Perfringens Strains Research Articles

Prevalence, toxin-typing, and antimicrobial susceptibility of Clostridium perfringens from retail meats in Seoul, Korea

Clostridium perfringens is a ubiquitous, Gram-positive, spore-forming bacterium. It can contaminate many types of retail meat products and cause food poisoning by producing enterotoxins in the small intestines of humans and domestic animals. We investigated the prevalence, toxin-encoding gene profile, and antimicrobial resistance of C. perfringens in beef, chicken, and pork meat purchased from retail markets in Seoul, Korea. C. perfringens was detected according to the International Organization for Standardization 7937, with some modifications, and confirmed using the Vitek 2 system. In total, 38 C. perfringens strains were isolated from 200 meat samples (38/200, 19%; thirty-three from chicken, and five from beef). Among the six toxins evaluated, including alpha, beta, epsilon, iota, enterotoxin (encoded in the cpe gene), and netB, only the cpa gene was detected in all isolates by polymerase chain reaction (PCR) amplification. The antimicrobial resistance of the isolates was evaluated using the agar dilution method and resistance to ampicillin (12/38, 31.6%), tetracycline (38/38, 100%), chloramphenicol (26/38, 68.4%), metronidazole (13/38, 34.2%), and imipenem (27/38, 71%) was observed. Interestingly, 30 of the 38 isolates (78.9%) were multiple-drug resistant, showing resistance to more than three different antimicrobial classes.

Interactive effect of 2 dietary calcium and phytase levels on broilers challenged with subclinical necrotic enteritis: part 1—broiler performance, gut lesions and pH, bacterial counts, and apparent ileal digestibility

This study investigated the hypothesis that high dietary calcium (Ca) would stimulate necrotic enteritis (NE) and reduce performance, gut health, and nutrient digestibility, and if increased, phytase would reduce NE. Ross 308 male broilers (n = 768) were randomly distributed to 8 treatments in a factorial arrangement. Factors were NE challenge (no or yes), phytase level (500 or 1,500 FTU/kg using 500 FTU/kg matrix values), and Ca level (0.6 or 1.0% starter, 0.5 or 0.9% grower, 0.4 or 0.8% finisher) with the same level of available P (0.40 S, 0.35 G, and 0.35 F). There were 48 pens, 16 birds per pen and 6 replications. Half of the birds were challenged with Eimeria spp on day 9 and 108 CFU per mL of Clostridium perfringens strain EHE-NE18 on day 14 and 15. Gain was higher in birds fed high phytase on day 14 (P < 0.01), day 21 (P < 0.01), day 28 (P < 0.01), and day 35 (P < 0.01). Birds fed high phytase had greater livability on day 21 (P < 0.01). Ca was more digestible in high-Ca diets on day 16, and an NE × Ca interaction (P < 0.05) showed this effect to be more pronounced in unchallenged than in challenged birds. A challenge × Ca interaction for apparent ileal digestibility (AID) of crude protein (CP) (P < 0.05) indicated lower AID of CP in challenged birds fed high Ca. The challenge decreased AID of Ca (P < 0.01). Ca level had no impact on C. perfringens count, but it decreased Lactobacillus (P < 0.05) and Bifidobacteria (P < 0.05) populations in the ceca. High dietary Ca decreased feed conversion ratio. Overall (42 D), the highest WG was observed in unchallenged birds fed high Ca and high phytase with the lowest WG observed in NE-challenged birds fed low Ca and low phytase. The results suggest that full matrix values for high doses of phytase may be appropriate during NE challenge.

Inside environmental Clostridium perfringens genomes: antibiotic resistance genes, virulence factors and genomic features.

Until recently, research has focused on Clostridium perfringens in clinical settings without considering environmental isolates. In this study, environmental genomes were used to investigate possible antibiotic resistance and the presence of virulence traits in C. perfringens strains from raw surface water. In silico assembly of three C. perfringens strains, DNA generated almost complete genomes setting their length ranging from 3.4 to 3.6 Mbp with GC content of 28.18%. An average of 3,175 open reading frames was identified, with the majority associated with carbohydrate and protein metabolisms. The genomes harboured several antibiotic resistance genes for glycopeptides, macrolide-lincosamide-streptogramin B, β-lactam, trimethoprim, tetracycline and aminoglycosides and also the presence of several genes encoding for polypeptides and multidrug resistance efflux pumps and 35 virulence genes. Some of these encode for haemolysins, sialidase, hyaluronidase, collagenase, perfringolysin O and phospholipase C. All three genomes contained sequences indicating phage, antibiotic resistance and pathogenic islands integration sites. A genomic comparison of these three strains confirmed high similarity and shared core genes with clinical C. perfringens strains, highlighting their health security risks. This study provides a genomic insight into the potential pathogenicity of C. perfringens present in the environment and emphasises the importance of monitoring this niche in the future.

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.

Over-processed meat and bone meal and phytase effects on broilers challenged with subclinical necrotic enteritis: Part 2. Inositol phosphate esters hydrolysis, intestinal permeability, hematology, jejunal gene expression and intestinal morphology.

This study investigated the hypothesis that feeding broilers over-processed meat and bone meal (MBM) would impair gut health in the absence of phytase and in turn, affect inositol phosphate (inositol x-phosphate, IPx: IP3, IP4, IP5 and IP6) ester hydrolysis, intestinal permeability, hematology, jejunal gene expression and intestinal morphology during necrotic enteritis (NE). Ross 308 male broilers (n = 768) were assigned to one of 8 dietary treatments in a 2 × 2 × 2 factorial arrangement, with 6 replicate pens per diet and 16 birds per pen in a completely randomized design. Factors were: NE challenge (no or yes), phytase level (500 or 5,000 FTU/kg) and MBM processing (as-received or over-processed). For the NE challenge, half of the birds were challenged with field strains of Eimeria spp. on d 9 and 108 CFU/mL of Clostridium perfringens strain EHE-NE18 on d 14 and 15. A 3-way challenge, phytase and MBM processing interaction was detected for IP5 (P < 0.05) and IP6 (P < 0.05) levels in the ileum. Birds fed low phytase had increased IP5 and IP6 in unchallenged birds only when diets contained over-processed MBM. Challenge with NE increased intestinal permeability as measured by serum fluorescein isothiocyanate dextran (FITC-d; P < 0.001), increased white blood cells (WBC; P < 0.001), decreased mean corpuscular volume (MCV; P < 0.001) and mean corpuscular hemoglobin (MCH; P < 0.05), and decreased crypt-to-villi ratio (P < 0.05). The over-processed MBM reduced the villi-to-crypt ratio (P < 0.05). A 3-way challenge × phytase × MBM processing interaction was detected for mucin 2 (MUC-2) expression (P < 0.05) where only in unchallenged birds fed over-processed MBM did high phytase reduce MUC-2 expression. A lower expression of aminopeptidase N (APN; P < 0.001) and vitamin D receptor (VDR; P < 0.001) were recorded in NE challenged birds. In conclusion, NE has a negative impact on the gut and hematology of broilers, but its effect on phytate hydrolysis is minimal.

Interactive effect of dietary calcium and phytase on broilers challenged with subclinical necrotic enteritis: 3. Serum calcium and phosphorus, and bone mineralization

Calcium is chelated by phytic acid and forms phytate-mineral complexes reducing Ca availability and the ability of phytase to hydrolyze phytate. An increased Ca concentration in the gut favors the activity of Clostridium perfringens (C. perfringens). Therefore, it was hypothesized that high dietary calcium with high dietary phytase would decrease serum Ca and P and bone mineralization during necrotic enteritis occurrence. A total of 768 one-day-old Ross 308 male chicks were randomly allocated to 8 treatments with 6 replicate pens, each housing 16 birds. A 2 × 2 × 2 factorial arrangement of treatments was applied: dietary Ca (0.6 or 1.0%), phytase (500 or 1,500 FTU/kg), and challenge (no or yes). Half of the birds (384) were challenged with Eimeria spp. on day 9 and C. perfringens strain EHE-NE18 on day 14 and 15. Blood was collected from 2 birds per pen to determine Ca, P, and parathyroid hormone in the serum. The middle toe, tibia, and femur were excised from 2 birds per pen on day 16 and 29 for determination of ash, breaking strength (BS), and mineral concentration. The challenge decreased (P < 0.05) serum Ca+ in birds regardless of dietary Ca level (day 16). There was a challenge × Ca interaction (P < 0.05) for tibial BS (day 16), with challenge being more severe in birds fed high Ca than low Ca diets. A challenge × phytase interaction (P < 0.05) was present for femur ash (day 16), with high phytase only increasing ash in challenged birds. The challenge decreased (P < 0.05) the BS of femur and tibia at each time point. Birds fed high dietary Ca had lower tibial Mg (P < 0.001), Fe (P < 0.001), Na (P < 0.001), and Zn (P < 0.05) concentrations (day 29). Altogether, high dietary Ca and phytase improved bone mineralization showing that attention to Ca and P nutrition and phytase matrix values is warranted when high levels of phytase are used.

Over-processed meat and bone meal and phytase effects on broilers challenged with subclinical necrotic enteritis: Part 1. Performance, intestinal lesions and pH, bacterial counts and apparent ileal digestibility.

This feeding study investigated the hypothesis that over-processing of meat and bone meal (MBM) would impair the performance, gut health and ileal digestibility of nutrients in birds challenged with necrotic enteritis (NE). The effect of phytase (500 vs. 5,000 FTU/kg) was also examined using manufacturers recommended matrix values for 500 FTU for both levels. Ross 308 male broilers (n = 768) were assigned to 8 diets, with 6 replicate pens per diet and 16 birds per replicate pen using a randomized design with a factorial arrangement of treatments. Factors were NE challenge (no or yes), MBM (as received or over-processed), and phytase level (500 or 5,000 FTU/kg). Half of the birds were challenged with 5,000 oocysts of field strains of Eimeria acervulina and Eimeria brunetti, and 2,500 oocysts of Eimeria maxima on d 9 and 108 CFU/mL of Clostridium perfringens strain EHE-NE18 on d 14 and 15 post-hatch. Challenge × MBM interactions were detected for weight gain (WG), feed conversion ratio (FCR) and feed intake (FI) at d 14, 21 and 28, showing that challenged birds fed over-processed MBM had decreased WG (P < 0.05) and FI (P < 0.05) at d 14, increased FCR (P < 0.05) at d 21 and decreased WG (P < 0.05) and FI (P > 0.05) at d 28. Birds fed low phytase had increased livability (P < 0.05) at d 42. The challenge increased the prevalence and severity of NE induced lesions in the jejunum (P < 0.05) and ileum (P < 0.05). The birds fed over-processed MBM had decreased pH in the jejunum (P < 0.05) and ileum (P < 0.05) at d 16. High phytase increased apparent ileal digestibility (AID) of Ca (P < 0.05) and P (P < 0.05), and over-processed MBM increased AID of carbon (C; P < 0.05) and Ca (P < 0.05) at d 29. The challenge increased the caecal counts of Lactobacillus spp. (P < 0.05) and C. perfringens (P < 0.05) at d 16. The results indicated that supplementation of diets with high phytase reduces the negative impact on performance from over-processed MBM during NE as a result of increased nutrient digestibility.

Persistence of a Clostridium perfringens Strain in a Broiler Chicken Farm over a Three-Year Period.

Clostridium perfringens, a commensal of the intestinal tract of many animal species, has been associated with necrotic enteritis (NE), an economically significant poultry disease. Clostridium perfringens is known to survive in the environment for extended periods of time through the formation of spores. These spores have the potential to be transmitted to subsequent flocks. Persistence of a single C. perfringens strain in a broiler chicken farm environment has, however, been poorly documented. The aim of this study was to compare multiple isolates of C. perfringens collected over time in a single farm with recurrent episodes of NE. Isolates were recovered from the intestines of chickens affected with NE (2014 and 2016 outbreaks) and from healthy chickens (2017), as well as from environmental samples (2016). PCR characterization of those isolates showed that all sampling groups contained netB-positive isolates except for the environmental samples. Moreover, results showed that all environmental isolates were positive for the cna adhesin whereas other groups had lower numbers of cna-positive isolates. Biofilm formation assays showed that most of the isolates were able to form biofilm. Pulsed-field gel electrophoresis analysis showed that one clone was present in every sampling group, with the exception of the 2014 outbreak. However, one clone found in the latter group was highly similar, having 94% similarity with the persistent C. perfringens clone. This study describes for the first time the persistence of a C. perfringens strain on a broiler chicken house over a 3-yr period.

Influence of meat and bone meal, phytase, and antibiotics on broiler chickens challenged with subclinical necrotic enteritis: 2. intestinal permeability, organ weights, hematology, intestinal morphology, and jejunal gene expression

Undigested proteins entering the hindgut may favor the proliferation of Clostridium perfringens. Using phytase to eliminate the need for meat and bone meal (MBM) as a P source may reduce potential infection with C. perfringens. A study was conducted to determine the impact of MBM, phytase, and antibiotics (AB) on intestinal permeability and morphology, organ weights, and jejunal gene expression in Ross 308 chickens challenged with subclinical necrotic enteritis (NE). Male Ross 308-day-old chicks (672 each) were randomly allocated to 8 treatments with 6 replicate pens each housing 14 birds. A 2 × 2 × 2 factorial arrangement of treatments was used: MBM (no or yes); AB (no or yes—Zn bacitracin 100 in S and 50 ppm in G/F and salinomycin Na 60 ppm in all phases); phytase (500 or 1,500 FTU/kg, both using 500 FTU matrix values) using wheat-SBM-canola meal diets. Birds were challenged with Eimeria spp on day 9, and C. perfringens strain EHE-NE18 on day 14 and 15. An AB × MBM interaction (P < 0.05) was detected for relative gizzard weight (with contents) being lower in birds fed MBM and AB compared to those fed MBM and no AB. A MBM × AB interaction (P > 0.01) was detected for lymphocyte counts being lower with MBM and AB compared to MBM without AB. A phytase × AB interaction (P < 0.05) was observed for villi length being increased with high phytase and no AB compared to with AB. Inclusion of MBM increased (P < 0.05) blood FICT-d concentration, whereas AB decreased it (P < 0.05). Antibiotics increased RBC (P < 0.05), Hgb (P < 0.05), and PCV (P < 0.05) and expression of Ca-binding protein, CALB1 (P > 0.05). Inclusion of MBM decreased expression of MUC2 (P < 0.05). Results indicate that dietary MBM has a detrimental effect on gut health of broilers but this may be counteracted using AB.

β-Lactam resistance development affects binding of penicillin-binding proteins (PBPs) of Clostridium perfringens to the fluorescent penicillin, BOCILLIN FL

Alteration in the binding of bacterial penicillin-binding proteins (PBPs) to β-lactams is important in the development of drug resistance. The PBPs of wild type Clostridium perfringens ATCC 13124 and three β-lactam-resistant mutants were compared for the ability to bind to a fluorescent penicillin, BOCILLIN FL. The binding of the high molecular weight protein PBP1, a transpeptidase, to BOCILLIN FL was reduced in all of the resistant strains. In contrast, the binding of BOCILLIN FL to a low molecular weight protein, PBP6, a D-alanyl-d-alanine carboxypeptidase that was more abundant in all three resistant strains, was substantially increased. A competition assay with β-lactams reduced the binding of all of the PBPs, including PBP6, to BOCILLIN FL. β-Lactams enhanced transcription of the putative gene for PBP6 in both wild type and resistant strains. This is the first report showing that mutations in a high molecular weight PBP and overexpression of a low molecular weight PBP in resistant C. perfringens strains affected their binding to β-lactams.

Influence of meat and bone meal, phytase, and antibiotics on broiler chickens challenged with subclinical necrotic enteritis: 1. growth performance, intestinal pH, apparent ileal digestibility, cecal microbiota, and tibial mineralization

This study investigated the influence of meat and bone meal (MBM), phytase, and antibiotics (AB) on the performance, intestinal pH, ileal digestibility, cecal microbiota, and tibial mineralization in Ross 308 broilers challenged with necrotic enteritis (NE). A total of 672-day-old male Ross 308 chicks were allocated to 8 treatments with 6 replicate pens, with 14 birds each. The study employed a 2 × 2 × 2 factorial arrangement of treatments: MBM (no or yes), AB (no or yes, zinc bacitracin + salinomycin), and phytase level (500 or 1,500 FTU/kg; both using 500 matrix recommendations). Diets were based on wheat–soybean meal–canola meal. All birds were challenged with Eimeria spp on day 9 and Clostridium perfringens (C. perfringens) strain EHE-NE18 on day 14 and day 15. On day 21 (postchallenge), birds fed MBM had reduced weight gain (WG; P < 0.05) relative to without MBM. A 2-way phytase × AB interaction for WG on day 14 (P < 0.001) and day 21 (P < 0.001) and feed conversion ratio on day 21 (P < 0.001) and day 42 (P < 0.01) indicated positive effects of high phytase on bird performance in the presence of AB. On day 42, a 3-way MBM × phytase × AB interaction (P < 0.01) was observed for WG, showing high phytase increased WG with AB, relative to the birds without AB in the presence of MBM. A 2-way MBM × phytase interaction (P < 0.01) was observed for apparent ileal digestibility of Ca and P on day 16, whereby there was a notable reduction in Ca and P digestibility in birds fed MBM-free diets and a low phytase level, but with the high phytase level, Ca and P digestibility was not influenced by MBM. In conclusion, in NE challenged birds, high phytase has a beneficial effect on leg health and mineral utilization to the extent that it can replace MBM and has beneficial effects on bird performance in the presence of AB.

Focal duodenal necrosis in chickens: attempts to reproduce the disease experimentally and diagnostic considerations.

Focal duodenal necrosis (FDN) is an intestinal disease of egg-layer chickens characterized by multifocal necrosis of the duodenal loop and proximal jejunum. Affected flocks usually have decreased egg weights and drops in egg production. Previous studies have associated this condition with Clostridium perfringens infection. We tried to reproduce FDN by experimental infection of egg-laying chickens using different netB-positive and netB-negative C. perfringens strains, and duodenal homogenate obtained from FDN lesions. Chickens challenged with C. perfringens and/or duodenal homogenate developed duodenitis after challenge. Gross lesions included mucosal erosions, hyperemia, mucosal hemorrhages, and watery intestinal content. Microscopic lesions included mild enterocyte degeneration and necrosis, and mild-to-moderate hemorrhage and lymphoplasmacytic and heterophilic infiltration of the lamina propria. Two netB-positive C. perfringens strains closely related to necrotic enteritis pathogenic strains, by genomic composition, were re-isolated from lesions. Necrosis of intestinal crypts was observed in chickens challenged with duodenal homogenate with or without C. perfringens coinfection. Characteristic microscopic FDN lesions with significant necrosis and loss of villus enterocytes were not reproduced.

Draft Genome Sequence of Clostridium perfringens Strain TAMU, Which Causes Necrotic Enteritis in Broiler Chickens.

Clostridium perfringens causes severe gastrointestinal diseases, which include necrotic enteritis (NE) in chickens, a deadly disease worldwide. We report here the draft genome sequence of Clostridium perfringens strain TAMU, which was used in developing an NE chicken challenge model. This C. perfringens TAMU genome sequence will aid in advancing potential intervention strategies to reduce NE pathogenesis.

Isolation and characterization of Clostridium perfringens strains isolated from ostriches (Struthio camelus) in Vietnam.

Background and Aim:Clostridium perfringens can cause enteritis in ostriches. The toxin release is believed to play a major role in determining pathogenesis properties of these pathogenic bacteria. This study was conducted to isolate and characterize C. perfringens strains from ostriches in Vietnam for identifying if particular virulence factors of these pathogenic bacteria are associated with enteritis progress in ostriches.Materials and Methods:The prevalence of cpa, cpb, iA, etx, cpe, and cpb2 genes among C. perfringens isolates was determined by a multiplex polymerase chain reaction (PCR) method. The NetB toxin-encoding gene was detected by PCR and then sequenced to observe their variation. The expression of NetB toxin was checked by SDS-PAGE.Results:A total of 116 C. perfringens isolates were obtained from 318 fecal samples and 105 intestinal organs. Of 80 isolates from fecal samples, 33 isolates were from healthy and 47 isolates were from diseased ostriches. The results of multiplex PCR showed that all 116 C. perfringens strains from healthy and enteric disordered ostriches were positive for the alpha toxin-encoding gene (cpa). The cpe and cpb2 genes were found in only one and five diseased ostriches, respectively. The netB gene was detected in 1/33 (3.03%) C. perfringens isolates from healthy ostriches, in 8/47 (17.05%) isolates from feces, and in 7/36 (19.44%) intestinal contents of diseased ostriches. The full-length sequences of 5 out of 15 netB-positive isolates from diseased ostriches showed 100% identity to each other as well as to the netB sequences available in GenBank. All of these five isolates produced NetB toxin in vitro.Conclusion:Type A is the most prevalent among C. perfringens isolates from ostriches in Vietnam. Especially, the study provides data emphasizing the role of NetB toxin in causing necrotic enteritis by C. perfringens in ostriches.

Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression

BackgroundClostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and a cause of necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of drug-resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains.ResultsAn Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) containing a polyhistidine tag was expressed in E. coli. In addition, The E. coli optimized endolysin gene was engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X (PVX)-based transient expression vector. PlyCP41p accumulated to ~ 1% total soluble protein (100μg/gm f. wt. leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal systemic expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue.ConclusionWe demonstrated that functionally active bacteriophage PlyCP41 endolysin can be produced in systemically infected plant tissue with potential for use of crude plant sap as an effective antimicrobial agent against C. perfringens.

Two different Clostridium perfringens strains produce different levels of necrotic enteritis in broiler chickens

Subclinical necrotic enteritis (NE) is primarily caused by the gram-positive bacterium, Clostridium perfringens (Cp). The trend towards removal of in-feed antimicrobials and subsequent increased emergence of infection in poultry has resulted in a wide interest in better understanding of the mechanism behind this disease. The virulence of NE, to a large extent, depends on the virulence of Cp strains. Thus, this study was to assess how 2 different strains of Cp affect performance and gut characteristics of broiler chickens. Ross 308 male broilers (n = 468) were assigned to a 2 × 3 factorial arrangement of treatments with antibiotics (Salinomycin at 72 ppm and zinc bacitracin at 50 ppm −, or +) and challenge (non-challenge, Cp EHE-NE18, or Cp WER-NE36). Oral administration of Eimeria oocysts (day 9) followed by inoculation with 1 mL 108 CFU Cp strains (day 14 and 15) were used to induce NE. Broiler performance was analyzed at day 10, 24, and 35. On day 16, intestinal lesion score and intestinal pH were evaluated and samples of cecal content were analyzed for bacterial counts and short-chain fatty acid concentrations (SCFA). Birds in both challenged groups showed higher feed conversion ratio (FCR), lower weight gain (P < 0.001), increased lesion scores in the jejunum (P < 0.01), and reduced pH in the ileum and cecum (P < 0.01), compared to the non-challenged birds. They also showed decreased numbers of Bacillus spp. (P < 0.001), and Ruminococcus spp. (P < 0.01) in the cecal content. On day 35, the NE36 challenged birds had a lower weight gain (P < 0.001) and higher FCR (P < 0.001) compared to the NE18 challenged birds. Interestingly, cecal Lactobacillus and lactate were increased by the NE challenge (P < 0.001), and to a greater extent in birds challenged with NE36 compared to the NE18 strain (P < 0.001). This study suggests that Cp strains varying in virulence produce different levels of disease in broiler chickens through modulating the gut environment, intestinal microbiota, and SCFA profile to different extents.

The antibiotic susceptibility pattern of gas gangrene-forming Clostridium spp. clinical isolates from South-Eastern Hungary

Introduction: Clostridium perfringens and other gas gangrene-forming clostridia are commensals of the human gut and vaginal microbiota, but can cause serious or even fatal infections. As there are relatively few published studies on antibiotic susceptibility of these bacteria, we decided to perform a 10-year retrospective study in a South-Eastern Hungarian clinical centre.Methods: A total of 372 gas gangrene-forming Clostridium spp. were isolated from clinically relevant samples and identified with rapid ID 32A (bioMérieux, France) and MALDI-TOF MS (Bruker Daltinics, Germany) methods. Antibiotic susceptibility was determined with E-tests.Results: We identified 313 C. perfringens, 20 C. septicum, 10 C. sordellii, 10 C. sporogenes, 9 C. tertium, 6 C. bifermentans, 4 C. histolyticum isolates. In C. perfringens isolates, the rate of penicillin resistance was 2.6% and the rate of clindamycin resistance 3.8%. Penicillin resistance was found in 6.8% and clindamycin resistance in 8.5% of the non-perfringens Clostridium spp. isolates.Conclusion: The antibiotic susceptibility of C. perfringens isolates was in good agreement with previous publications. The rates of resistance to penicillin and clindamycin were very low. The resistance rates of non-perfringens Clostridium spp. isolates were higher than those of C. perfringens strains, but lower than those published in the literature.

In silico design and in vitro analysis of a recombinant trivalent fusion protein candidate vaccine targeting virulence factor of Clostridium perfringens

Necrotic enteritis (NE) is a multifactorial disease in broiler that is caused by colonization of Clostridium perfringens in their gastrointestinal tract. Recently several immunogenic proteins from virulent C. perfringens have been considered as vaccines to provide protection against NE. In this study, a novel trivalent fusion protein including immunogenic epitopes of three virulence factors of, NetB, alpha toxin and a metallopeptidase protein (NAM) was designed using in silico studies. Circular dichroism spectra was applied for determination of secondary structure and folding properties of the purified recombinant NAM (rNAM) expressed in E. coli. The antigenicity of rNAM was confirmed by induction of immune response in rabbit and neutralization experiments of the toxins in cell culture studies. To this end, anti-rNAM antisera neutralized the crude toxins produced by a wild type virulent C. perfringens strain using chicken hepatocellular carcinoma (LMH) cell lines. The cells were exposed to a mixture of anti-rNAM antisera and 2 × LD50 doses of the toxins. The result showed 94% viability of the cells against the crude toxins, in the presence of anti-rNAM antisera. Our study suggests that combination of metallopeptidase protein along with alpha toxin and NetB toxins is a potent immunogen which is able to neutralize the toxicity of crude extracellular toxins. The recombinant chimeric NAM could be a suitable and effective subunit vaccine candidate to prevent NE disease caused by C. perfringens.

Genomic analysis on broiler-associated Clostridium perfringens strains and exploratory caecal microbiome investigation reveals key factors linked to poultry necrotic enteritis

BackgroundClostridium perfringens is a key pathogen in poultry-associated necrotic enteritis (NE). To date there are limited Whole Genome Sequencing based studies describing broiler-associated C. perfringens in healthy and diseased birds. Moreover, changes in the caecal microbiome during NE is currently not well characterised. Thus, the aim of this present study was to investigate C. perfringens virulence factors linked to health and diseased chickens, including identifying putative caecal microbiota signatures associated with NE.ResultsWe analysed 88 broiler chicken C. perfringens genomes (representing 66 publicly available genomes and 22 newly sequenced genomes) using different phylogenomics approaches and identified a potential hypervirulent and globally-distributed clone spanning 20-year time-frame (1993–2013). These isolates harbored a greater number of virulence genes (including toxin and collagen adhesin genes) when compared to other isolates. Further genomic analysis indicated exclusive and overabundant presence of important NE-linked toxin genes including netB and tpeL in NE-associated broiler isolates. Secondary virulence genes including pfoA, cpb2, and collagen adhesin genes cna, cnaA and cnaD were also enriched in the NE-linked C. perfringens genomes. Moreover, an environmental isolate obtained from farm animal feeds was found to encode netB, suggesting potential reservoirs of NetB-positive C. perfringens strains (toxinotype G). We also analysed caecal samples from a small sub-set of 11 diseased and healthy broilers for exploratory microbiome investigation using 16S rRNA amplicon sequencing, which indicated a significant and positive correlation in genus Clostridium within the wider microbiota of those broilers diagnosed with NE, alongside reductions in beneficial microbiota members.ConclusionsThese data indicate a positive association of virulence genes including netB, pfoA, cpb2, tpeL and cna variants linked to NE-linked isolates. Potential global dissemination of specific hypervirulent lineage, coupled with distinctive microbiome profiles, highlights the need for further investigations, which will require a large worldwide sample collection from healthy and NE-associated birds.

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.