The presence of differentiated C2C12 muscle cells enhances toxin production and growth by Clostridium perfringens type A strain ATCC3624.

Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R, agrB/D, and eutV/W. The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.

Association between Insurance Status and Outcomes of Hospitalizations for Necrotizing Soft Tissue Infections.

Background: Lack of insurance is associated with poorer outcomes in hospitalized patients. However, few studies have explored this association in hospitalizations for necrotizing soft tissue infections (NSTIs). This study examined the impact of insurance status on the outcome of NSTI admissions. Methods: All adult hospitalizations for necrotizing fasciitis, gas gangrene, and Fournier gangrene between 2016 and 2018 were examined using the Nationwide Inpatient Sample database. Insurance status was categorized as insured (including Medicare, Medicaid, and Private, including Health maintenance organization (HMO) or uninsured (Self-pay). Outcome measures included mortality rates, limb loss, length of hospital stay, prolonged hospital stay, and critical care admissions. Statistical analysis included weighted sample analysis, chi-square tests, multivariate regression analysis, and negative binomial regression modeling. Results: Approximately 29,705 adult hospitalizations for NSTIs were analyzed. Of these, 57.4% (17,065) were due to necrotizing fasciitis, 22% (6,545) to gas gangrene, and 20.5% (6,095) to Fournier gangrene. Approximately 9.7% (2,875) were uninsured, whereas 70% (26,780) had insurance coverage. Among the insured, Medicare covered 39.6% (10,605), Medicaid 29% (7,775), and private insurance 31.4% (8,400). After adjustments, Medicare insurance was associated with greater odds of mortality (adjusted odds ratio [aOR]: 1.81; 95% confidence interval [CI]: 1.33-2.47; p = 0.001). Medicaid insurance was associated with increased odds of amputation (aOR: 1.81; 95% CI: 1.33-2.47; p < 0.001), whereas private insurance was associated with lower odds of amputation (aOR: 0.70; 95% CI: 0.51-0.97; p = 0.030). Medicaid insurance was associated with greater odds of prolonged hospital stay (aOR: 1.34; 95% CI: 1.09-1.64; p < 0.001). No significant association was observed between the lack of insurance or self-pay and the odds of primary or secondary outcomes. Conclusion: Medicare insurance was correlated with greater odds of mortality, whereas Medicaid insurance was associated with increased odds of amputation and longer hospital stay. Uninsured status was not associated with significant differences in NSTI outcomes.

Salvage radiofrequency ablation for microwave ablation treated hepatocellular carcinoma complicated by gas gangrene: case report.

AIM: Gas gangrene (GG) is a rare severe infection with a very high mortality rate mainly caused by Clostridium species. It develops suddenly, often as a complication of abdominal surgery or liver transplantation. We report a case of GG of the liver occurred after percutaneous microwave (MW) ablation of an hepatocellular carcinoma (HCC) successfully treated with percutaneous Radiofrequency ablation (RFA). CASE PRESENTATION: A 76-year-old female patient was treated with MW ablation for a large HCC in the VIII segment; 2 days later she developed fever, weakness, abdominal swelling and was hospitalized with diagnosis of anaerobic liver abscess. Despite antibiotic therapy, the patient conditions worsened, and she was moved to the intensive care unit (ICU). Percutaneous drainage was attempted, but was unsuccessful. The surgeon and the anesthesiologist excluded any indication of surgical resection. We performed RFA of the GG by 3 cool-tip needles into the infected area. The procedure was well tolerated by the patient, who left the hospital for follow-up. CONCLUSION: Percutaneous RFA could be a valuable therapy of focal GG of the liver in patients refractory to antibiotics and when surgery and OLT are not feasible. A fast and early indication is needed in case of rapid worsening of the patient's conditions.

Absent in Melanoma 2 Mediates Inflammasome Signaling Activation against Clostridium perfringens Infection.

Absent in melanoma 2 (AIM2), a key component of the IFI20X/IFI16 (PYHIN) protein family, is characterized as a DNA sensor to detect cytosolic bacteria and DNA viruses. However, little is known about its immunological role during pathogenic Clostridium perfringens (C. perfringens) infection, an extracellular bacterial pathogen. In a pathogenic C. perfringens gas gangrene model, Aim2-/- mice are more susceptible to pathogenic C. perfringens soft tissue infection, revealing the importance of AIM2 in host protection. Notably, Aim2 deficiency leads to a defect in bacterial killing and clearance. Our in vivo and in vitro findings further establish that inflammasome signaling is impaired in the absence of Aim2 in response to pathogenic C. perfringens. Mechanistically, inflammasome signaling downstream of active AIM2 promotes pathogen control. Importantly, pathogenic C. perfringens-derived genomic DNA triggers inflammasome signaling activation in an AIM2-dependent manner. Thus, these observations uncover a central role for AIM2 in host defense and triggering innate immunity to combat pathogenic C. perfringens infections.

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.

Clostridium perfringens phospholipase C, an archetypal bacterial virulence factor, induces the formation of extracellular traps by human neutrophils.

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.

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.

A 52-Year-Old Diabetic Man with Poor Dental Hygiene, Right Dental Abscess, and Parapharyngeal Abscess with Gas Gangrene Due to Klebsiella pneumoniae Infection, Presenting with Septic Shock and Multiorgan Failure, Who Recovered After Surgical Neck Debridement.

BACKGROUND Gas gangrene is a rapidly progressive and potentially fatal infection that is common in the extremities. Gas gangrene of the head and neck is a very rare condition, and non-clostridial gas-forming neck abscess caused by Klebsiella pneumoniae is unusual. This report is of a diabetic man with poor dental hygiene, a right dental abscess, and parapharyngeal abscess with gas gangrene due to Klebsiella pneumoniae infection, presenting with septic shock and multiorgan failure, who recovered after surgical neck debridement. CASE REPORT A 52-year-old man with diabetes mellitus lost consciousness and collapsed on a curbside. He presented with painful swelling of the right-side neck, associated with spiking fever, confusion, dyspnea, and stridor. He had right submandibular and supraclavicular swelling with crepitus, multiple dental caries, and multiorgan dysfunction, and was intubated. A computed tomography scan showed a gas-forming abscess in the right parapharyngeal, retropharyngeal, and paralaryngeal spaces and dense infiltration with pleural effusion in the upper lobes. Neck exploration was performed for drainage. Necrotic tissue and foul-smelling pus were debrided and drained. Gram stain showed gram-negative bacilli. Necrotic tissue, pus, and blood culture showed Klebsiella pneumoniae. He remained on intravenous meropenem for 14 days and was frequently debrided with irrigation until the infection subsided. Finally, normal physiologic functions of the failing organ system were restored. CONCLUSIONS We present a rare case of Klebsiella pneumoniae infection causing gas gangrene in the deep neck spaces, leading to septic shock and multiorgan failure, who recovered after surgical neck debridement. This is a potentially fatal condition that requires emergency drainage because of its high mortality rate.

Phagocyte extracellular traps formation contributes to host defense against Clostridium perfringens infection.

Clostridium perfringens (C. perfringens) is an important Gram-positive anaerobic spore-forming pathogen that provokes life-threatening gas gangrene and acute enterotoxaemia, although it colonizes as a component of the symbiotic bacteria in humans and animals. However, the mechanisms by which C. perfringens is cleared from the host remains poorly understood, thereby impeding the development of novel strategies for control this infection. Here, we uncover a beneficial effect of extracellular traps (ETs) formation on bacterial killing and clearance by phagocytes. C. perfringens strain ATCC13124, and wild-type isolates CP1 and CP3 markedly trigger ETs formation in macrophages and neutrophils. As expected, visualization of DNA decorated with histone, myeloperoxidase (MPO) and neutrophils elastase (NE) in C. perfringens-triggered classical ETs structures. Notably, the bacteria-induced ETs formation is an ERK1/2-, P38 MAPK-, store-operated calcium entry (SOCE)-, NADPH oxidase-, histone-, NE-, and MPO-dependent process, and is independent of LDH activity. Meanwhile, the defect of bactericidal activity is mediated by impairing ETs formation in phagocytes. Moreover, In vivo studies indicated that degradation of ETs by DNase I administration leads to a defect in the protection against experimental gas gangrene, with higher mortality rates, exacerbated tissue damage, and more bacterial colonization. Together, these results suggest that phagocyte ETs formation is essential for the host defense against C. perfringens infection.

Postpartum clostridial gangrenous metritis in 12 dairy goats in France.

Clostridial infections in goats have been associated frequently with enteric diseases or gas gangrene but very rarely with the reproductive system. We describe here 12 cases of fatal postpartum gangrenous metritis in does associated with infection by several clostridial species. Clinically, these cases were characterized by rapid onset of hyperthermia followed by death after kidding. On postmortem examination, the uteri appeared to be necrotic and were hemorrhagic and edematous. Microscopically, the uteri had diffuse coagulative necrosis, edema, hemorrhage, and fibrinous thrombi with intralesional gram-positive rods. Clostridium perfringens was isolated from 7 of 9 uterine samples cultured, and C. perfringens, C. septicum, C. novyi, or C. chauvoei were demonstrated by immunohistochemistry (IHC) in the 5 cases examined. IHC for Paeniclostridium sordellii was negative in all 5 cases. PCR performed on 3 of the C. perfringens isolates was positive for alpha toxin and perfringolysin, identifying these isolates as type A. Clostridial infection should be considered in cases of postpartum gangrenous metritis of does.

Gas-Producing Infections in the Foot at a Large Academic Medical Center: A 10-Year Retrospective Review.

Gas-producing infections, such as clostridial and nonclostridial gas gangrene, crepitant cellulitis, and necrotizing fasciitis, are characterized in the literature by a variety of initial presentations, microbial burdens and surgical outcomes-ranging from debridement to amputation to death. The primary aim of this study was to identify the organisms cultured in gas-producing infections of the foot in patients that presented to a large academic medical center over a 10-year period. Our secondary aims were to report the prevalence of sepsis in this population upon presentation, and patient outcomes upon discharge. After a retrospective chart review of 207,534 procedures, 70 surgical cases met inclusion criteria. The most common organisms that grew in operating room cultures were Staphylococcus aureus, Group B Beta Streptococcus, and Enterococcus species. Just over half of the population presented with sepsis. After an average of 2 or more operations, 64% of patients underwent amputation. One death occurred. Gas-producing infections, or "gas gangrene," are primarily polymicrobial infections, rarely due to Clostridium perfringens, that warrant surgical exploration for optimal outcomes.

Transtentorial herniation syndrome from meningococcal meningitis in a young woman: the case for neurocritical care.

We report a case of a previously healthy early adolescent female who presented with meningococcal meningitis. While in hospital, she had marked neurologic deterioration with clinical herniation from malignant cerebral oedema. She was transferred to a neurocritical care centre where she underwent invasive intracranial pressure (ICP) and brain tissue oxygen (PbtO2) monitoring. Early in her course, she demonstrated a compete absence of autoregulation, with pressure passive cerebral blood flow. As a result, maintaining a mean arterial pressure between 50 mm Hg and 60 mm Hg, which ensured adequate cerebral oxygenation, while avoiding increases in ICP. Although her course was initially complicated by bilateral optic neuropathy, she has subsequently made a full neurologic recovery and is now undertaking postsecondary education. This case highlights that access to specialist neurocritical care, guided by neurophysiologic monitoring of ICP and PbtO2, may help improve outcomes, even among those patients with catastrophic cerebral oedema from bacterial meningitis.

Piceatannol Alleviates Clostridium perfringens Virulence by Inhibiting Perfringolysin O.

Clostridium perfringens (C. perfringens) is an important foodborne pathogen that can cause diseases such as gas gangrene and necrotizing enteritis in a variety of economic animals, seriously affecting public health and the economic benefits and healthy development of the livestock and poultry breeding industry. Perfringolysin O (PFO) is an important virulence factor of C. perfringens and plays critical roles in necrotic enteritis and gas gangrene, rendering it an ideal target for developing new drugs against infections caused by this pathogen. In this study, based on biological activity inhibition assays, oligomerization tests and computational biology assays, we found that the foodborne natural component piceatannol reduced pore-forming activity with an inhibitory ratio of 83.84% in the concentration of 16 µg/mL (IC50 = 7.83 µg/mL) by binding with PFO directly and changing some of its secondary structures, including 3-Helix, A-helix, bend, and in turn, ultimately affecting oligomer formation. Furthermore, we confirmed that piceatannol protected human intestinal epithelial cells from the damage induced by PFO with LDH release reduced by 38.44% at 16 µg/mL, based on a cytotoxicity test. By performing an animal experiment, we found the C. perfringens clones showed an approximate 10-fold reduction in infected mice. These results suggest that piceatannol may be a candidate for anti-C. perfringens drug development.