Genomic analysis of Clostridium perfringens type D isolates from goat farms.

C. perfringens type D strains are the leading cause of enterotoxaemia in ruminants such as goats, sheep, and cattle. However, there has been no prior research on the genomic characteristics of C. perfringens type D strains from various regions in China. Here, we investigated the antibiotic resistance, genomic characteristics, and phylogenetic relationship of C. perfringens type D isolates recovered from goat farms in Shaanxi, Gansu, and Ningxia provinces. The antibiotic resistance test indicated that the isolates displayed high minimum inhibitory concentration (MIC) values to sulfafurazole, whereas the other antibiotics tested, such as penicillin, enrofloxacin, and florfenicol, worked well on them. Additionally, only tetracycline resistance genes [tetA(P) and tetB(P)] were identified from the isolates. A collective of 13 toxin genes, including etx and cpe were detected among the isolates. Sequence comparison revealed that the etx and cpe genes shared high sequence identities, and they could coexist on a pCW3-like plasmid, representing a potential risk to both animal breeding and public health. Phylogenetic analysis using core genome multi-locus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (SNPs) revealed the close genetic relationship and potential regional/transregional transmission of the C. perfringens type D isolates in Shaanxi and Gansu provinces. Furthermore, pan-genomic analysis suggested the functional differences at the protein-coding gene level, although isolates from the same source shared a close genetic relationship. In conclusion, this study indicated the antibiotic resistance, virulence markers, potential transregional transmission, and genomic diversity of C. perfringens type D strains from various regions in China, which could provide references for the prevention of C. perfringens foodborne diseases and further research.

Identification and structure-based engineering of a dipeptidase CpPepD from Clostridium perfringens for the synthesis of l-carnosine.

l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94 mM (14.46 g L-1) or 67.02 mM (15.16 g L-1) l-Car was produced at the substrate concentrations of 6 M ß-Ala and 0.2 M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.

Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors.

Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.

Overexpressing the cpr1953 Orphan Histidine Kinase Gene in the Absence of cpr1954 Orphan Histidine Kinase Gene Expression, or Vice Versa, Is Sufficient to Obtain Significant Sporulation and Strong Production of Clostridium perfringens Enterotoxin or Spo0A by Clostridium perfringens Type F Strain SM101.

The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan-Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production.

The impact of indole and mucin on sporulation, biofilm formation, and enterotoxin production in foodborne Clostridium perfringens.

AIMS: Indole and mucin are compounds found in the host environment as they are produced by the host or by the host-associated microbiota. This study investigated whether indole and mucin impact Clostridium perfringens growth and sporulation, as well as enterotoxin production and biofilm formation. METHODS AND RESULTS: There was no impact on growth of Cl. perfringens for up to 400 µM indole and 240 mg/l mucin, and neither indole nor mucin affected sporulation. Reverse-transcriptase qPCR showed that mucin strongly upregulated the expression of Cl. perfringens enterotoxin (up to 121-fold increase), whereas indole had a much more modest effect (2-fold). This was also reflected in increased Cl. perfringens enterotoxin levels in mucin-treated Cl. perfringens (as assessed by a reversed passive latex agglutination assay). Finally, mucin and indole significantly increased biofilm formation of Cl. perfringens, although the effect size was relatively small (less than 1.5 fold). CONCLUSION: These results indicate that Cl. perfringens can sense its presence in a host environment by responding to mucin, and thereby markedly increased enterotoxin production.

Prevalence, toxin-genotype distribution, and transmission of Clostridium perfringens from the breeding and milking process of dairy farms.

This study aimed to elucidate the distribution, transmission, and cross-contamination of Clostridium perfringens during the breeding and milking process from dairy farms. The prevalence of 22.3% (301/1351) yielded 494 C. perfringens isolates; all isolates were type A, except for one type D, and 69.8% (345/494) of the isolates carried atyp. cpb2 and only 0.6% (3/494) of the isolates carried cons. cpb2. C. perfringens detected throughout the whole process but without type F. 150 isolates were classified into 94 pulsed-field gel electrophoresis (PFGE) genotypes; among them, six clusters contained 34 PFGE genotypes with 58.0% isolates which revealed epidemic correlation and genetic diversity; four PFGE genotypes (PT57, PT9, PT61, and PT8) were the predominant genotypes. The isolates from different farms demonstrated high homology. Our study confirmed that C. perfringens demonstrated broad cross-contamination from nipples and hides of dairy cattle, followed by personnel and tools and air-introduced raw milk during the milking process. In conclusion, raw milk could serve as a medium for the transmission of C. perfringens, which could result in human food poisoning. Monitoring and controlling several points of cross-contamination during the milking process are essential as is implementing stringent hygiene measures to prevent further spread and reduce the risk of C. perfringens infection.

Research Note: Clostridium perfringens NetB and CnaA neutralizing nanobodies in feed reduce the incidence of poultry necrotic enteritis.

Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed.

Clostridium perfringens Epsilon Toxin Mutant I51C as a Recombinant Vaccine Candidate Against Enterotoxemia.

BACKGROUND: Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which can lead to enterotoxemia, an extremely lethal disease that has significant consequences for the farming of domestic ruminants, specifically sheep and goats. The bacterin-toxoids/toxoids enterotoxemia vaccines need time-consuming detoxification steps. Genetically derived toxoids (GTs) can be the alternative vaccines against ETX-associated enterotoxemia. This study was aimed to design, synthesize, and evaluate of five epsilon toxin mutants of C. perfringens by site-directed mutagenesis (SDM). METHODS: In this study, five ETX mutants (H106P, I51C, V56C, A114C, and F118C), as ETX-GTs, were designed and synthesized by SDM, which were then cloned in pET-26b (+) and expressed in Escherichia coli /BL21 (DE3). The expression of recombinant ETX-GTs was evaluated by SDS-PAGE, blotting, and ELISA and their toxicity was evaluated by the residual toxicity test based on BP Pharmacopoeia, 2021. RESULTS: The findings showed that the ETX-GTs could be considered alternative vaccine candidates against ETX-associated enterotoxemia. CONCLUSIONS: These data suggest that I51C mutant could form the basis of an improved recombinant vaccine against enterotoxemia.

Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model.

BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

Determination of the virulence status of Clostridium perfringens strains using a chicken intestinal ligated loop model is important for understanding the pathogenesis of necrotic.

Necrotic enteritis (NE) is a poultry intestinal disease caused by virulent strains of the bacterium Clostridium perfringens (C. perfringens). This anaerobic bacterium produces a wide range of enzymes and toxins in the gut which leads to NE development. It is generally accepted by the poultry veterinarians that netB-positive C. perfringens strains are virulent and netB-negative strains do not cause NE. However, NE pathogenesis remains unclear as contradictory results have been reported. The use of experimental in vivo models is a valuable tool to understand the pathogenesis of a disease. In this study, a chicken ligated loop model was used to determine the virulence status of 79 C. perfringens strains from various geographical locations, sources, and genotype profiles. According to our model and based on histologic lesion scoring, 9 C. perfringens strains were classified as commensal, 35 as virulent, and 34 as highly virulent. The virulence of only 1 C. perfringens strain could not be classified as its lesion score was variable (from <10 to >15). In general, NE lesions were more severe in intestinal loops inoculated with netB-positive C. perfringens strains than those inoculated with netB-negative strains. The prevalence of netB among strains classified as commensal, virulent, and highly virulent was 56% (5/9), 54%, (19/35), and 59% (20/34). These results suggest that NetB is not required to cause NE lesions and that other factors are also involved. The classification of the virulence status of C. perfringens strains should not be based solely on the presence or absence of this toxin. Therefore, the use of an in vivo model is essential to distinguish commensal from virulent strains of C. perfringens.

In silico mutation of aromatic with aliphatic amino acid residues in Clostridium perfringens epsilon toxin (ETX) reduces its binding efficiency to Caprine Myelin and lymphocyte (MAL) protein receptors.

Enterotoxaemia (ET) is a severe disease that affects domestic ruminants, including sheep and goats, and is caused by Clostridium perfringens type B and D strains. The disease is characterized by the production of Epsilon toxin (ETX), which has a significant impact on the farming industry due to its high lethality. The binding of ETX to the host cell receptor is crucial, but still poorly understood. Therefore, the structural features of goat Myelin and lymphocytic (MAL) protein were investigated and defined in this study. We induced the mutations in aromatic amino acid residues of ETX and substituted them with aliphatic residues at domains I and II. Subsequently, protein-protein interactions (PPI) were performed between ETX (wild)-MAL and ETX (mutated)-MAL protein predicting the domain sites of ETX structure. Further, molecular dynamics (MD) simulation studies were performed for both complexes to investigate the dynamic behavior of the proteins. The binding efficiency between 'ETX (wild)-MAL protein' and 'ETX (mutated)-MAL protein complex' interactions were compared and showed that the former had stronger interactions and binding efficiency due to the higher stability of the complex. The MD analysis showed destabilization and higher fluctuations in the PPI of the mutated heterodimeric ETX-MAL complex which is otherwise essential for its functional conformation. Such kind of interactions with mutated functional domains of ligands provided much-needed clarity in understanding the pre-pore complex formation of epsilon toxin with the MAL protein receptor of goats. The findings from this study would provide an impetus for designing a novel vaccine for Enterotoxaemia in goats.Communicated by Ramaswamy H. Sarma.

Characterization and multilocus sequence typing of Clostridium perfringens isolated from patients with diarrhoea and food poisoning in Tai'an region, China.

OBJECTIVES: Clostridium perfringens (C. perfringens) is a significant opportunistic pathogen. This study aims to examine the occurrence of C. perfringens in patients with diarrhoea and food poisoning and compare the genetic similarities with strains found in poultry retail markets and poultry farms in the same city (Tai'an, China). METHODS: Clostridium perfringens was isolated from 30 human faecal samples and genotyped using multiplex PCR. The antimicrobial susceptibility test was conducted using the Kirby-Bauer disk diffusion method. Genetic relationships were analysed through Multi-locus sequence typing (MLST) and Phylogenetic analysis. RESULTS: The positive rate of C. perfringens was found to be 96.67%. Among the positive samples, 91.67% of the faecal samples from patients with food poisoning contained type F strains of C. perfringens, while only 16.67% of the samples from diarrhoea cases contained type F. The drug susceptibility test revealed that the majority of isolates displayed broad-spectrum antimicrobial resistance. Out of the 57 isolates tested for drug susceptibility, 89.47% demonstrated resistance to at least three antibiotics. The MLST results indicated that strains originating from the same host and environment tended to be more closely related. However, certain strains associated with food poisoning and diarrhoea in patients shared the same ST and CC as some strains found in the retail market. These strains were also found to be phylogenetically similar to some retail market strains, suggesting potential risks to human health. CONCLUSIONS: Therefore, it is crucial to enhance the management of poultry retail markets in order to mitigate these associated risks.

Isolation and Molecular Characterization of Clostridium perfringens Toxinotypes F & G in Diarrhoeic Sheep (Ovis aries) Flocks in Southeast of Iran.

Clostridial enteric diseases, called enterotoxemia, are caused by Clostridium perfringens toxinotypes in sheep and other ruminants. This study aimed to describe the molecular characterization of C. perfringens isolates in diarrhoeic sheep (Ovis aries) flocks in the southeast of Iran. Fecal/intestinal samples were collected from diarrhoeic (n=116), dead (n= 13), and healthy (n=63) sheep over four years (2016-2020) and subjected to bacteriological and molecular examinations. The C. perfringens isolates were typed by polymerase chain reaction targeting genes, namely 16SrRNA, CPA, CPB, ETX, IAP, CPE, and NetB. The overall prevalence of C. perfringens was 28.6% among the studied sheep, and there was a significant relationship between its isolation rate and diarrhea (P<0.001). The C. perfringens isolation rate also decreased with animal age (P=0.012) and was significantly higher in late winter and spring (P=0.000). The most prevalent toxinotypes were types A (52.4%), D (22.2%), and F (18.5%), in that order. Moreover, C, G, and B types were found in 4.2%, 1.6%, and 1.1% of the isolates, respectively, and no type E was detected. The CPE gene was detected in 32.3% of all isolates, and the diarrhoeic sheep were most likely to yield CPE+ strains of C. perfringens (93.1%). These findings highlight the importance of CPE+ strains of C. perfringens in sheep enteritis and suggest that the high presence of type F needs to be considered in new clostridial vaccines containing this toxinotype. It is noteworthy that the present study reported the isolation of C. perfringens type F, type G, and the CPE+ strains of type B from diarrhoeic sheep for the first time.

Baicalin-aluminum alleviates necrotic enteritis in broiler chickens by inhibiting virulence factors expression of Clostridium perfringens.

Clostridium perfringens type A is the main cause of necrotic enteritis (NE) in chickens. Since the use of antibiotics in feed is withdrawn, it is imperative to find out suitable alternatives to control NE. Baicalin-aluminum complex is synthesized from baicalin, a flavonoid isolated from Scutellaria baicalensis Georgi. The present study investigated the effects of baicalin-aluminum on the virulence-associated traits and virulence genes expression of C. perfringens CVCC2030, it also evaluated the in vivo therapeutic effect on NE. The results showed that baicalin-aluminum inhibited bacterial hemolytic activity, diminished biofilm formation, attenuated cytotoxicity to Caco-2 cells, downregulated the expression of genes encoding for clostridial toxins and extracellular enzymes such as alpha toxin (CPA), perfringolysin O (PFO), collagenase (ColA), and sialidases (NanI, NanJ). Additionally, baicalin-aluminum was found to negatively regulate the expression of genes involved in quorum sensing (QS) communication, including genes of Agr QS system (agrB, agrD) and genes of VirS/R two-component regulatory system (virS, virR). In vivo experiments, baicalin-aluminum lightened the intestinal lesions and histological damage, it inhibited pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) expression in the jejunal and ileal tissues. Besides, baicalin-aluminum alleviated the upregulation of C. perfringens and Escherichia coli and raised the relative abundance of Lactobacillus in the ileal digesta. This study suggests that baicalin-aluminum may be a potential candidate against C. perfringens infection by inhibiting the virulence-associated traits and virulence genes expression.

Acquisition site-based remodelling of Clostridium perfringens- and Clostridioides difficile-related gut microbiota.

INTRODUCTION: Clostridium perfringens is a gram-positive, anaerobic sporulating bacillus which can infect several hosts, thereby being considered the causative agent of many gut illnesses. Some studies have suggested that C. perfringens's virulence factors may negatively affect gut microbiota homeostasis by decreasing beneficial bacteria; however, studies have failed to evaluate the simultaneous presence of other pathogenic bacteria, such as C. difficile (another sporulating bacillus known to play a role in gut microbiota imbalance). Conscious of the lack of compelling data, this work has ascertained how such microorganisms' coexistence can be associated with a variation in gut microbiota composition, compared to that of C. perfringens colonisation. METHODS: PCR was thus used for identifying C. perfringens and C. difficile in 98 samples. Amplicon-based sequencing of 16S- and 18S-rRNA genes' V4 hypervariable region from such samples was used for determining the microbiota's taxonomical composition and diversity. RESULTS: Small differences were observed in bacterial communities' taxonomic composition and diversity; such imbalance was mainly associated with groups having hospital-acquired diarrhoea. CONCLUSION: The alterations reported herein may have been influenced by C. difficile and diarrhoea acquisition site, despite C. perfringens' ability to cause alterations in microbiota due to its virulence factors. Our findings highlight the need for a holistic view of gut microbiota.

Effect of feeding dairy calves with milk fermented with selected probiotic strains on occurrence of diarrhoea, carriage of pathogenic and zoonotic microorganisms and growth performance.

Calf-diarrhoea is a major health problem in dairy calves and a primary reason for use of antimicrobials. We aimed to investigate the effect of feeding milk fermented with a combination of four probiotic bacterial strains to young-calves on; occurrence of diarrhoea and associated-pathogens (bacteria, virus and parasites), shedding of Salmonella Dublin and Campylobacter, occurrence of virulence genes linked to Clostridium perfringens, Enterotoxigenic Escherichia coli and shiga-toxin producing E. coli (STEC), as well as growth performance. For this, 143 new-born calves from three Danish dairy-farms were allocated into Treatment- (fed the fermented milk for the first 8-weeks-of-life) and Control-groups (fed regular farm-milk). Diarrhoea was observed in 18.6 % (Farm 1), 22.4 % (Farm 2) and 15.7 % (Farm 3) of the total registrations mainly within the first 3-weeks-of-life. C. perfringens was the most frequently detected pathogen. The treatment did not affect the occurrence of virulence genes linked to STEC and C. perfringens and, overall, their detection levels were very low/undetected. The statistical model applied found no significant effect of the treatment on prevalence of early-diarrhoea (≤ 3 weeks), late-diarrhoea (>3 weeks), occurrence of C. perfringens and Cryptosporidium parvum or levels of Campylobacter spp. Limited detection of the other pathogens and associated virulence-genes under study, did not allow for assessment of the impact of the treatment on their occurrence. Notably, the feeding-approach showed a significant detrimental effect on daily-weight-gain. The inefficacy of the treatment may be associated with the complexity of influencing factors under field conditions including management practices.

[Study on biofilm formation and heterogeneity in Clostridium perfringens].

Many bacteria form biofilms and survive in the actual environment. Biofilms are not only a major form of bacteria but are also involved in tolerance to environmental stresses and antibiotics, suggesting the association with bacterial pathogenesis. Cells within biofilms display phenotypic heterogeneity; thus, even bacteria, unicellular organisms, can functionally differentiate and show multicellular behavior. Therefore, it is necessary to understand bacteria as a population to control their survival and pathogenesis in the actual environment. Previously, we found that Clostridium perfringens, an anaerobic pathogenic bacterium, form different structures in different temperatures and phenotypic heterogeneity on biofilm matrix gene expression within the biofilm. In this article, I summarize the results of our research on biofilms and their heterogeneity, the mechanisms of post-transcriptional gene expression regulation of virulence genes, and bacteria-host interactions mediated by extracellular membrane vesicles.

Gut-targeted nanoparticles deliver specifically targeted antimicrobial peptides against Clostridium perfringens infections.

Specifically targeted antimicrobial peptides (STAMPs) are novel alternatives to antibiotics, whereas the development of STAMPs for colonic infections is hindered by limited de novo design efficiency and colonic bioavailability. In this study, we report an efficient de novo STAMP design strategy that combines a traversal design, machine learning model, and phage display technology to identify STAMPs against Clostridium perfringens. STAMPs could physically damage C. perfringens, eliminate biofilms, and self-assemble into nanoparticles to entrap pathogens. Further, a gut-targeted engineering particle vaccine (EPV) was used for STAMPs delivery. In vivo studies showed that both STAMP and EPV@STAMP effectively limited C. perfringens infections and then reduced inflammatory response. Notably, EPV@STAMP exhibited stronger protection against colonic infections than STAMPs alone. Moreover, 16S ribosomal RNA sequencing showed that both STAMPs and EPV@STAMP facilitated the recovery of disturbed gut microflora. Collectively, our work may accelerate the development of the discovery and delivery of precise antimicrobials.

Autolysin as a fibronectin receptor on the cell surface of Clostridium perfringens.

OBJECTIVE: Clostridium perfringens causes food poisoning and gas gangrene, a serious wound-associated infection. C. perfringens cells adhere to collagen via fibronectin (Fn). We investigated whether the peptidoglycan hydrolase of C. perfringens, i.e., autolysin (Acp), is implicated in Fn binding to C. perfringens cells. METHODS: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA. RESULTS: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells. CONCLUSIONS: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells.