Isolation of a Virulent Clostridium perfringens Strain from Elaphurus davidianus and Characterization by Whole-Genome Sequence Analysis.

Clostridium perfringens (C. perfringens) is an important veterinary pathogen and a noteworthy threat to human and animal health. Recently, there has been a significant rise in the number of moose fatalities caused by this rare, endemic species in China. Currently, there is an increasing trend in conducting whole-genome analysis of C. perfringens strains originating from pigs and chickens, whereas fewer studies have been undertaken on Elaphurus davidianus-originating strains at the whole-genome level. Our laboratory has identified and isolated five C. perfringens type A from affected Elaphurus davidianus. The current study identified the most potent strain of C. perfringens, which originated from Elaphurus davidianus, and sequenced its genome to reveal virulence genes and pathogenicity. Our findings show that strain CX1-4 exhibits the highest levels of phospholipase activity, hemolytic activity, and mouse toxicity compared to the other four isolated C. perfringens type A strains. The chromosome sequence length of the CX1-4 strain was found to be 3,355,389 bp by complete genome sequencing. The current study unveils the genomic characteristics of C. perfringens type A originating from Elaphurus davidianus. It provides a core foundation for further investigation regarding the prevention and treatment of such infectious diseases in Elaphurus davidianus.

STING-dependent trained immunity contributes to host defense against Clostridium perfringens infection via mTOR signaling.

Clostridium perfringens (C. perfringens) infection is recognized as one of the most challenging issues threatening food safety and perplexing agricultural development. To date, the molecular mechanisms of the interactions between C. perfringens and the host remain poorly understood. Here, we show that stimulator of interferon genes (STING)-dependent trained immunity protected against C. perfringens infection through mTOR signaling. Heat-killed Candida albicans (HKCA) training elicited elevated TNF-α and IL-6 production after LPS restimulation in mouse peritoneal macrophages (PM). Although HKCA-trained PM produced decreased levels of TNF-α and IL-6, the importance of trained immunity was demonstrated by the fact that HKCA training resulted in enhanced bacterial phagocytic ability and clearance in vivo and in vitro during C. perfringens infection. Interestingly, HKCA training resulted in the activation of STING signaling. We further demonstrate that STING agonist DMXAA is a strong inducer of trained immunity and conferred host resistance to C. perfringens infection in PM. Importantly, corresponding to higher bacterial burden, reduction in cytokine secretion, phagocytosis, and bacterial killing were shown in the absence of STING after HKCA training. Meanwhile, the high expression levels of AKT/mTOR/HIF1α were indeed accompanied by an activated STING signaling under HKCA or DMXAA training. Moreover, inhibiting mTOR signaling with rapamycin dampened the trained response to LPS and C. perfringens challenge in wild-type (WT) PM after HKCA training. Furthermore, STING­deficient PM presented decreased levels of mTOR signaling-related proteins. Altogether, these results support STING involvement in trained immunity which protects against C. perfringens infection via mTOR signaling.

Efficacy of Bacillus subtilis probiotic in preventing necrotic enteritis in broilers: a systematic review and meta-analysis.

Probiotics can enhance broiler chicken health by improving intestinal microbiota, potentially replacing antibiotics. They protect against bacterial diseases like necrotic enteritis (NE) in poultry. Understanding their role is crucial for managing bacterial diseases, including NE. This study conducted a meta-analysis to assess the effects of Bacillus subtilis probiotic supplementation on feed conversion ratio (FCR), NE lesion score, and mortality. Additionally, a systematic review analysed gut microbiota changes in broilers challenged with Clostridium perfringens with or without the probiotic supplementation. Effect sizes from the studies were estimated in terms of standardized mean difference (SMD). Random effect models were fitted to estimate the pooled effect size and 95% confidence interval (CI) of the pooled effect size between the control [probiotic-free + C. perfringens] and the treatment [Bacillus subtilis supplemented + C. perfringens] groups. Overall variance was computed by heterogeneity (Q). The meta-analysis showed that Bacillus subtilis probiotic supplementation significantly improved FCR and reduced NE lesion score but had no effect on mortality rates. The estimated overall effects of probiotic supplementation on FCR, NE lesion score and mortality percentage in terms of SMD were -0.91 (CI = -1.34, -0.49; P < 0.001*); -0.67 (CI = -1.11, -0.22; P = 0.006*), and -0.32 (CI = -0.70, 0.06; P = 0.08), respectively. Heterogeneity analysis indicated significant variations across studies for FCR (Q = 69.66; P < 0.001*) and NE lesion score (Q = 42.35; P < 0.001*) while heterogeneity was not significant for mortality (Q = 2.72; P = 0.74). Bacillus subtilis probiotic supplementation enriched specific gut microbiota including Streptococcus, Butyricicoccus, Faecalibacterium, and Ruminococcus. These microbiotas were found to upregulate expression of various genes such as TJ proteins occluding, ZO-1, junctional adhesion 2 (JAM2), interferon gamma, IL12-ß and transforming growth factor-ß4. Moreover, downregulated mucin-2 expression was involved in restoring the intestinal physical barrier, reducing intestinal inflammation, and recovering the physiological functions of damaged intestines. These findings highlight the potential benefits of probiotic supplementation in poultry management, particularly in combating bacterial diseases and promoting intestinal health.

Exploring the Interactive Impacts of Citronellol, Thymol, and Trans-Cinnamaldehyde in Broilers: Moving towards an Improved Performance, Immunity, Gastrointestinal Integrity, and Clostridium perfringens Resistance.

AIMS: This study aimed to explore the effectiveness of dietary citronellol, thymol, and trans-cinnamaldehyde (CTC) essential oils blend on broilers` growth performance, immunity, intestinal microbial count, gut integrity, and resistance against Clostridium perfringens utilizing the necrotic enteritis (NE) challenge model. METHODS AND RESULTS: A total of 200 Ross 308 male broiler chicks received either a control diet or diet supplemented with three graded levels of CTC blend including 300, 600, and 900 mg of CTC blend/Kg diet and experimentally infected with C. perfringens strain at 23 days of age. Herein, dietary CTC blend fortifications significantly improved the broilers` growth performance, which was supported by upregulating the expression levels of MUC-2, occludin, and JAM-2 genes. Moreover, dietary CTC blend inclusion significantly enhanced the levels of blood phagocytic percentage and serum IgA, IgG, and MPO, and reduced the values of serum CRP, and NO at 5 days pre-infection, 10-, and 15 days post-infection (dpi) with C. perfringens. At 15 dpi, CTC blend inclusion significantly reduced the intestinal digesta pH, coliforms and C. perfringens loads, and the expression levels of genes related to C. perfringens virulence (cpe, cnaA, and nanI), proinflammatory cytokines (IL-1ß and TNF-α), and chemokines (CCL20), in addition to increasing the count of beneficial total Lactobacillus and total aerobic bacteria, and the expression levels of genes related to anti-inflammatory cytokines (IL-10) and chemokines (AvBD6, and AvBD612). CONCLUSION: Our results point to the growth-provoking, immunostimulant, antibacterial, anti-inflammatory, and antivirulence characteristics of the CTC blend, which improves the broilers' resistance to C. perfringens and ameliorates the negative impacts of NE.

Emergence of genetic diversity and multi-drug resistant Clostridium perfringens from wild birds.

BACKGROUND: Clostridium perfringens (C. perfringens) is an important zoonotic microorganism that can cause animal and human infections, however information about the prevalence status in wild birds of this pathogenic bacterium is currently limited. RESULT: In this study, 57 strains of C. perfringens were isolated from 328 fecal samples of wild birds. All the isolates were identified as type A and 70.18% of the isolates carried the cpb2 gene. Antimicrobial susceptibility testing showed that and 22.80% of the isolates were classified as multidrug-resistant strains. The MLST analysis of the 57 isolates from wild birds was categorized into 55 different sequence types (STs) and clustered into eight clonal complexes (CCs) with an average of 20.1 alleles and the Simpson Diversity index (Ds) of 0.9812, and revealed a high level of genetic diversity within the C. perfringens populations. Interestingly, the isolates from swan goose were clustered in the same CC while isolates from other bird species were more scattered suggesting that a potential difference in genetic diversity among the C. perfringens populations associated with different bird species. CONCLUSION: C. perfringens exhibits a wide range of host adaptations, varying degrees of antimicrobial resistance, and a high degree of genetic diversity in wild birds. Understanding the prevalence, toxin type, antimicrobial resistance, and genetic diversity of C. perfringens in wildlife populations is essential for developing effective strategies for disease control and management.

Clostridium and Cryptosporidium outbreak linked to a splash pad.

BACKGROUND: . Splash pads for recreational purposes are widespread. Using these pads can pose a health risk if they lack installation regulation and water quality supervision. Our aim was to describe a waterborne disease outbreak caused by Clostridium perfringens and Cryptosporidium spp. in a Barcelona district and the measures taken for its control. METHODS: . On August 2018, 71 cases of acute gastroenteritis were detected, affecting people who used a splash pad or were in contact with a user. Microbiological and environmental investigations were carried out. A descriptive analysis of the sample and Poisson regression models adjusted for age and sex were performed, obtaining frequencies, median values, and adjusted prevalence ratios with their 95% confidence intervals. RESULTS: The median age of the cases was 6.7 years, 27 (38%) required medical care, and three (4.2%) were hospitalized. The greater the number of times a person entered the area, the greater the number of symptoms and their severity. Nineteen (76%) of the 25 stool samples collected from cases showed the presence of one or both pathogens. Environmental investigations showed deficiencies in the facilities and identified the presence of both species in the splash pad. Health education and hygiene measures were carried out, and 14 days after the closure of the facilities, no more cases related to the pad were recorded. CONCLUSIONS: . Specific regulations are needed on the use of splash pads for recreational purposes. Until these regulations are in place, these types of facility should comply with the regulations that apply to swimming pools and spas, including those related to the design of the tanks, water recirculation systems, and adequate disinfection systems.

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.

Approaching toxigenic Clostridia from a One Health perspective.

Spore-forming pathogens have a unique capacity to thrive in diverse environments, and with temporal persistence afforded through their ability to sporulate. Their prevalence in diverse ecosystems requires a One Health approach to identify critical reservoirs and outbreak-associated transmission chains, given their capacity to freely move across soils, waterways, foodstuffs and as commensals or infecting pathogens in human and animal populations. Among anaerobic spore-formers, genomic resources for pathogens including C. botulinum, C. difficile, and C. perfringens enable our capacity to identify common and unique factors that support their persistence in diverse reservoirs and capacity to cause disease. Publicly available genomic resources for spore-forming pathogens at NCBI's Pathogen Detection program aid outbreak investigations and longitudinal monitoring in national and international programs in public health and food safety, as well as for local healthcare systems. These tools also enable research to derive new knowledge regarding disease pathogenesis, and to inform strategies in disease prevention and treatment. As global community resources, the continued sharing of strain genomic data and phenotypes further enhances international resources and means to develop impactful applications. We present examples showing use of these resources in surveillance, including capacity to assess linkages among clinical, environmental, and foodborne reservoirs and to further research investigations into factors promoting their persistence and virulence in different settings.

The small acid-soluble proteins of spore-forming organisms: similarities and differences in function.

The small acid-soluble proteins are found in all endospore-forming organisms and are a major component of spores. Through their DNA binding capabilities, the SASPs shield the DNA from outside insults (e.g., UV and genotoxic chemicals). The absence of the major SASPs results in spores with reduced viability when exposed to UV light and, in at least one case, the inability to complete sporulation. While the SASPs have been characterized for decades, some evidence suggests that using newer technologies to revisit the roles of the SASPs could reveal novel functions in spore regulation.

Mechanisms of intestinal epithelial cell damage by Clostridiumperfringens.

Clostridium perfringens, a Gram-positive bacterium, causes intestinal diseases in humans and livestock through its toxins, related to alpha toxin (CPA), beta toxin (CPB), C. perfringens enterotoxin (CPE), epsilon toxin (ETX), Iota toxin (ITX), and necrotic enteritis B-like toxin (NetB). These toxins disrupt intestinal barrier, leading to various cell death mechanisms such as necrosis, apoptosis, and necroptosis. Additionally, non-toxin factors like adhesins and degradative enzymes contribute to virulence by enhancing colonization and survival of C. perfringens. A vicious cycle of intestinal barrier breach, misregulated cell death, and subsequent inflammation is at the heart of chronic inflammatory and infectious gastrointestinal diseases. Understanding these mechanisms is essential for developing targeted therapies against C. perfringens-associated intestinal diseases.

Injury of Macrophages Induced by Clostridium perfringens Type C Exotoxins.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

Antimicrobial Activity of Synthetic Enterocins A, B, P, SEK4, and L50, Alone and in Combinations, against Clostridium perfringens.

Multidrug-resistant Clostridium perfringens infections are a major threat to the poultry industry. Effective alternatives to antibiotics are urgently needed to prevent these infections and limit the spread of multidrug-resistant bacteria. The aim of the study was to produce by chemical synthesis a set of enterocins of different subgroups of class II bacteriocins and to compare their spectrum of inhibitory activity, either alone or in combination, against a panel of twenty C. perfringens isolates. Enterocins A, P, SEK4 (class IIa bacteriocins), B (unsubgrouped class II bacteriocin), and L50 (class IId leaderless bacteriocin) were produced by microwave-assisted solid-phase peptide synthesis. Their antimicrobial activity was determined by agar well diffusion and microtitration methods against twenty C. perfringens isolates and against other pathogens. The FICINDEX of different combinations of the selected enterocins was calculated in order to identify combinations with synergistic effects. The results showed that synthetic analogs of L50A and L50B were the most active against C. perfringens. These peptides also showed the broadest spectrum of activity when tested against other non-clostridial indicator strains, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Streptococcus suis, Streptococcus pyogenes, Enterococcus cecorum, Enterococcus faecalis, as well as Gram-negative bacteria (Campylobacter coli and Pseudomonas aeruginosa), among others. The selected synthetic enterocins were combined on the basis of their different mechanisms of action, and all combinations tested showed synergy or partial synergy against C. perfringens. In conclusion, because of their high activity against C. perfringens and other pathogens, the use of synthetic enterocins alone or as a consortium can be a good alternative to the use of antibiotics in the poultry sector.

The Effects of Encapsulation on the In Vitro Anti-Clostridial Activity of Olive Mill Wastewater Polyphenolic Extracts: A Promising Strategy to Limit Microbial Growth in Food Systems.

Despite the technologies applied to food production, microbial contamination and chemical deterioration are still matters of great concern. In order to limit these phenomena, new natural approaches should be applied. In this context, the present study aimed to assess the antioxidant and anti-Clostridial effects of two different polyphenolic extracts derived from olive mill vegetation water, one liquid (LE) and one encapsulated (EE). The extracts have been preliminary characterized using Liquid Chromatography Quadrupole Time-Of Flight spectrometry. The Oxygen Radical Absorbance Capacity method was used to determine the antioxidant capacity, registering a higher value for EE compared to that for LE (3256 ± 85 and 2446 ± 13 µgTE/g, respectively). The antibacterial activity against C. perfringens, C. botulinum and C. difficile was studied by the agar well diffusion method, MIC and MBC determination and a time-kill test. The results confirm that EE and LE are able to limit microbial growth, albeit with minor effects when the phenolic compounds are encapsulated. Further studies are needed to evaluate the possible application of these extracts in food systems.

Dietary supplementation of prebiotic yeast Saccharomyces cerevisiae cell wall promotes growth performance and intestinal health in broiler chickens challenged with Clostridium perfringens.

1. This study evaluated the effectiveness of yeast (Saccharomyces cerevisiae) cell wall (YCW) supplementation on the growth performance, carcase characteristics, serum biomarkers, liver function, ileal histology and microbiota of broiler chickens challenged with Clostridium perfringens (C. perfringens).2. In a 35-d trial, 240 chicks aged 1-d-old were randomly assigned to one of four treatment groups, each with 10 replicates: control (CON) with no challenge or additives, challenged with C. perfringens (CHAL), CHAL and supplemented with YCW at either 0.25 g/kg (YCW0.25) or 0.5 g/kg (YCW0.5).3. In comparison to CON, the CHAL birds had reduced growth performance, survival rate, dressing percentage, breast meat yield, levels of total protein (TP), globulin (GLO), glucose (GLU), total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD), as well as a decreased Lactobacillus population (P < 0.01). Additionally, this group showed elevated levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and C. perfringens count (P < 0.01). Compared to CHAL, the YCW0.25 or YCW0.5 groups had improved growth performance, survival rate, dressing percentage, breast meat yield, levels of TP, GLO, GLU, and T-AOC, as well as the activities of T-SOD, GOT, and GPT, villus height, villus surface area, villus height to crypt depth ratio, and the populations of both Lactobacillus and C. perfringens; (P < 0.01).4. The data suggested that YCW supplementation at either 0.25 or 0.50 g/kg can restore the growth performance of broiler chickens during a C. perfringens challenge.

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 sepsis after comprehensive multicourse treatment of hepatocellular carcinoma: A case report and review.

Introduction: Clostridium perfringens sepsis is a rare but serious clinical syndrome that is typically triggered by gastrointestinal disorders. We present a case of bloodstream infection caused by Clostridium perfringens in a liver cancer patient after comprehensive multicourse treatment. Case presentations: The patient, a 68-year-old male, experienced nausea, decreased appetite, and abdominal distension on the 15th day after receiving comprehensive multicourse treatment and transcatheter arterial chemoembolization (TACE). During admission, he developed fever, and blood culture results confirmed the presence of Clostridium perfringens. The patient was discharged with improved symptoms. Conclusion: Our findings underscore the rarity of Clostridium perfringens sepsis. For liver cancer patients, particularly those who have undergone TACE or radiofrequency ablation and who experience post treatment fever, vigilance for Clostridium perfringens bloodstream infection is crucial. Timely diagnostic assessments and proactive treatment can significantly enhance the survival prospects of these patients.

Successful Conservative Management of Second Trimester Spontaneous Abortion Complicated by Clostridial Sepsis.

Clostridium perfringens is responsible for 5% of septic abortions. Emergent hysterectomy is often required for patient survival. This can be devastating to patients desiring future fertility. We report a 15-year-old female patient at 17-week 6 day gestation with diagnosis of sepsis on admission. She was managed with broad spectrum antibiotics followed by immediate evacuation of the retained placenta. Blood and placental cultures confirmed Clostridial species. Successful conservative management allowed for a term pregnancy two years later resulting in a healthy newborn. Few reports describe effective conservative management resulting in uterine sparing and good subsequent pregnancy outcomes.

Pulmonary Clostridium perfringens: Seeding Beyond the Gastrointestinal Tract.

Clostridia perfringens infection outside the gastrointestinal system is rare. Here, we report on a 75-year-old man with history of end-stage renal disease presenting after a syncopal event with lactic acidosis, leukocytosis, and mild hyper-bilirubinemia. Chest imaging revealed a loculated, left-sided pleural effusion; diagnostic thoracentesis identified Clostridia perfringens, consistent with an empyema. Video-assisted thoracic left lung decortication was performed; tissue culture also speciated Clostridia perfringens. Further imaging revealed concomitant acute cholecystitis, suggesting hematogenous seeding or transdiaphragmatic extension of Clostridia perfringens to pleural space from an abdominal source. The patient was successfully managed with laparoscopic cholecystectomy and discharged on a one-month course of amoxicillin-sulbactam. This case highlights the potential for Clostridia perfringens to produce a pleuropulmonary infection, necessitating timely diagnosis and intervention, to improve patient outcomes.

Spontaneous gas gangrene of the pancreas: extremely rare condition.

Acute pancreatitis is a common condition, only occasionally leading to necrosis of the pancreas. In instances where abscess formation takes place, the predominant microbial profile involves both aerobic and anaerobic enteric species. We present the case of a patient with clostridial emphysematous pancreatitis who developed pneumoperitoneum without associated visceral perforation.

Hematological investigations in a case of intravascular hemolysis due to Clostridium perfringens infection.

A patient presented with fever, severe pain and edematous tight due to hip trauma and was scheduled for urgent fasciotomy. Following physical examination, laboratory analyses were requested, and results revealed anemia and severe infection. As the patient's condition was serious, a new set of samples was sent to the laboratory four hours later. Following centrifugation, severely hemolyzed dark-colored serum and plasma samples were obtained and in vitro hemolysis was suspected. The collection of samples was repeated, but a new set of samples was also hemolyzed with a significant decrease in the hemoglobin value. At that point, in vivo hemolysis was suspected, and samples were processed according to standard laboratory procedures for hemolytic samples. Following confirmation of the gas gangrene diagnosis by clinicians, the cause of hemolysis was attributed to the cytotoxic activity of α-toxin produced by the anaerobic gram-positive bacterium Clostridium perfringens. An insight into the laboratory procedure that could help to narrow down the causes of hemolysis and single out C. perfringens as a cause of intravascular hemolysis was given.