The impact of existing total anti-toxin B IgG immunity in outcomes of recurrent Clostridioides difficile infection.

Late anti-toxin-B humoral immunity acquired after treatment is important for preventing recurrent Clostridioides difficile infection. We prospectively-measured anti-toxin-B IgG and neutralization titers at diagnosis as potential early predictors of recurrence. High anti-toxin-B-IgG/neutralizing antibodies were associated with short-lasting protection within 6-weeks, however, no difference in recurrence risk was observed by 90-days post-infection.

Clindamycin suppresses virulence expression in inducible clindamycin-resistant Staphylococcus aureus strains.

Clindamycin is a protein synthesis inhibitory agent that has the ability to suppress the expression of virulence factors in Staphylococcus aureus. Recent guidelines recommend the use of clindamycin for the treatment of toxin-mediated infections. Clindamycin modulates virulence expression at sub-inhibitory concentrations (sub-MICs) in clindamycin-susceptible S. aureus strains but previous report shown that this effect was supressed for constitutive clindamycin resistant strains. However, no data are currently available on the impact of clindamycin at sub-MICs on the virulence of inducible clindamycin-resistant S. aureus strains. Here, we show that sub-MICs of clindamycin decrease Panton-Valentine leucocidin, toxic-shock-staphylococcal toxin (TSST-1) and alpha-haemolysin (Hla) expression in six inducible clindamycin-resistant isolates cultivated in vitro in CCY medium. These results suggest that the clindamycin anti-toxin effect is retained for inducible clindamycin-resistant S. aureus isolates; therefore, its usage should be considered within the treatment regimen of toxin related infections for inducible clindamycin-resistant S. aureus.

Effective concentration of intravenous immunoglobulin for neutralizing Panton-Valentine leukocidin in human blood.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infects healthy individuals, although the precise cause remains unclear. CA-MRSA produces Panton-Valentine leukocidin (PVL), which often causes severe invasive infection; however, antitoxin drugs against PVL are limited. Intravenous immunoglobulin (IVIg) possesses antitoxin activity, but unfortunately, the optimal dose is unknown. Here, we measured the PVL neutralizing antibody titer in the plasma of Japanese individuals and sera of American donors. Next, we compared the cytotoxic effects of PVL on neutrophils in phosphate buffered saline (PBS) or whole blood to determine the effect of the neutralizing antibody. Finally, we evaluated the effective concentration of IVIg required to neutralize PVL in PBS and whole blood. We observed that the titer of PVL neutralizing antibody in healthy individuals polarized as high and low/none group. Additionally, the PVL neutralizing antibody titer considerably affected the concentration at which IVIg elicited its effect. This suggests that PVL-producing CA-MRSA might be involved in determining the severity of infection in healthy individuals without neutralizing antibody against PVL. The neutralizing effect of IVIg was observed in both PBS and whole blood. However, the optimal concentration of IVIg required for neutralizing PVL varied between PBS and whole blood. In addition, since the PVL-neutralizing activity of IVIg also largely depends on blood composition, such as neutralizing antibody concentration, the optimal dosage of IVIg as an antitoxin drug should be decided in a timely manner after considering the patient's medical background.

Induction of antitoxin responses in Clostridium-difficile-infected patients compared to healthy blood donors.

According to the literature Clostridium difficile antitoxins are present in up to 66% of humans. In a survey of ∼400 plasma samples from healthy blood donors we found that less than 6% were positive for anti-TcdA or anti-TcdB antitoxins. Using the same standard immunoassay protocol, we looked for IgG and IgA antitoxins in the blood and stool samples from 25 patients with C. difficile infection (CDI). Some patients with CDI had no antitoxin detected at all, while others had high levels of specific IgG- and IgA-antitoxins against both TcdA and TcdB in blood and IgA-anti-TcdA and -anti-TcdB antibodies in stool. Systemic responses to TcdB and mucosal responses to TcdA predominated. Among patients infected with the NAP1/027/BI strain, systemic IgG-anti-TcdB responses were particularly elevated. In contrast, patients infected with non-027 strains had more elevated mucosal IgA-anti-TcdA responses. Furthermore, high titer sera did not correlate with high neutralizing potential. We hypothesize that paradoxical killing of primed B-cells by antibody-mediated endosomal uptake of the Large Clostridial Toxins, TcdA and TcdB leads to clonal elimination of the fittest B-cells. If this hypothesis is confirmed, immune suppression rather than protective humoral immunity might be the consequence in some patients infected with toxigenic C. difficile.

Foodborne Botulism Type F: A Rare Presentation of Neurologic Manifestation and Successful Management.

Botulism is a neuroparalytic syndrome resulting from the systemic effects of an exoneurotoxin produced by gram-positive, rod-shaped, spore-forming, obligate anaerobic bacterium Clostridium botulinum. Here, we present the case of a 40-year-old male, presenting with a sudden onset of abdominal pain associated with vomiting. He was admitted for conservative management once the CT of the abdomen and pelvis revealed partial small bowel obstruction with no signs of bowel perforation or ischemia. However, the next day, the patient had a cardiac arrest thought to be secondary to respiratory arrest. The return of spontaneous circulation was achieved after two cycles of cardiopulmonary resuscitation. The patient developed quadriplegia, areflexia, and bilateral ophthalmoplegia. He was empirically treated with pyridostigmine, intravenous immunoglobulin (IVIG), and botulinum antitoxin. Stool polymerase chain reaction (PCR) testing resulted positive for C. botulinum toxin type F. The patient ultimately recovered with botulinum antitoxin and a month of physical and speech therapy. Our case highlights that clinicians should consider botulism as a differential and emphasize the importance of early diagnosis for effective management and prognosis.

Staphylococcus aureus Toxins: An Update on Their Pathogenic Properties and Potential Treatments.

Staphylococcus aureus is a clinically important pathogen that causes a wide range of human infections, from minor skin infections to severe tissue infection and sepsis. S. aureus has a high level of antibiotic resistance and is a common cause of infections in hospitals and the community. The rising prevalence of community-acquired methicillin-resistant S. aureus (CA-MRSA), combined with the important severity of S. aureus infections in general, has resulted in the frequent use of anti-staphylococcal antibiotics, leading to increasing resistance rates. Antibiotic-resistant S. aureus continues to be a major health concern, necessitating the development of novel therapeutic strategies. S. aureus uses a wide range of virulence factors, such as toxins, to develop an infection in the host. Recently, anti-virulence treatments that directly or indirectly neutralize S. aureus toxins have showed promise. In this review, we provide an update on toxin pathogenic characteristics, as well as anti-toxin therapeutical strategies.

A Review of the Efficacy of FDA-Approved B. anthracis Anti-Toxin Agents When Combined with Antibiotic or Hemodynamic Support in Infection- or Toxin-Challenged Preclinical Models.

Anti-toxin agents for severe B. anthracis infection will only be effective if they add to the benefit of the two mainstays of septic shock management, antibiotic therapy and titrated hemodynamic support. Both of these standard therapies could negate benefits related to anti-toxin treatment. At present, three anthrax anti-toxin antibody preparations have received US Food and Drug Administration (FDA) approval: Raxibacumab, Anthrax Immune Globulin Intravenous (AIGIV) and ETI-204. Each agent is directed at the protective antigen component of lethal and edema toxin. All three agents were compared to placebo in antibiotic-treated animal models of live B. anthracis infection, and Raxibacumab and AIGIV were compared to placebo when combined with standard hemodynamic support in a 96 h canine model of anthrax toxin-associated shock. However, only AIG has actually been administered to a group of infected patients, and this experience was not controlled and offers little insight into the efficacy of the agents. To provide a broader view of the potential effectiveness of these agents, this review examines the controlled preclinical experience either in antibiotic-treated B. anthracis models or in titrated hemodynamic-supported toxin-challenged canines. The strength and weaknesses of these preclinical experiences are discussed.

Emergency response to a cluster of suspected food-borne botulism in Abuja, Nigeria: challenges with diagnosis and treatment in a resource-poor setting.

Food-borne botulism is a rare, acute and potentially fatal neurologic disorder that results from ingestion of food contaminated by botulinum toxin released from the anaerobic, spore-forming, gram-positive bacterium Clostridium botulinum. We reported an unusual cluster of botulism outbreak with high case fatality affecting a family following ingestion of home-made fish. A suspected outbreak of botulism affecting three patients in a family of six was reported to the Nigeria Centre for Disease Control. A rapid response team investigated by line-listing all the family members, interviewed extended family members, caregivers, clinicians, and nurses to collect socio-demographic and clinico epidemiological information using a semi-structured questionnaires. We collected blood from patients and food samples and locally made drink from the family home for laboratory testing. All family members ingested the same home-made food within the 48hrs before onset of symptoms in the index case. The clinical presentation of the three affected cases (AR=50.0%) was consistent with botulinum poisoning. Two of the affected cases died (CFR=66.7%) within 48hrs of admission, before antitoxin was made available. The third case had a milder presentation and survived, after administration of appropriate antitoxin. The remaining three children developed no symptoms. None of the samples cultured Clostridium botulinum. The blood samples were negative for mouse lethality test. Our report describes the challenges of diagnosis and management of rare emerging infectious disease outbreaks in resource-constrained settings.

A Zinc-Dependent Metalloproteinase of Brucella abortus Is Required in the Intracellular Adaptation of Macrophages.

Brucella abortus is a pathogen that survives in macrophages. Several virulence factors participate in this process, including the open reading frame (ORF) BAB1_0270 codifying for a zinc-dependent metalloproteinase (ZnMP). Here, its contribution in the intracellular adaptation of B. abortus was analyzed by infecting RAW264.7 macrophages with the mutant B. abortus Δ270 strain. Results showed that this ZnMP did not participated in either the adherence or the initial intracellular traffic of B. abortus in macrophages. Nevertheless, its deletion significantly increased the co-localization of B. abortus Δ270 with phagolysosomal cathepsin D and reduced its co-localization with calnexin present in endoplasmic reticulum (RE)-derived vesicles. Although B. abortus Δ270 showed an upregulated expression of genes involved in virulence (vjbR, hutC, bvrR, virB1), it was insufficient to reach a successful intracellular replication within macrophages. Furthermore, its attenuation favored in macrophages infected the production of high levels of cytokines (TNF-α and IL-6) and co-stimulatory proteins (CD80 and CD86), signals required in T cell activation. Finally, its deletion significantly reduced the ability of B. abortus Δ270 to adapt, grow and express several virulence factors under acidic conditions. Based on these results, and considering that this ZnMP has homology with ImmA/IrrE proteases, we discuss its role in the virulence of this pathogen, concluding that ZnMP is required in the intracellular adaptation of B. abortus 2308 during the infection of macrophages.

Botulism in pregnancy - a clinical approach to diagnosis and management.

Botulism is a life-threatening toxin mediated disease that often presents with a sudden rapid onset of paralysis of their skeletal muscles with subsequent respiratory compromise and sudden death. Given the natural physiological changes during pregnancy, pregnant women with botulism may experience a more exaggerated course and have worse outcomes compared to nonpregnant women. Medical providers caring for such patients should not only maintain a high level of suspicion for botulism, but administration of an antitoxin early in their care can help reduce morbidity and mortality, while awaiting confirmatory laboratory results. It is not uncommon for a medical provider caring for these women to mistakenly associate the signs and symptoms associated with botulism with pregnancy-related findings; therefore, this article illustrates a clinical algorithmic approach to caring for these women, including a systematic approach to establishing a diagnosis and management plan for pregnant women with botulism.

Antibacterial and antivirulence activities of auranofin against Clostridium difficile.

Clostridium difficile is a deadly, opportunistic bacterial pathogen. In the last two decades, C. difficile infections (CDIs) have become a national concern because of the emergence of hypervirulent mutants with increased capability to produce toxins and spores. This has resulted in an increased number of infections and deaths associated with CDI. The scarcity of anticlostridial drugs has led to unsatisfactory cure rates, elevated recurrence rates and permitted enhanced colonization with other drug-resistant pathogens (such as vancomycin-resistant enterococci) in afflicted patients. Therefore, both patients and physicians are facing an urgent need for more effective therapies to treat CDI. In an effort to find new anticlostridial drugs, we investigated auranofin, an FDA-approved oral antirheumatic drug that has recently been found to possess antibacterial activity. Auranofin exhibited potent activity against C. difficile isolates, inhibiting growth at a concentration of 1 µg/mL against 50% of all tested isolates. Auranofin inhibited both toxin production and spore formation, a property lacking in both vancomycin and metronidazole (the primary agents used to treat CDI). Auranofin had a direct protective activity against C. difficile toxin-mediated inflammation and inhibited the growth of vancomycin-resistant enterococci. Auranofin is a promising candidate that warrants further investigation as a treatment option for C. difficile infections.

Comparison of the immunogenicity of Dukoral® oral cholera vaccine between renal transplant recipients on either a calcineurin inhibitor or mycophenolate - A controlled trial.

BACKGROUND: The evidence for recommendations regarding vaccination in solid organ transplant recipients is sparse. There is little data comparing vaccine responses between groups on different immunosuppressive drugs. This study was conducted to evaluate the antibody response to Dukoral® oral cholera vaccine in renal transplant recipients (RTR). METHODS: In a single-center non-randomized controlled clinical trial, healthy volunteers (n = 21) and renal transplant recipients (n = 30) were vaccinated with the oral whole cell/recombinant B subunit cholera vaccine Dukoral® (Valneva Inc., Vienna, Austria). The RTR were stratified according to their maintenance immunosuppressive therapy: either prednisone and a calcineurin inhibitor (cyclosporine A or tacrolimus; P/CNI group; n = 15) or prednisone and mycophenolate (P/MMF group; n = 15). All volunteers ingested Dukoral® at baseline and at day 14. Serum samples were drawn at day 0 and day 21. The primary outcome was seroconversion, defined as either a 3-fold IgA serum titer increase in anti-cholera toxin B antibodies and/or a 4-fold rise in the serum vibriocidal titer. RESULTS: Follow-up was complete. Seroconversion after vaccination was 57% (standard error, SE 9%) in RTR and 81% (SE 9%) in healthy controls (Relative Risk, RR 0.70; 95% CI 0.48-1.02). When stratified according to maintenance immunosuppression, the seroconversion rate was 67% (SE 12%) in the P/CNI group (RR compared with controls 0.82; 95% CI 0.55-1.25) and 47% (SE 13%) in the P/MMF group (RR compared with controls 0.58; 95% CI 0.32-1.03). CONCLUSION: Adverse events were mild to moderate and transient. The response to Dukoral was weaker and the seroconversion rate was lower in renal transplant recipients than in healthy controls. In particular, those using mycophenolate had a poor response. Nevertheless, more than half of the transplant recipients seroconverted. Therefore oral vaccines should not be discarded as a potential tool for protection of solid organ transplant recipients. This trial is registered in clinicaltrials.gov under NCT01109914.

Bezlotoxumab: an emerging monoclonal antibody therapy for prevention of recurrent Clostridium difficile infection.

Clostridium difficile infection (CDI) is the most common health care-acquired infection associated with high hospital expenditures. The incidence of subsequent recurrent CDI increases with prior episodes of CDI, 15%-35% risk after primary CDI to 35%-65% risk after the first recurrent episode. Recurrent CDI is one of the most challenging and a very difficult to treat infections. Standard guidelines provide recommendations on treatment of primary CDI. However, treatment choices for recurrent CDI are limited. Recent research studies have focused on the discovery of newer alternatives for prevention of recurrent CDI targeting prime virulence factors involved in C. difficile pathogenesis. Bezlotoxumab is a human monoclonal antibody directed against C. difficile toxin B. Multiple in vitro and in vivo animal studies have demonstrated direct binding of bezlotoxumab to C. difficile toxin B preventing intestinal epithelial damage and colitis. Furthermore, this monoclonal antibody mediates early reconstitution of gut microbiota preventing risk of recurrent CDI. Randomized placebo-controlled trials showed concomitant administration of a single intravenous dose of 10 mg/kg of bezlotoxumab, in patients on standard-of-care therapy for CDI, had no substantial effect on clinical cure rates but significantly reduced the incidence of recurrent CDI (~40%). It shows efficacy against multiple strains, including the epidemic BI/NAP1/027 strain. Bezlotoxumab is a US Food and Drug administration-approved, safe and well-tolerated drug with low risk of serious adverse events and drug-drug interactions. Bezlotoxumab has emerged as a novel dynamic adjunctive therapy for prevention of recurrent CDI. Further studies on real-world experience with bezlotoxumab and its impact in reducing rates of recurrent CDI are needed.

Liposomal Therapy Attenuates Dermonecrosis Induced by Community-Associated Methicillin-Resistant Staphylococcus aureus by Targeting α-Type Phenol-Soluble Modulins and α-Hemolysin.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), typified by the pulse-field type USA300, is an emerging endemic pathogen that is spreading rapidly among healthy people. CA-MRSA causes skin and soft tissue infections, life-threatening necrotizing pneumonia and sepsis, and is remarkably resistant to many antibiotics. Here we show that engineered liposomes composed of naturally occurring sphingomyelin were able to sequester cytolytic toxins secreted by USA300 and prevent necrosis of human erythrocytes, peripheral blood mononuclear cells and bronchial epithelial cells. Mass spectrometric analysis revealed the capture by liposomes of phenol-soluble modulins, α-hemolysin and other toxins. Sphingomyelin liposomes prevented hemolysis induced by pure phenol-soluble modulin-α3, one of the main cytolytic components in the USA300 secretome. In contrast, sphingomyelin liposomes harboring a high cholesterol content (66 mol/%) were unable to protect human cells from phenol-soluble modulin-α3-induced lysis, however these liposomes efficiently sequestered the potent staphylococcal toxin α-hemolysin. In a murine cutaneous abscess model, a single dose of either type of liposomes was sufficient to significantly decrease tissue dermonecrosis. Our results provide further insights into the promising potential of tailored liposomal therapy in the battle against infectious diseases.

Characterization of serum anti-diphtheria antibody activity following administration of equine anti-toxin for suspected diphtheria.

There is a global shortage of equine-derived diphtheria anti-toxin (DAT) for diphtheria treatment. There are few existing data on serum antibody concentrations and neutralizing activity post-treatment to support development of new therapeutics. Antibody concentrations were quantified by ELISA and anti-toxin neutralizing activity by cytotoxicity assay in serum from 4 patients receiving DAT for suspected diphtheria. Using linear mixed effects modeling, estimated mean (SE) half-life was 78.2 (20.0) hours. Maximum serum neutralizing activity ranged from 28.42-38.64 AU/mL with an estimated mean AUC1-72 of 1396.7 (399.3) AU/mL*hr. These data provide a standard of comparison for development of novel anti-toxins to replace DAT.

Purification and Anti-pathogenic Properties of Immunoglobulin Concentrates from Porcine Blood.

During slaughtering, animal blood is typically discarded, resulting in water pollution. However, this discarded blood has valuable components, such as immunoglobulin (Ig). Although several studies have been conducted to develop methods for effective recycling of slaughterhouse blood, they have not been commercially utilized in Korea. Here, we extracted an Ig-rich fraction from porcine blood that was then subjected to various in vitro tests, including pathogen growth inhibition, antigenic cross-reactivity, and anti-toxin activity. The porcine immunoglobulin concentrate (PIC) was effectively purified by eliminating other components, such as albumin, and consisted of approximately 63.2±2.9% IgG and 7.2±0.4% IgM on a protein basis. The results showed that it significantly suppressed the growth of pathogenic bacteria, and bound to all tested pathogens, including both gram-positive and gram-negative species, although the degree of activity differed according to strain. The PIC bound to two types of lipopolysaccharide (LPS) obtained from Escherichia coli O111:B4 and Salmonella enterica serotype typhimurium in a concentration-dependent manner. In addition, the PIC restored the proliferation activity of the lymphoblast K-562 cells when co-incubated with pathogenic LPS. These results confirm that the PIC prepared in this study is a potentially valuable functional food material or diet supplement as an alternative to antibiotics that can protect animals from pathogenic bacteria.

Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model.

Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans.

Engineered nanoparticles mimicking cell membranes for toxin neutralization.

Protein toxins secreted from pathogenic bacteria and venomous animals rely on multiple mechanisms to overcome the cell membrane barrier to inflict their virulence effect. A promising therapeutic concept toward developing a broadly applicable anti-toxin platform is to administer cell membrane mimics as decoys to sequester these virulence factors. As such, lipid membrane-based nanoparticulates are an ideal candidate given their structural similarity to cellular membranes. This article reviews the virulence mechanisms employed by toxins at the cell membrane interface and highlights the application of cell-membrane mimicking nanoparticles as toxin decoys for systemic detoxification. In addition, the implication of particle/toxin nanocomplexes in the development of toxoid vaccines is discussed.

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain.

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (~30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.

The roles of toxin A and toxin B in Clostridium difficile infection: insights from the gnotobiotic piglet model.

We recently published our findings indicating that anti-TcdB antibodies were effective as treatment for C. difficile infection, but that anti-TcdA actually worsened prognosis in the gnotobiotic piglet model. To further investigate the roles of the two toxins, we administered purified toxins separately or together, systemically, to piglets and found that both toxins, either alone or together, are able to elicit severe lesions systemically and are also able to cross into the gut lumen and cause large intestinal lesions typical of infection. We also found that anti-TcdA administered before systemic challenge with TcdA again did not protect from development of disease, but, in this case, did not appear to worsen prognosis. Further work is still needed, but these findings add to the growing knowledge regarding the roles of the C. difficile toxins.