Beta Toxin Research Articles

Effects of Clostridium perfringens beta2 toxin on apoptosis, inflammation, and barrier function of intestinal porcine epithelial cells

Clostridium perfringens beta2 (CPB2) toxin is an important virulence factor that causes enteric diseases in both humans and animals. To investigate the underlying mechanism in CPB2-induced inflammation and damage in the small intestinal epithelium, intestinal porcine epithelial cells (IPEC-J2) were treated with recombinant CPB2 (rCPB2) toxin. The results showed that IPEC-J2 cell viability was decreased by rCPB2 toxin treatment in a dose- and time-dependent manner. Analysis of cell morphology and Annexin V-FTIC/PI staining revealed that rCPB2 toxin induces cell apoptosis. Indeed, the expression of caspase-3, caspase-8, and caspase-9 was significantly increased at both the mRNA and protein levels in IPEC-J2 cells treated with rCPB2 toxin. The caspase-3 inhibitor Ac-DEVD-CHO reduced rCPB2 toxin-induced cell apoptosis. Moreover, exposure to the toxin increased the expression of interleukin (IL)-6, IL-7, IL-12, and IL-1β, while decreasing that of transforming growth factor beta 1 (TGFβ1). Additionally, rCPB2 toxin treatment also induced intestinal barrier dysfunction, as evidenced by the degradation of zonula occludens (ZO)-1, claudin-1, and E-cadherin, as well as an increase in paracellular permeability. Overall, the results indicated that rCPB2 toxin induces apoptosis and inflammation, in addition to impairing intestinal barrier function in IPEC-J2 cells. Our findings provide a foundation to better understand the pathogenesis of C. perfringens infection and inform strategies to effectively prevent and treat C. perfringens-induced enteric diseases.

Immunization of mice against alpha, beta, and epsilon toxins of Clostridium perfringens using recombinant rCpa-b-x expressed by Bacillus subtilis

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.

Multilocus sequence typing of Clostridium perfringens strains from neonatal calves, dairy workers and associated environment in India

Clostridium perfringens is a globally recognized zoonotic pathogen. We report isolation and genotyping of C. perfringens from neonatal calves, dairy workers and their associated environment in India. A total of 103 fecal samples from neonatal calves, 25 stool swabs from the dairy workers and 50 samples from their associated environment were collected from two dairy farms. C. perfringens was detected in 26 out of 103 (25.2%) neonatal calf samples, 7 out of 25 (28%) human stool samples and 17 out of 50 (34%) environmental samples. C. perfringens type A strains were predominant in neonatal calves (24/26; 92.3%) and associated environment (15/17; 88.2%). In contrast, strains from dairy workers mostly belonged to type F (5/7; 71.4%), which also carried the beta2 toxin gene. Seventeen strains were analyzed by multilocus sequence typing (MLST) for studying genotypic relationship along with 188 C. perfringens strains available from public databases. A total of 112 sequence types (STs) were identified from 205 C. perfringens strains analyzed. A Clonal complex (CC) represented by three STs (ST 98, ST 41 and ST 110) representing predominantly type F (18/20 strains) were mostly associated with human illnesses. Among predominant STs, ST 54 was associated with enteritis cases in foals and dogs and ST 58 associated with necrotic enteritis in poultry. Seventeen Indian strains were assigned to 13 STs. Genetic relatedness among strains of calves, dairy worker and associated environments indicate inter-host transfers and zoonotic spreads.

Genotyping of Clostridium perfringens Isolates from Domestic Livestock in Saudi Arabia.

The present study was undertaken to confirm the genetic identity of Clostridium perfringens isolates from domestic livestock in Saudi Arabia and to characterize the genes encoding to alpha, beta, epsilon, and iota (α-, β-, ε-, and ι-) toxins. C. perfringens were confirmed in 104 out of 136 isolates on multiplex polymerase chain reaction using specific primers amplifying genes related to toxins produced by C. perfringens. Genes encoding α-toxins were detected in 104 samples. Of the isolates, 80.8% were diagnosed as type A, 15.4% as type D, 2.9% as type C, and 0.96% as type B. None of the isolates has genes encoding iota (ι-) toxin. All isolates investigated yielded enterotoxin (cpe) products and none yielded β2 (cpb2-toxin) or NetB products. PLC gene sequences encoding α-toxin showed >96.7% similarity. Isolates which had α-toxins as well as enterotoxin (cpe) are regarded as type F. Phylogenetic analysis using maximum likelihood analysis yielded two clades, and the majority of the isolates were in one group while only two isolates clustered on the second clade. Within the Kingdom of Saudi Arabia strains, 54 variable positions and 23 polymorphic amino acids were noticed. Isolates with ε- and β-toxins were variable and were found to be close to those published for C. perfringens. ETX gene sequences encoding ε-toxins were found to be related to CPE sequences.

Dynamics of toxigenic Clostridium perfringens colonisation in a cohort of prematurely born neonatal infants

BackgroundClostridium perfringens forms part of the human gut microbiota and has been associated with life-threatening necrotising enterocolitis (NEC) in premature infants. Whether specific toxigenic strains are responsible is unknown, as is the extent of diversity of strains in healthy premature babies. We investigated the C. perfringens carrier status of premature infants in the neonatal intensive care unit, factors influence this status, and the toxic potential of the strains.MethodsC. perfringens was isolated by culture from faecal samples from 333 infants and their toxin gene profiles analysed by PCR. A survival analysis was used to identify factors affecting probability of carriage. Competitive growth experiments were used to explore the results of the survival analysis.Results29.4% of infants were colonized with C. perfringens before they left hospital. Three factors were inversely associated with probability of carriage: increased duration of maternal milk feeds, CPAP oxygen treatment and antibiotic treatment. C. perfringens grew poorly in breast milk and was significantly outperformed by Bifidobacterium infantis, whether grown together or separately. Toxin gene screening revealed that infants carried isolates positive for collagenase, perfringolysin O, beta 2, beta, becA/B, netB and enterotoxin toxin genes, yet none were observed to be associated with the development of NEC.ConclusionsApproximately a third of preterm infants are colonised 3 weeks after birth with toxin gene-carrying C. perfringens. We speculate that increased maternal breast milk, oxygen and antibiotic treatment creates an environment in the gut hostile to growth of C. perfringens. Whilst potentially toxigenic C. perfringens isolates were frequent, no toxin type was associated with NEC.Trial registrationclinicaltrials.govNCT01102738, registered 13th April 2010.

Death of a neonatal lamb due to Clostridium perfringens type B in New Zealand

ABSTRACTCase history and clinical findings: A flock of 20 sheep was kept within three paddocks on a single property. None of the animals in the flock had been vaccinated against any disease for at least three years. Abdominal bloating and haemorrhagic diarrhoea were observed in Lamb 1 at 24 hours-of-age. The lamb subsequently died within an hour of the onset of clinical signs. Lamb 2 was 3-days-old when observed to be recumbent with opisthotonus. The lamb was treated with dextrose, vitamins B1 and B12, and penicillin G, but died 4 hours later.Pathological findings: Examination of Lamb 1 revealed markedly increased gas within the peritoneum and within dilated loops of intestine. The intestines were dark red and contained large quantities of haemorrhagic fluid. Histology of the intestines revealed peracute mucosal necrosis with minimal accompanying inflammation. The intestinal lumen contained cell debris, haemorrhage, and myriad large Gram-positive bacilli. The intestines of Lamb 2 did not appear bloated or reddened. However, multiple fibrin clots were visible within the pericardial sac. Histopathological examination revealed small foci of necrosis within the mucosa of the distal intestine. The necrotic foci were often associated with large numbers of large Gram-positive bacilli.Immunohistochemsitry and molecular biology: Intestinal samples from Lamb 1 were processed for Clostridium perfringens immunohistochemistry, which revealed large numbers of intralesional, positively immunostained rods. Fragments corresponding to the expected sizes for genes encoding alpha, beta, and epsilon C. perfringens typing toxins were amplified by PCR from DNA extracted from formalin-fixed sections of intestine.Diagnosis: Lamb dysentery due to C. perfringens type B.Clinical relevance: C. perfringens bacteria have a worldwide distribution, but disease due to C. perfringens type B has only been diagnosed in a small number of countries and has never been reported in New Zealand or Australia. C. perfringens type B produce both beta toxin and epsilon toxins, therefore both haemorrhagic enteritis and systemic vascular damage can develop. As many animals are exposed to C. perfringens without developing disease, there must be additional unknown factors that resulted in disease in these particular sheep. Vaccines that specifically protect against C. perfringens type B are available and may be recommended for use in smaller non-commercial flocks, as in the present case.

MOLECULAR CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS ISOLATED FROM TURKEYS

A total of 82 C. perfringensisolates (41%) were recovered from 200 samples collected from 100 turkeys (20 apparently healthy, 40 clinically diseased and 40 freshly dead), 4-6 weeks old. The clinical signs of diseased birds were sudden mortality, depression, ruffled feathers, diarrhea, dehydration, emaciation and decrease in feed consumption with increase in water consumption. While, postmortem findings were consistent with necrotic enteritis (NE), the small intestine was severely affected. Small and demarcated lesions were found in the duodenal loop, hepatitis and cholecystitis were also observed. Unopened intestines were gas-filled and enlarged with thin wall. The intestinal contents were dark due to necrotic material. The opened intestines showed necrotic lesions of varying severity of the mucosa. Isolation and biochemical identification of C.perfringenswere done.Five C.Perfringens field isolates were analyzed by PCR assay to determine the presence of some toxin genes.In all tested isolates, the Cpa gene (alpha toxin) was detected (100%) confirming the isolates were C.perfringens. While, Cpb (beta toxin) gene was detected in two samples (40%). But the Etx gene (epsilon toxin) was not detected in any isolate (0%). NetB gene was detected in two isolates (40%). Antimicrobial susceptibility of 40 C.perfringens field isolates recovered from turkeys revealed that C.perfringens isolates were sensitive to Ampicillin (85%), Amoxicillin (85%), Penicillin (82.5%), Florfenicol (72.5%), Enrofloxacin (67.5%), Vancomycin (65%), Bacitracin (60%), Oxytetracyclin (32.5%), Lincomycin (22.5%) and Clindamycin (15%).These five C. perfringens isolates were also screened by PCR which detected the presence of tetracycline resistance gene tet(K) in three isolates (60%) and Lincomycin resistance gene lin(B) in three isolates (60%).

Production, immunogenicity, stability, and safety of a vaccine against Clostridium perfringens beta toxins.

Background and Aim:The beta toxin is causing the most severe Clostridium perfringens-related diseases. This work was dedicated to developing a vaccine against beta toxin using C. perfringens type C (NCTC 3180).Materials and Methods:The crude toxoid harvest contained 710 limits of flocculation (Lf)/mL. The vaccine was formulated. Each 1 mL of the final vaccine product contained at least 50 Lf/mL of beta toxoids, 0.2 mL 3% aluminum hydroxide gel (equivalent to 5.18 mg of aluminum), <0.001% W/V thiomersal, formaldehyde <0.05% W/V, and ~0.7 mL phosphate-buffered saline (pH 7.2). The efficacy of the vaccine was evaluated by potency, stability, and safety tests.Results:The vaccine demonstrated 24.36 IU/mL (standard deviation, ±0.56) and 14.74 IU/mL (±0.36) of neutralizing antibodies in rabbits and cattle, respectively. Indeed, these levels were above the minimum recommended by international protocols since the obtained antibody levels had 2.43- and 1.47-fold increase in both rabbits and cattle, respectively, over the minimum antitoxin level suggested by the United States Department of Agriculture. Interestingly, our formulation was capable of inducing 1.65-fold higher immune responses in rabbits than that stimulated in cattle (65% increase) with a significant difference (p<0.0001). The vaccine was stable up to 30 months. The vaccinated rabbits were suffered from a temporarily slight increase in temperatures in the first 10 h without any significant difference (p>0.05).Conclusion:The research showed a procedure for the manufacturing process of the vaccine against C. perfringens beta toxins with a feasible quantity and the vaccine described here showed to be effective in eliciting levels of neutralizing antibodies higher than required by international standards. In addition, The vaccine was stable up to 30 months. Thus, it may represent an effective and safe for preventing C. perfringens-related diseases in rabbits and cattle, although further studies to prove its efficacy in the field on other farm animals are still needed.

Hemorrhagic bowel syndrome in cattle

Hemorrhagic bowel syndrome is relatively common disease in dairy and beef cattle which has high fatality rate. It is classified as acute, sporadic and necrohemorrhagic enteritis. It is commonly seen in highly productive dairy cattle. The cause of hemorrhagic bowel syndrome is unclear and the pathogenesis is not well-understood. As a primary etiologic agent Clostridium perfringens type A has been strongly proposed and its alpha and beta2 toxin are suggested to have an important role in occurence of the disease. Disease especially occur in lactating dairy cows in the first 3 months of lactation. Ration is seem to be the most important predisposing factor in the development of hemorrhagic bowel syndrome. Severe toxemia (enterotoxemia) and intense hemorrhage in the small intestine cause pathological changes in the intestine. The blood clots in the intestinal lumen, a specific finding of hemorrhagic bowel syndrome can only be demonstrated in 19% of affected cows. Diagnosis is often based on the clinical, ultrasound and necropsy findings and also with differential diagnosis of other haemorrhagic enteritis caused by salmonellosis, Bovine Viral Diarrhea and coccidiosis etc. Prognosis is poor and fatality rate is nearly 100% despite intensive medical and surgical therapy. The use of feed additives, good nutritional management and vaccine administration are recommended for disease control.

Respuesta inmune humoral contra toxoides de Clostridium perfringens en vaquillonas Holando seropositivas al virus de la Leucosis Bovina Enzoótica

Enzootic bovine leukosis is the main virosis that affects dairy cattle in Uruguay. The subclinical infection causes importants dysfunctions in the immune system impacting directly in the animal health. The aim of this study was to evaluate the immune response of Holstein heifers with positive serology against Leukosis bovine virus (BLV) when they were ordinary immunized against Clostridium perfringens. It was evaluated a seropositive group to BLV animals (n=29) and other seronegative group (n=19) during twelve months. The animals recived three immunizations with a commercial polyvalent vaccine against several Clostridios (days 0, 30, 173). Blood samples were taken in days 0, 30, 60, 90, 180 y 360 post vaccination and then the humoral immune response was analyzed through the use of the ELISA technique to quantify total antibodies against C. perfringens toxins alfa, beta and epsilon. Besides it was in vitro analyzed the amount of neutralizing antibodies against epsilon toxin using the reference technique, seroneutralization. The results demonstrated a low response of neutralizing antibodies to epsilon toxin by SN technique showing no correlation with the results obtained by the commercial ELISA kit. Total antibodies response evaluated using commercial ELISA Kit was variable according to the studied toxoid. No significant differences were found in the immune response against Clostridium perfringens vaccination between BLV seropositive and seronegative animals using the techniques mentioned. The commercial ELISA kit use to analyze the response to vaccination against Clostridiosis, must be discussed and analyzed according to the results of this work.

Clostridium perfringens type A Causing Necrotic Enteritis Outbreaks among Chickens in Egypt

Undoubtedly, necrotic enteritis is identified as one of the most threatening diseases which face poultry industry in Egypt and need radical solution to avoid huge economic losses. The present work was designed for typing Clostridium perfringens isolated from diseased chickens suspected to have necrotic enteritis from different outbreaks in six Governorates in Egypt (Gharbia, Dakhalia, Sharkia, Ismailia, North Sinai and Kafr El Sheikh) during the period from August to December 2018. Intestine and liver samples were obtained from eighty six diseased broiler chickens representing 60 flocks. C. perfringens was isolated and toxigenicity of the recovered isolates was determined by Nagler's and dermonecrotic reactions. Furthermore, multiplex polymerase chain reaction (PCR) targeting alpha, beta, epsilon and iota toxins' genes was performed for result confirmation and typing of the toxigenic isolates. Sixty-six C.perfringens isolates (38.37%) were recovered from 172 intestine and liver samples and twenty isolates (30.3%) were toxigenic and typed as C. perfringens type A producing alpha toxin only. These findings established the fact that alpha toxin is the only main toxin of C. perfringens type A which is basically responsible for its pathogenicity and virulence. In addition, most of the positively toxigenic isolates were isolated from hepatic lesions (15 isolates) rather than intestinal lesion (5 isolates). In conclusion, alpha toxin is a major toxin for NE development in chickens. Genotyping of Clostridium perfringens by multiplex PCR is a useful adjunct to diagnosis of necrotic enteritis in chickens.

Isolation and Genotyping Study of Clostridium Perfringens From Broiler Farms Infected with Necrotic Enteritis in Sulaimania Province

The study is conducting to isolate and toxinotype the suspected cases of Clostridium perfringens infections of broiler farms in Sulaimania province. A total of 108 samples were collected from intestinal contents, mucosal scraping, and hemorrhagic lymphoid nodules from suspected cases of necrotic enteritis in broilers. The result of isolated and identified bacteria were revealed that 63 (58%) out of 108 samples were positive for C. perfringens. The results revealed that the isolates were only positive for alpha and beta2 toxin genes. Phylogenetic and DNA sequence analysis of cpa and cpb2 gene showed that cpa genes were highly identical to isolates from broiler in Iran, poultry stool and broiler in Brazil, and blue calves in Belgium. While cpb2 gene is closely related to the isolates of broiler in Iran, India and isolates of goat in Pakistan. The results indicated that the causative agent of necrotic enteritis in broiler farms in the region was mainly due to C. perfringens type A infection

Pathobiology and diagnosis of clostridial hepatitis in animals.

Clostridia can cause hepatic damage in domestic livestock, and wild and laboratory animals. Clostridium novyi type B causes infectious necrotic hepatitis (INH) in sheep and less frequently in other species. Spores of C. novyi type B can be present in soil; after ingestion, they reach the liver via portal circulation where they persist in phagocytic cells. Following liver damage, frequently caused by migrating parasites, local anaerobic conditions allow germination of the clostridial spores and production of toxins. C. novyi type B alpha toxin causes necrotizing hepatitis and extensive edema, congestion, and hemorrhage in multiple organs. Clostridium haemolyticum causes bacillary hemoglobinuria (BH) in cattle, sheep, and rarely, horses. Beta toxin is the main virulence factor of C. haemolyticum, causing hepatic necrosis and hemolysis. Clostridium piliforme, the causal agent of Tyzzer disease (TD), is the only gram-negative and obligate intracellular pathogenic clostridia. TD occurs in multiple species, but it is more frequent in foals, lagomorphs, and laboratory animals. The mode of transmission is fecal-oral, with ingestion of spores from a fecal-contaminated environment. In affected animals, C. piliforme proliferates in the intestinal mucosa, resulting in necrosis, and then disseminates to the liver and other organs. Virulence factors for this microorganism have not been identified, to date. Given the peracute or acute nature of clostridial hepatitis in animals, treatment is rarely effective. However, INH and BH can be prevented, and should be controlled by vaccination and control of liver flukes. To date, no vaccine is available to prevent TD.

Incidence of Clostridium perfringens and its toxin genes in the gut of children with autism spectrum disorder

This study was designed to determine the incidence of Clostridium perfringens and their toxin genes in children with autism spectrum disorder (ASD), and determine the antimicrobial susceptibility of C. perfringens isolates. A hundred and fourteen fecal samples were obtained from children aged 3–12 years old (57 samples from ASD children and 57 from healthy controls). Children were divided into four groups based on their gastrointestinal (GI) symptoms as follows: ASD group with and without GI symptoms, and control group with and without GI symptoms. Selective anaerobic media and VITEK®2 ANC ID card were used for isolation and identification of C. perfringens from fecal samples. Antimicrobial susceptibility of C. perfringnes isolates were performed using (E-Test) strips against clindamycin, penicillin and metronidazole antibiotics. Conventional PCR was used to detect the alpha toxin gene (Cpa) and the beta-2 toxin gene (Cpb2). Genetic Analyzer 3130Xi was used to confirm the sequencing of Cpb2 gene. Our findings indicated that 38.6% of ASD group samples had a significantly (p = 0.003) higher incidence of C. perfringens than that of the control group (14%). The highest incidence of C. perfringens was found in the ASD group with GI symptoms (53.8%; p = 0.001). C. perfringens isolated from the ASD group (54.5%) were significantly (p = 0.047) more resistant to clindamycin than those isolated from the control group (12.5%). The C. perfringens isolates from the ASD and the control group showed 95.5% and 100% susceptibility to penicillin, respectively. All C. perfringens isolates of ASD and control group were susceptible to metronidazole. The Cpa toxin gene was also detected in all the C. perfringens isolates of ASD and control group, both with and without GI symptoms. Cpb2 toxin gene showed 100% incidence in ASD samples with GI symptoms and in the control groups both with or without GI symptoms, while it was present at significantly lower levels (25%) in the ASD samples without GI symptoms (p = 0.001). Our findings suggests that a high incidence of C. perfringens and its toxin gene (Cpb2) are associated with the GI complications in ASD which may affect the severity of the disease.

Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens in England and Wales over a 7-year period indicates distribution of clonal toxigenic strains in multiple outbreaks and extensive involvement of enterotoxin-encoding (CPE) plasmids.

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated C. perfringens strains. We performed phylogenomic analysis on 109 C. perfringens isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin cpe gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that three major cpe-encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal-cpe strains. Our findings substantially expanded our knowledge on type F C. perfringens involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly.

Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China

Clostridium perfringens is an important zoonotic microorganism, which can cause diseases in animal and human under certain conditions. Contamination of C. perfringens in chicken and pork meat has been reported worldwide, but it is rarely reported in duck products. The current study was undertaken to investigate C. perfringens contamination in duck products from a large retail market in Tai'an region, China and the serotype distribution, antimicrobial resistance and genetic relatedness of the isolates. In total, 173 samples of duck products, 10 samples of environmental origins and 7 samples of fresh faeces from healthy shopkeepers were collected between March and November 2018, of which, 58 (31.69%), 10 (100%) and 7 (100%) samples were determined to be positive for C. perfringens, respectively. Ninety-nine isolates of C. perfringens were recovered, all of which were identified as type A. Beta2 (cpb2) toxin gene was found in 54.30% and 33.30% of the isolates from duck products and healthy shopkeepers, respectively. Antimicrobial susceptibility testing revealed that 90.10% of the isolates from duck products and environment showed multiple antibiotic resistance, among which, 49.40% were resistant to at least 6 classes of commonly used antibiotics. Multilocus sequence typing (MLST) showed that 58 representative isolates were divided into 41 sequences types (STs), among which, ST11 (8.60%) was the most common; 37.90% of all isolates were classified into four clonal complexes (CC1-CC4). The most prolific clonal complex (CC1), accounting for 24.13% of all isolates, contained isolates mainly from carcass, animal intestinal contents and environment of four retail stores. A portion of human isolates and duck isolates was distributed in the same CC or ST. In conclusion, C. perfringens contamination in some duck products in Tai'an retail market was relatively high, and most of the isolates exhibited broad-spectrum antimicrobial resistance. Although all the isolates belong to type A, considerable genetic diversity was observed, and a portion of the strains from human and duck was found to be phylogenetically close. The results indicated that antimicrobial-resistance strains of duck origin pose a potential threat to humans by spreading through the food chain.

Prevalence and Genetic Diversity of Toxin Genes in Clinical Isolates of Clostridium perfringens: Coexistence of Alpha-Toxin Variant and Binary Enterotoxin Genes (bec/cpile).

Clostridium perfringens (C. perfringens) is responsible for food-borne gastroenteritis and other infectious diseases, and toxins produced by this bacterium play a key role in pathogenesis. Although various toxins have been described for C. perfringens isolates from humans and animals, prevalence of individual toxins among clinical isolates has not yet been well explored. In the present study, a total of 798 C. perfringens clinical isolates were investigated for prevalence of eight toxin genes and their genetic diversity by PCR, nucleotide sequencing, and phylogenetic analysis. Besides the alpha-toxin gene (plc) present in all the isolates, the most common toxin gene was cpe (enterotoxin) (34.2%), followed by cpb2 (beta2 toxin) (1.4%), netB (NetB) (0.3%), and bec/cpile (binary enterotoxin BEC/CPILE) (0.1%), while beta-, epsilon-, and iota-toxin genes were not detected. Genetic analysis of toxin genes indicated a high level of conservation of plc, cpe, and netB. In contrast, cpb2 was revealed to be considerably divergent, containing at least two lineages. Alpha-toxin among 46 isolates was classified into ten sequence types, among which common types were distinct from those reported for avian isolates. A single isolate with bec/cpile harbored a plc variant containing an insertion of 834-bp sequence, suggesting its putative origin from chickens.

Diversity of toxin genotypes and antimicrobial susceptibility of Clostridium perfringens isolates from feces of infants.

This study investigated the most important epidemiological characteristics of C. perfringens strains colonizing the intestine of Jordanian infants. A total of 302 fecal samples were collected from Jordanian infants aged ≤ 1 year from patients hospitalized in the neonatal intensive care unit and from the outpatient department. Samples were cultured for detection of C. perfringens and evaluation of their antimicrobial resistance; identification of their potential toxins genes was performed using PCR. Overall the C. perfringens colonization rate was 27.2% (82/302). Infants aged ≤ 6 months showed significantly higher (p<0.004) colonization than older infants. The occurrence rates of C. perfringens isolates carrying potential specific toxin genes were as follows: alpha toxin 95.1% (78/82), beta-2 toxin 69.5% (57/82), beta toxin 14.6% (12/82), and only 2.4% (2/82) epsilon toxin. No isolate carried the iota toxin. Genotype A was the most prevalent among C. perfringens isolates (78.1%). The antimicrobial resistance rates of C. perfringens isolates were 20% to metronidazole and erythromycin, 16.7% to levofloxacin, and 6.7% to vancomycin. This study demonstrates that the majority of C. perfringens isolates from feces of Jordanian infants were classified as type A, few isolates were classified as type C and type D, and all were negative for potential enterotoxin genes causing diarrhea.

Characterization of a bacteriophage from avian Staphylococcus aureus associated with innate immune evasion

Staphylococcus aureus is an important human and livestock pathogen. An S. aureus phage (φAvβ) inserted into the chromosome at the beta toxin gene (beta-converting phage) is present in approx. 90 % of human strains and contributes to human-specific innate immune evasion. Comparative genomic analysis of S. aureus isolates from infected poultry has revealed an avian-specific subfamily of beta-converting phages represented by multiple variants with distinct integrase gene alleles. To investigate the role of the avian beta-converting phages in host-pathogen interactions, a φAvβ-deficient strain with non-functional beta-toxinand a φAvβ-deficient strain with restored beta-toxin were constructed by allele replacement in an avian pathogenic S. aureus strain. Compared to the wild type, both φAvβ-deficient strains have reduced net extracellular growth in vitro in chicken bone-marrow derived macrophages. Further investigation using GFP-tagged bacteria has revealed that both φAvβ-deficient strains show reduced initial phagocytosis and intracellular survival compared to the wild type. Absence of φAvβ is also associated with decreased killing of the chicken bone-marrow derived macrophages. We are currently investigating the mechanism underlying this phenotype using deletion mutants of the candidate phage effector genes. Ongoing work also involves using RNAseq to investigate differential host transcriptional response of the macrophages to S. aureus in presence/absence ofφAvβ. Overall, these data will contribute new information relating to the evolution of avian S. aureus and mechanisms of bacterial host-adaptation.

Deciphering Potential Drug Targets in Clostridium Perfringens through Metabolic Pathway Analysis

Background: In our day-to-day life, we are facing many dreadful diseases caused by many infectious pathogens. These pathogens invade the living organisms (host) and lethally damaging them. These dreadful pathogens were also be used as bioweapons. Among them, Clostridium perfringens is taken for the study. Clostridium perfringens is an anaerobic, rod shaped, gram positive bacteria capable of forming spores. It is prevalent in the environment and in the intestine of humans and other animals. It is the causative agent for a wide range of diseases including food borne diseases, gas gangrene and flesh eating disease called necrotizing fasciitis. C. perfringens is commonly found on raw meat and poultry that espouse to grow in conditions with very little or no oxygen, and under ideal conditions can multiply very rapidly. These conditions are occasionally lethal due to the substantial number of toxins such as alpha toxin, beta toxin, epsilon toxin and iota toxin produced by C. perfringens. It is significantly important to analyze the Drug targets of the pathogen in order to destroy them. Objective: The present work aims in identifying potential drug targets in C. perfringens through metabolic pathway analysis. Method: Primarily, the metabolic pathways of the host and pathogen are compared to identify unique pathways in the bacteria. Among the enzymes that catalyze unique metabolic pathways, the essential ones for the survival of the pathogen are identified. The druggability of the essential enzymes are predicted through identification of its sub cellular localization and other druggable parameters. Results: The comparative metabolic pathway analysis result shows that, among the 98 metabolic pathways of C.perfringens, 25 pathways were unique that they did not have a counterpart with Human. There were 113 enzymes involved in these unique pathways. The NCBI’s protein Blast search against human was done to identify the non-homologous proteins. There were 93 non-homologous proteins. Among the 93 non-homologous proteins, 47 proteins were found to be essential. Based on their sub-cellular localization, 32 proteins were identified as potential drug targets and 15 are probable vaccine candidates. Conclusion: The present work which started with 25 different pathways with more than a hundred different enzymes, resulted in the identification of 32 putative drug targets against C.perfringens infection. All these 32 identified targets did not have any human homolog and are highly essential for the survival of the organism. They were concluded as potential drug targets. Designing of compounds to inhibit these enzymes would be successful for treating the life threatening infections caused by this pathogen.