Standardization of the Japanese National Standard, Equine Botulinum Antitoxin Type A, and Factors Affecting Standardization.

Equine botulinum antitoxin is one of the most popular countermeasures for human botulism. The unitage of the antitoxin product is defined according to national minimum requirement or pharmacopoeia in each country by referring to national standard antitoxins for four types (A, B, E, and F). With the expected depletion of the national standard antitoxins, replacement national standard antitoxins are produced and standardized through collaboration of the National Control Laboratory and other participants, including manufacturer(s). Therefore, Japanese National Standard Botulinum Antitoxin Type A, Equine, was replaced according to the results of a collaborative study involving the National Institute of Infectious Diseases and KM Biologics Co., Ltd. The unitage of the replacement material was determined through mouse neutralization tests, which involved toxin-antitoxin mixture injection at pH 7.0. Potency value of 440 units/vial was obtained. However, the Japanese Minimum Requirement for Biological Products was revised, and the neutralization reactions were repeated at pH 6.0, for which considerably different potency value (656 units/vial) and survival profile of mice were obtained. In September 2021, the replacement material, Japanese National Standard Botulinum Antitoxin Type A, Equine, lot 2, was established with potency value of 656 Units/vial. The impact of pH-dependent change in potency on antitoxin quality control is discussed.

Clinical Characteristics of Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans is a closely related bacterium to the diphtheria bacterium C. diphtheriae, and some C. ulcerans strains produce toxins that are similar to diphtheria toxin. C. ulcerans is widely distributed in the environment and is considered one of the most harmful pathogens to livestock and wildlife. Infection with C. ulcerans can cause respiratory or nonrespiratory symptoms in patients. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness in Japan. To clarify the overall clinical characteristics, treatment-related factors, and outcomes of C. ulcerans infection, we analyzed 34 cases of C. ulcerans that occurred in Japan during 2001-2020. During 2010-2020, the incidence rate of C. ulcerans infection increased markedly, and the overall mortality rate was 5.9%. It is recommended that adults be vaccinated with diphtheria toxoid vaccine to prevent the spread of this infection.

Complete Genome Sequence of Corynebacterium ulcerans Strain TSU-28, Harboring Two Diphtheria Toxin Genes, Isolated from a Patient with Diphtheria-Like Symptoms.

Diphtheria toxin-producing Corynebacterium ulcerans is an emerging zoonotic pathogen that causes severe disease in humans. Here, we report the complete genome sequence of C. ulcerans strain TSU-28, harboring two diphtheria toxin genes, which was isolated from the throat of a patient with diphtheria-like symptoms in 2019 in Japan.

An ELISA system for tetanus toxoid potency tests: An alternative to lethal challenge.

For a long time, a widely used method for tetanus toxoid (Ttd) potency has been the challenge test, in which animals are immunized and then challenged with tetanus toxin in lethal or non-lethal way. In the context of animal welfare, an alternative is desired because the method causes unsustainable distress to animals. We aimed to replace the system for describing test results, in which scores are assigned to symptoms exhibited by challenged animals, with scores assigned to antibody ELISA titers in immunized mouse sera. The potency values and confidence intervals calculated by the absorbance score system were equivalent to those calculated by the symptom score system. We also attempted to utilize the raw ELISA absorbance instead of the assigned absorbance score and obtained similar results. ELISA may serve as an alternative to the lethal challenge for Ttd potency tests, not only in Japan but also in other countries in which mouse challenge tests are employed.

Seroepidemiology and Carriage of Diphtheria in Epidemic-Prone Area and Implications for Vaccination Policy, Vietnam.

In 2019, a community-based, cross-sectional carriage survey and a seroprevalence survey of 1,216 persons 1-55 years of age were conducted in rural Vietnam to investigate the mechanism of diphtheria outbreaks. Seroprevalence was further compared with that of an urban area that had no cases reported for the past decade. Carriage prevalence was 1.4%. The highest prevalence, 4.5%, was observed for children 1-5 years of age. Twenty-seven asymptomatic Coerynebacterium diphtheriae carriers were identified; 9 carriers had tox gene-bearing strains, and 3 had nontoxigenic tox gene-bearing strains. Child malnutrition was associated with low levels of diphtheria toxoid IgG, which might have subsequently increased child carriage prevalence. Different immunity patterns in the 2 populations suggested that the low immunity among children caused by low vaccination coverage increased transmission, resulting in symptomatic infections at school-going age, when vaccine-induced immunity waned most. A school-entry booster dose and improved infant vaccination coverage are recommended to control transmissions.

Potential risk of SARS-CoV-2 infection among people handling linens used by COVID-19 patients before and after washing.

The risk of SARS-CoV-2 infection when people handle linens is uncertain. We examined the presence of SARS-CoV-2 on linens, in the air, and on personal protective equipment (PPE) to assess potential infection risk among individuals who handle linens used by SARS-CoV-2-infected people. Patients in a hospital and an accommodation facility who tested positive for SARS-CoV-2 participated in this study in 2020. Linen samples before washing or disinfection, rinse water after washing or disinfection, air in the workplace at the hospital and an accommodation facility, and the PPE worn by linen-handling people were tested for SARS-CoV-2 RNA and viable viruses. Among 700 samples from 13 SARS-CoV-2-infected participants and their surrounding environment, SARS-CoV-2 RNA was detected from 14% (52/362) of the linens used by COVID-19 patients (cycle threshold [Ct] value: 33-40). SARS-CoV-2 RNA was detected from 8% (2/26) of rinse water after washing or disinfection, from 15% (16/104) of air samples in the workspace, and from 10% (5/52) of gowns worn by linen-handling people, all with high Ct values (> 36). No SARS-CoV-2 was isolated from any samples. The potential risk of SARS-CoV-2 infection from handling linens used by SARS-CoV-2-infected people exists but appears to below.

Rapid detection and discrimination of potentially toxigenic Corynebacterium ulcerans and Corynebacterium pseudotuberculosis by multiplex real-time PCR and amplicon melting curve analysis.

We developed a multiplex real-time PCR assay with amplicon melting curve analysis to rapidly discriminate Corynebacterium ulcerans from Corynebacterium pseudotuberculosis and detect the bacterial diphtheria toxin gene. This assay should be a valuable tool for identification of potentially toxigenic C. ulcerans.

Transmission of toxigenic Corynebacterium ulcerans infection with airway obstruction from cats to a human.

BACKGROUND: Infections caused by Corynebacterium ulcerans, a zoonotic pathogen, have been reported worldwide. This microorganism is known to produce the diphtheria toxin and cause diphtheria-like illness. CASE PRESENTATION: A 63-year-old woman with a history of diabetes and hypertension developed cold and flu-like symptoms, which gradually progressed into respiratory distress. Therefore, the patient was intubated for dyspnea with pseudomembrane formation. A toxin-producing strain of C. ulcerans was identified, also detected in the patient's domestic cats. Multilocus sequence typing confirmed all strains, including the patient's isolate, as ST337. CONCLUSION: Multilocus sequence typing revealed zoonotic transmission of C. ulcerans from domestic cats to a human.

Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae.

Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.

Limitations of Ribotyping as Genotyping Method for Corynebacterium ulcerans.

We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate's ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.

Development of vaccine for Clostridium difficile infection using membrane fraction of nontoxigenic Clostridium difficile.

Although standard antibiotic therapy is performed for diarrhea and pseudomembranous colitis caused by Clostridium difficile, a high recurrence rate of C. difficile infection (CDI) remains a major problem. We previously showed that a membrane fraction of nontoxigenic C. difficile (ntCDMF) was effective as a vaccine antigen by in vitro experiments. In this study, we examined whether ntCDMF had an in vivo effect in animal challenge experiments. By intrarectal immunization with ntCDMF, the number of C. difficile cells in feces of mice was decreased approximately 99% compared to the control mice. In addition, survival rate of C. difficile-challenged hamsters was increased almost 30% by immunization with ntCDMF. These results showed that ntCDMF could be a practical vaccine candidate.

Severe Pneumonia Caused by Toxigenic Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans infection was recently recognized as a zoonosis. We present 2 cases of severe pneumonia complicated by diffuse pseudomembrane formation on the bronchus caused by C. ulcerans-producing diphtheria toxin. Our purpose is to alert medical professionals to the virulence of Corynebacterium species other than C. diphtheriae.

The first fatal case of Corynebacterium ulcerans infection in Japan.

Introduction.Corynebacterium ulcerans (C. ulcerans) is a zoonotic pathogen that occasionally causes diphtheria-like symptoms in humans. Cases of C. ulcerans infection have been increasing in recent years, and C. ulcerans has been recognized as an emerging pathogen. Case presentation. Here we report a case of asphyxia death due to pseudomembrane caused by diphtheria toxin (DT)-producing C. ulcerans. This is, to our knowledge, the first fatal case of C. ulcerans infection in Japan. A strain of C. ulcerans was obtained from the patient's pet cat and was confirmed to be identical to the patient's isolate by sequencing of the 16S rRNA gene and the DT gene, by pulsed-field gel electrophoresis (PFGE) and by ribotyping. In the same way, it was revealed that the isolate in this case belonged to the same molecular type as the C. ulcerans 0102 isolated from the first case in Japan in a distant prefecture 15 years earlier, in 2001. Conclusion. DT-producing C. ulcerans can be contracted from a companion animal and causes human death if the appropriate treatment is delayed. The finding indicates that this molecular type of virulent C. ulcerans is currently widespread in Japan.

Genome Sequence of Clostridium botulinum Strain Adk2012 Associated with a Foodborne Botulinum Case in Tottori, Japan, in 2012.

We report here a draft genome sequence of Clostridium botulinum Adk2012 responsible for a foodborne botulism case that occurred in Tottori, Japan, in 2012. Its genome size was 2,904,173 bp, with 46 rRNAs and 54 tRNAs, at a coverage of 14.5×.

Toxigenic Corynebacterium ulcerans isolated from a wild bird (ural owl) and its feed (shrew-moles): comparison of molecular types with human isolates.

BACKGROUND: Corynebacterium ulcerans is a pathogen causing diphtheria-like illness to humans. In contrast to diphtheria by Corynebacterium diphtheriae circulating mostly among humans, C. ulcerans infection is zoonotic. The present study aimed to clarify how a zoonotic pathogen C. ulcerans circulates among wild birds and animals. RESULTS: By screening 380 birds, a single strain of toxigenic C. ulcerans was isolated from a carnivorous bird, ural owl (Strix uralensis). The bacterium was also isolated from two individuals of Japanese shrew-mole (Urotrichus talpoides), a food preference of the owl. Analysis by ribotyping showed that the owl and mole isolates were classified in a group, suggesting that C. ulcerans can be transmissible among wild birds and their prey animals. Moreover, our isolates were found to belong to a group of previously reported C. ulcerans isolates from dogs and a cat, which are known to serve as sources for human infection. CONCLUSION: The findings suggest that the shrew-mole may be a potential reservoir of a zoonotic pathogen C. ulcerans.

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

A novel experimental platform for toxigenic and non-toxigenic Corynebacterium ulcerans infection in mice.

Corynebacterium ulcerans is a zoonotic pathogen that can produce diphtheria toxin and causes an illness categorized as diphtheria in the European Union because its clinical appearance is similar to that of diphtheria caused by Corynebacterium diphtheriae. Despite the importance of the pathogen in public health, the organism's mechanism of infection has not been extensively studied, especially in experimental animal models. Therefore in the present study we constructed an intranasal infection system for mice. Mice are insensitive to diphtheria toxin and this has the advantage of excluding the cytotoxic effect of the toxin that might interfere with the analysis of the early stage of infection. Both the toxigenic and non-toxigenic C. ulcerans strains were capable of killing mice within 3 days after inoculation at 10(7) colony-forming units per mouse. In experimentally infected animals, C. ulcerans was detected in the respiratory tract but not in the intestinal tract. The bacterium was also detected in peripheral blood and it disseminated into the lung, kidney and spleen to produce a systemic infection. This experimental infection system provides a platform for analyzing the virulence of C. ulcerans in future studies.

Adenylate cyclase toxin-mediated delivery of the S1 subunit of pertussis toxin into mammalian cells.

The adenylate cyclase toxin (ACT) of Bordetella pertussis internalizes its catalytic domain into target cells. ACT can function as a tool for delivering foreign protein antigen moieties into immune effector cells to induce a cytotoxic T lymphocyte response. In this study, we replaced the catalytic domain of ACT with an enzymatically active protein moiety, the S1 (ADP-ribosyltransferase) subunit of pertussis toxin (PT). The S1 moiety was successfully internalized independent of endocytosis into sheep erythrocytes. The introduced polypeptide exhibited ADP-ribosyltransferase activity in CHO cells and induced clustering typical to PT. The results indicate that ACT can act as a vehicle for not only epitopes but also enzymatically active peptides to mammalian cells.

Retrospective survey to evaluate the safety and efficacy of Japanese botulinum antitoxin therapy in Japan.

Japanese botulinum antitoxins have been used for more than 50 years; however, their safety and therapeutic efficacy are not clear. In order to analyze the available data on botulinum antitoxin therapy in Japan, we surveyed published reports about botulism cases in which botulinum antitoxins were used, and retrospectively analyzed the safety and efficacy of the therapy. A total of 134 patients administered botulinum antitoxins were identified from published reports. Two cases of side effects (1.5%) were detected after antitoxin administration, both not fatal. The fatality rate was 9.4%, and more than 70% of the patients showed improvement in their symptoms and better clinical conditions than those not treated with antitoxins. These data suggest that the therapy with Japanese antitoxins is safe and highly effective.

Inhibition of adhesion of Clostridium difficile to human intestinal cells after treatment with serum and intestinal fluid isolated from mice immunized with nontoxigenic C. difficile membrane fraction.

Diarrhea and pseudomembrane colitis caused by Clostridium difficile infection is a global health concern because of the high recurrence rate after standard antibiotic therapy. Vaccination presents a powerful countermeasure against disease recurrence. In this study, mice vaccinated with the nontoxigenic C. difficile membrane fraction generated a marked immune response to the antigen, as demonstrated by the serum IgG and intestinal fluid IgA levels. Significantly, pretreatment with harvested IgG- and IgA-containing fluids was sufficient to prevent in vitro adhesion of C. difficile to human Caco-2 intestinal cells. These results highlight the potential of nontoxigenic C. difficile membrane fraction as a vaccine candidate for C. difficile infection.