimal transfer of pCW3 between cells of different strain backgrounds. Based on these studies we postulate that CnaC, SrtD, TcpN and Dam are involved in enhancing the transfer frequency of pCW3. These studies have led to a significant expansion of the tcp conjugation locus, which now encompasses a 19 kb region.

Successful Use of MTA Fillapex as a Sealant for Feline Root Canal Therapy of 50 Canines in 37 Cats.

Intentional or unintentional pulp exposure of cat canines can lead to periapical disease, osteomyelitis, and oral pain. Root canal therapy (RCT) allows the retention of cat canines with pulp exposure by removing the infected pulp and replacing it with an inert material. This study used MTA Fillapex™ as a root canal sealant with gutta percha single cone obturation in 37 cats (50 canine teeth). Roots were classified as "successful," "no evidence of failure (NEF)," or "failed" at 6-month radiographic reviews. Therapy was considered "successful" if a preoperative periapical lucency had healed or not formed after treatment and any preoperative external inflammatory root resorption (EIRR) had stabilized without progression. Therapy was categorized as "NEF" if a periapical lucency had remained the same or decreased in size but not completely resolved and any preoperative EIRR had stabilized without progression. "Failed" if a periapical lucency had occurred or increased in size posttreatment or if EIRR had developed or progressed posttreatment. Thirty-two canine teeth (64%) were classified as "successful," 14 canine teeth (28%) were classified as "NEF," and 4 canine teeth (8%) were classified as "failed". The study concluded that RCT using MTA Fillapex as a root canal sealant is a suitable endodontic treatment for fractured cat canines, especially those that are periodontally or endodontically challenged.

The EngCP endo α-N-acetylgalactosaminidase is a virulence factor involved in Clostridium perfringens gas gangrene infections.

Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance.

Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.

Body weight at 10 years of age and change in body composition between 8 and 10 years of age were related to survival in a longitudinal study of 39 Labrador retriever dogs.

BACKGROUND: Overweight and obesity have been adversely associated with longevity in dogs but there is scarce knowledge on the relation between body composition and lifespan. We aimed to investigate the effects of body composition, and within-dog changes over time, on survival in adult Labradors using a prospective cohort study design. The dogs had a median age of 6.5 years at study start and were kept in similar housing and management conditions throughout. The effects of the various predictors, including the effect of individual monthly-recorded change in body weight as a time varying covariate, were evaluated using survival analysis. RESULTS: All dogs were followed to end-of-life; median age at end-of-life was 14.0 years. Body composition was measured annually with dual-energy x-ray absorptiometer (DEXA) scans between 6.2 and 17.0  years. All 39 dogs had DEXA recorded at 8, 9 and 10 years of age. During the study the mean (± SD) percent of fat (PF) and lean mass (PL) was 32.8 (± 5.6) and 64.2 (± 5.5) %, respectively, with a mean lean:fat ratio (LFR) of 2.1 (± 0.6); body weight (BW) varied from 17.5 to 44.0 kg with a mean BW change of 9.9 kg (± 3.0). There was increased hazard of dying for every kg increase in BW at 10 years of age; for each additional kg of BW at 10 years, dogs had a 19% higher hazard (HR = 1.19, P = 0.004). For the change in both lean mass (LM) and LFR variables, it was protective to have a higher lean and/or lower fat mass (FM) at 10 years of age compared to 8 years of age, although the HR for change in LM was very close to 1.0. For age at study start, older dogs had an increased hazard. There was no observed effect for the potential confounders sex, coat colour and height at shoulders, or of the time-varying covariate. CONCLUSIONS: These results suggest that even rather late-life control efforts on body weight and the relationship between lean and fat mass may influence survival in dogs. Such "windows of opportunity" can be used to develop healthcare strategies that would help promote an increased healthspan in dogs.

Virulence Plasmids of the Pathogenic Clostridia.

The clostridia cause a spectrum of diseases in humans and animals ranging from life-threatening tetanus and botulism, uterine infections, histotoxic infections and enteric diseases, including antibiotic-associated diarrhea, and food poisoning. The symptoms of all these diseases are the result of potent protein toxins produced by these organisms. These toxins are diverse, ranging from a multitude of pore-forming toxins to phospholipases, metalloproteases, ADP-ribosyltransferases and large glycosyltransferases. The location of the toxin genes is the unifying theme of this review because with one or two exceptions they are all located on plasmids or on bacteriophage that replicate using a plasmid-like intermediate. Some of these plasmids are distantly related whilst others share little or no similarity. Many of these toxin plasmids have been shown to be conjugative. The mobile nature of these toxin genes gives a ready explanation of how clostridial toxin genes have been so widely disseminated both within the clostridial genera as well as in the wider bacterial community.

pCP13, a representative of a new family of conjugative toxin plasmids in Clostridium perfringens.

Conjugative transfer is a major contributor to the dissemination of antibiotic resistance and virulence genes in the human and animal pathogen, Clostridium perfringens. The C. perfringens plasmid pCW3 is the archetype of an extensive family of highly related conjugative toxin and antibiotic resistance plasmids found in this bacterium. These plasmids were thought to constitute the only conjugative plasmid family in C. perfringens. Recently, another series of C. perfringens plasmids, the pCP13-like family, have been shown to harbour important toxin genes, including genes that encode the novel binary clostridial enterotoxin, BEC. Based on early bioinformatics analysis this plasmid family was thought to be non-conjugative. Here we demonstrate that pCP13 is in fact conjugative, transfers at high frequency and that the newly defined Pcp conjugation locus encodes putative homologues of a type 4 secretion system (T4SS), one of which, PcpB4, was shown to be essential for transfer. The T4SS of pCP13 also appears to be evolutionarily related to conjugative toxin plasmids from other clostridia-like species, including Paeniclostridium (formerly Clostridium) sordellii, Clostridioides (formerly Clostridium) difficile and Clostridium botulinum. Therefore, it is clear that there are two distinct families of conjugative plasmids in C. perfringens: the pCW3 family and the pCP13 family. This study has significant implications for our understanding of the movement of toxin genes both within C. perfringens, but also potentially to other pathogenic clostridia.

Crystal structure of TcpK in complex with oriT DNA of the antibiotic resistance plasmid pCW3.

Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution. Furthermore, TcpK specifically binds to a nine-nucleotide sequence that is present as tandem repeats within the pCW3 origin of transfer (oriT). The X-ray crystal structure of the TcpK-TcpK box complex reveals a binding mode centered on and around the ß-wing, which is different from what has been previously shown for other wHTH proteins. Structure-guided mutagenesis experiments validate the specific interaction between TcpK and the DNA molecule. Additional studies highlight that the TcpK dimer is important for specific DNA binding.