Epidemiological investigation of a food-borne outbreak in a kindergarten, Jeju Province, Korea.

OBJECTIVES: On Monday, September 6, 2021, at a kindergarten in Jeju Province, a large number of children vomited and developed food poisoning symptoms, and this necessitated an epidemiological investigation. METHODS: The team surveyed symptoms and food intake history of kindergarten children, teachers, and workers who ate lunch between September 2 (Thursday) and September 6 (Monday), excluding weekends. In addition to rectal swabs, environmental samples from preserved foods, cooking utensils, drinking water, and refrigerator handles were collected. Pulsed field gel electrophoresis (PFGE) for genetic fingerprint analysis was also performed. RESULTS: There were 19 cases among 176 subjects, which indicated an attack rate of 10.8%. The epidemic curve showed a unimodal shape, and the average incubation period was 2.6 hours. While no food was statistically significant in food intake history, the analysis of 35 rectal smear samples detected Bacillus cereus in 7 children, 4 teachers, and 1 cooking staff. Enterotoxins were also detected in 12 samples. Out of 38 environmental samples, B. cereus and enterotoxins were detected in the morning snack cereal, lunch bean sprouts, and afternoon snack steamed potatoes on Monday, September 6th. The result of the PFGE test on 10 isolates of B. cereus showed that there was no genetic homology. CONCLUSIONS: Our results indicated that this outbreak was simultaneously caused by various strains of B. cereus from the environment.

Methicillin-resistant Staphylococcus aureus contamination of high-touched surfaces in a university campus.

AIM: Methicillin-resistant Staphylococcus (MRSA) is a public and occupational health concern, both in community and healthcare settings. In recent years, community-acquired MRSA (CA-MRSA) has emerged as a major causative agent of infections in individuals with no health care exposure or any of the classical risk factors associated with infections. Environmental surfaces frequently touched by hands play a role in the transmission of CA-MRSA, where inanimate objects are considered potential reservoirs and the source of MRSA infections. The purpose of this study was to examine the prevalence of MRSA on environmental surfaces inside a university campus. METHODS AND RESULTS: A total of 1078 high-touch surface samples were collected from door handles, light switches, desks, keyboards and restroom surfaces. MRSA isolates were identified and confirmed by PCR, utilizing the Staph. aureus nuc and mecA genes. Antibiotic resistance profiles were determined using disc diffusion and minimum inhibitory concertation methods. In addition, the ability to form biofilms was investigated by the 96-well plate microdilution technique. PCR assays were performed to detect enterotoxin and antibiotic-resistant genes. The genetic diversity of MRSA was determined through multi-locus sequence typing (MLST), spa and agr typing methods. The overall contamination of Staph. aureus and MRSA was 14.6% (157/1078) and 2.8% (30/1078), respectively. The highest rate of MRSA contamination was detected in restroom sinks and door handles. All MRSA isolates were MDR, with the highest resistance observed was against trimethoprim-sulfamethoxazole. Most MRSA isolates (29/30, 97%) carried at least one gene encoding for staphylococcal enterotoxins (SE), with 10 different SE genotypes were observed. A total of 16 different spa types were detected among the 30 MRSA isolates. Multi-locus sequence typing revealed that 21 MRSA isolates belonged to eight known sequence types (ST), while nine isolates were novel strains. The most detected ST and spa types were ST22 and t223, respectively. Agr types I and III were represented in 28 out of the 30 isolates. The majority of the isolates carried SCCmec type IV, but only one isolate was positive for PVL. CONCLUSIONS: Our findings signify the potential of the high-touch surfaces in harbouring and transmitting MRSA to campus staff and students. Thus, the implementation of effective prevention measures outside the healthcare setting is needed to reduce the risk of acquiring CA-MRSA infections. SIGNIFICANCE AND IMPACT: MRSA infections impose a profound economic burden due to illness and productivity loss. The results of this study not only help us to better understand the environmental reservoirs of this pathogen, but also provide information about its transmission pathways and healthcare settings entry routs.

An outbreak of food poisoning due to Escherichia coli serotype O7:H4 carrying astA for enteroaggregative E. coli heat-stable enterotoxin1 (EAST1).

In June 2020, a large-scale food poisoning outbreak involving about 3000 elementary and junior high school students occurred in Yashio, Saitama, Japan. A school lunch was the only food stuff ingested by all of the patients. Escherichia coli serotype O7:H4 carrying the astA gene for enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1) was detected in faecal specimens from the patients, and sample inspection revealed its presence in a seaweed salad and red seaweed (Gigartina tenella) as one of the raw materials. Analysis of the antibiotic sensitivity of the isolates revealed resistance to ampicillin and cefotaxime. All isolates were confirmed to be of the same origin by pulsed-field gel electrophoresis after digestion with the restriction enzyme XbaI, and single nucleotide polymorphism analysis using whole genome sequencing. To our knowledge, this is the first report of a large-scale food poisoning caused by E. coli O7:H4, which lacks well-characterized virulence genes other than astA.

Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile.

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile), we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl2 and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of >99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile.

Crystal Structure of Exotoxin A from Aeromonas Pathogenic Species.

Aeromonas exotoxin A (AE) is a bacterial virulence factor recently discovered in a clinical case of necrotising fasciitis caused by the flesh-eating Aeromonas hydrophila. Here, database mining shows that AE is present in the genome of several emerging Aeromonas pathogenic species. The X-ray crystal structure of AE was solved at 2.3 Å and presents all the hallmarks common to diphthamide-specific mono-ADP-ribosylating toxins, suggesting AE is a fourth member of this family alongside the diphtheria toxin, Pseudomonas exotoxin A and cholix. Structural homology indicates AE may use a similar mechanism of cytotoxicity that targets eukaryotic elongation factor 2 and thus inhibition of protein synthesis. The structure of AE also highlights unique features including a metal binding site, and a negatively charged cleft that could play a role in interdomain interactions and may affect toxicity. This study raises new opportunities to engineer alternative toxin-based molecules with pharmaceutical potential.

Presence of genes encoding enterotoxins in Staphylococcus aureus isolates recovered from food, food establishment surfaces and cases of foodborne diseases.

The aim of this study was to describe the microbiological characteristics and profile of genes encoding enterotoxins in 95 Staphylococcus aureus isolates obtained between April 2011 and December 2014 from foodstuffs, persons and surfaces of retail food stores. After microbiological identification and antimicrobial susceptibility testing, polymerase chain reactions (PCR) were performed, targeting sea, seb, sec, sed and see genes that code for classical enterotoxins (ET) A-E, and three additional genes: seg , seh and sei , coding for so-called "new enterotoxins" G, H and I. The isolates were characterized by Pulsed Field Gel Electrophoresis (PFGE), and five selected isolates were further analyzed through Multi Locus Sequence Typing (MLST). It is noteworthy that 54.7% of the examined isolates harbored one or more of the investigated ET gene types. Most positive isolates carried more than one ET gene up to five types; seg was the most frequent ET gene, followed by sei. Five enterotoxin-coding isolates also coded for some antimicrobial resistance genes. Two of them, and four additional non-enterotoxic isolates carried erm genes expressing inducible clindamycin resistance. PFGE-types were numerous and diverse, even among enterotoxin-coding strains, because most isolates did not belong to known foodborne outbreaks and the sampling period was long. MLST profiles were also varied, and a new ST 3840 was described within this species. ST 88 and ST 72 enterotoxin-coding isolates have been identified in other regions in association with foodborne outbreaks. This manuscript reports the first systematic investigation of enterotoxin genes in S. aureus isolates obtained from foodstuffs and infected people in Uruguay.

Short communication: Enterotoxigenic potential of coagulase-negative staphylococci isolated from bovine milk in Poland.

Our objective was to assess the enterotoxigenic potential of coagulase-negative staphylococci (CNS) isolated from bovine milk in Poland. We analyzed CNS isolates collected from 133 bovine milk batches from dairy farms in the Western Pomerania district during 2 milking seasons. A total of 163 isolates were screened by multiplex/duplex PCR for the presence of 18 of 25 enterotoxin genes identified so far in Staphylococcus aureus. The CNS strains presumed to be potentially enterotoxigenic were identified at the species level based on MALDI-TOF mass spectrometry analysis. Based on the presence of an amplicon matching D, G, or O enterotoxin genes, we initially identified 32 of the 163 CNS isolates tested as potentially enterotoxigenic. However, only 8 of these strains were confirmed as such. All 8 of these CNS strains, identified as Staphylococcus haemolyticus, harbored the seg genes, a prerequisite for enterotoxin G production, but so far not connected with staphylococcal foodborne poisoning cases. None of the CNS bovine milk isolates tested was a potential producer of classical A to E staphylococcal enterotoxins. Results of our surveys revealed a low prevalence of enterotoxigenic CNS among the milk isolates from dairy farms in the Western Pomerania district, Poland, suggesting that they pose only a mild health risk in milk. In our opinion, confirmed formation of nonspecific amplicons leading to false-positive results excludes multiplex/duplex PCR as the sole method for assessing the enterotoxigenic potential of CNS.

Storage procedures and time influence the detectability of Clostridium difficile toxin A but not toxin B in porcine fecal specimens.

Storage procedures are known to affect the detectability of Clostridium difficile toxins in equine and human feces. We assessed the impact of different storage conditions on the detectability of C. difficile toxins in swine feces. Specimens were inoculated with toxins, 112 ng/g of toxin A (TcdA) and 16 ng/g of toxin B (TcdB) and subjected to the following 3 storage treatments: 4°C, -30°C, repetitive freezing at -30°C and thawing. Toxin determination was assessed at 1, 2, 7, 14, and 21 d with ELISA. A decrease in concentrations of TcdA with time was observed for samples stored at 4°C and repetitive freezing-thawing (p ≤0.05). On day 14, storage at 4°C resulted in decreased TcdA concentration as opposed to storage at -30°C and repetitive freezing-thawing (p ≤0.05). On day 21, storage at 4°C resulted in decreased TcdA detectability compared with storage at -30°C (p ≤0.05). The TcdB concentration was unaffected. These results on toxin detectability in swine feces should be carefully considered in in vitro studies on toxigenic C. difficile. Our results also offer valuable information for microbiologists and veterinarians monitoring the presence of virulent C. difficile in pigs.

Prevalence of netB-positive Clostridium perfringens in Italian poultry flocks by environmental sampling.

Clostridium perfringens type G is one of the pathogens involved in enteric diseases in poultry. NetB, a pore-forming toxin, is considered the main virulence factor responsible for necrotic enteritis during C. perfringens infection. We carried out a field study involving 14 farms to evaluate the occurrence of netB-positive C. perfringens and the impact of infection in Italian poultry flocks. Environmental samples (n = 117) and 50 carcasses were screened by microbiologic and molecular methods. Microbiologic investigations yielded 82 C. perfringens isolates. DNA was extracted from all samples and screened for α-toxin and NetB encoding genes by real-time PCR. The C. perfringens α-toxin gene was detected in 151 of 167 extracts (90.4%), and 31 of 151 (20.5%) were netB gene positive also. Sixteen isolates from a turkey flock with mild enteric disorders were also netB positive, demonstrating their occurrence not only in broiler but also in turkey flocks. A pulsed-field gel electrophoresis protocol was optimized to evaluate the diversity among isolates and revealed high genetic heterogeneity. The complete NetB toxin-coding gene of 2 C. perfringens isolates from turkey and broiler flocks were analyzed and showed very high relatedness with analogous sequences worldwide.

Core-Shell Gold/Silver Nanoparticles for Localized Surface Plasmon Resonance-Based Naked-Eye Toxin Biosensing.

The localized surface plasmon resonance (LSPR) phenomenon provides a versatile property for biodetection. Herein, this unique feature was employed to build a homogeneous optical biosensor to detect staphylococcal enterotoxin A (SEA) in solution down to very low levels by naked-eye readout. If the initial position of the LSPR band is located in the cyan region, even a small red shift (∼2-3 nm) induced by a refractive index change close to the surface of nanoparticles (NPs) could make the light absorption transit from cyan to green and become visually detectable via a concomitant change in the complementary colors. In this work, we aimed at synthesizing two types of NPs based on compositionally complex core-shell NPs-Ag shells on AuNPs (Au@AgNPs) and Ag inside gold nanoshells (Ag@AuNPs). By controlling the thickness of the shells and their surface chemistry with anti-SEA antibody (Ab), the LSPR band was tuned to near 495 and 520 nm for Ag@AuNPs and Au@AgNPs, respectively. The two particle systems were subsequently applied to spectroscopically and visually detect anti-SEA Ab-SEA interactions. Upon the addition of SEA, large red shifts of the LSPR band were observed spectroscopically and the limits of detection (LODs) were estimated to be 0.2 and 0.4 nM for Au@AgNPs and Ag@AuNPs, respectively. Although the two sets of NPs gave almost identical LODs, the Ag@AuNPs whose initial position of the LSPR band was tuned in the cyan to green region (∼500 nm) displayed a substantially more distinct color change from orange to red, as revealed by the naked eye. We foresee significant potential to this strategy in medical diagnostics and environmental monitoring, especially when basic laboratory infrastructure is sparse or nonexistent.

Microbial safety status of Serro artisanal cheese produced in Brazil.

Considering the growing consumption of artisanal foods worldwide, we aimed to evaluate the microbial safety of Serro artisanal cheese (SAC), produced in Minas Gerais State, Brazil. This cheese is produced with raw milk using 1 of 2 natural starter cultures: "pingo" and "rala." A total of 53 SAC samples (pingo = 8; rala = 45) were obtained from different farmers and subjected to conventional and molecular assays to detect and enumerate Listeria monocytogenes, Salmonella spp., coagulase-positive staphylococci (CPS), diarrheagenic Escherichia coli, Mycobacterium tuberculosis, and Brucella abortus. The SAC samples were also subjected to an ELISA to detect classical staphylococcal enterotoxins (CSE: SEA, SEB, SEC, SED, SEE) and to PCR assays to detect staphylococcal enterotoxin-related genes (sea, seb, sec, sed, see). Coagulase-positive staphylococci isolates were obtained and tested by the same assays to detect their potential in CSE production and presence of CSE-related genes. None of the SAC samples showed any of the screened food-borne pathogens and zoonotic agents, and none showed the presence of CSE by phenotypic and genotypic approaches. Despite the absence of microbial hazards, mean counts of CPS in SAC samples were 5.2 log cfu/g (pingo starter) and 4.6 log cfu/g (rala starter), indicating poor hygiene practices during production. None of the tested CPS isolates (n = 116) produced CSE or presented CSE-related genes. Despite the relative microbial safety, hygienic conditions during SAC production must be improved to meet official guidelines established in Brazil.

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay.

Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0 pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity+/CCCNA-) became CCCNA-, and 5/26 (19.2%) Clarity+/CCCNA- samples became CCCNA+, resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We evaluated the clinical performance of ultrasensitive single-molecule counting technology for detection of C. difficile toxins A and B. Stool specimens from 298 patients with suspected CDI were tested with the nucleic acid amplification test (NAAT; BD MAX Cdiff assay or Xpert C. difficile assay) and Singulex Clarity C. diff toxins A/B assay. Specimens with discordant results were tested with the cell cytotoxicity neutralization assay (CCNA), and the results were correlated with disease severity and outcome. There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity- and 4 NAAT-/Clarity+ samples, there were 26 CCNA- and 4 CCNA- samples, respectively. CDI relapse was more common in NAAT+/toxin+ patients than in NAAT+/toxin- and NAAT-/toxin- patients. The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was more than three times more common in NAAT+/toxin- than in NAAT+/toxin+ patients. The Clarity assay was superior to NAATs for the diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and the presence of toxins was associated with negative patient outcomes.

Rapid Detection of Clostridium difficile Toxins in Stool by Raman Spectroscopy.

BACKGROUND: Clinical practice guidelines define Clostridium difficile infections (CDI) as diarrhea (≥3 unformed stools in 24 h) with either a positive C difficile stool test or detection of pseudomembranous colitis. Diagnostic modalities such as toxigenic culture and nucleic acid amplification testing can identify the presence of toxigenic C difficile in stools. But these tests are confounded by the presence of asymptomatic colonization of toxigenic C difficile and lead to overdiagnosis of CDI. The presence of two large toxins, toxin A and B (TcdA and TcdB) is necessary for pathogenicity. Detection of toxins using toxin enzyme immunoassay is difficult as it has low sensitivity and moderate specificity. Raman spectroscopy (RS) is a novel technology that is used to detect bacteria and their toxins. RS does not require any reagents for detection such as antibodies, enzymes, primers, or stains. We hypothesize that RS is a sensitive method to detect C difficile toxins in stool and will solve the problem of overdiagnosis of CDI. MATERIALS AND METHODS: CDI negative stool samples were spiked with concentrations (1 ng/mL, 100 pg/mL, 1 pg/mL, and 0.1 pg/mL) of TcdA and TcdB. RS was performed on air-dried smeared samples of stool supernatant on a mirror-polished stainless-steel slide. As RS of feces is difficult because of confounding background material and autofluorescence, samples were photo-bleached before spectral acquisition to reduce autofluorescence. Raman spectra were obtained, background corrected, and vector normalized. The data were split into training (70%) and test (30%) datasets. The machine learning methods used on the training data set were Support Vector Machine with Linear and Radial Kernels, Random Forest, Stochastic Gradient Boosting Machine, and Principle Component Analysis-Linear Discriminant Analysis. Results were validated using a test data set. The best model was chosen, and its accuracy, sensitivity, and specificity were determined. RESULTS: In our preliminary results, at all concentrations (1 ng/mL, 100 pg/mL, 1 pg/mL, and 0.1 pg/mL), TcdA or TcdB spiked stool was distinguished from unspiked stool by all models with accuracies ranging from 64% to 77%. Gradient Boosting Machine, Principle Component Analysis-Linear Discriminant Analysis, and Support Vector Machine Linear Kernel performed best with sensitivities ranging from 69% to 90% and specificities ranging from 43% to 78%. CONCLUSIONS: Using RS, we successfully detected TcdA and TcdB in stool samples albeit with moderate-to-high sensitivity and low-to-moderate specificity. Sensitivity and specificity could be further increased with the implementation of deep learning methods, which require large sample sizes. In terms of sensitivity, RS performs better than toxin enzyme immunoassay and has the potential to rapidly detect C difficile toxins in stool at clinically relevant concentrations and thereby help mitigate overdiagnosis of CDI.

Development of isothermal amplification methods for rapid and sensitive detection of heat-labile enterotoxin producing Escherichia coli.

The objective of this study was to establish a novel isothermal amplification method for detection of heat-labile enterotoxin (LT-I)-producing Escherichia coli. Loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and isothermal multiple-self-matching-initiated amplification (IMSA) were developed and evaluated. Optimal conditions, specificity, and sensitivity tests were performed and compared to qPCR findings. All three methods could produce ladder-like products with LT-I positive samples, while no products were generated with the negative controls. The amplified products could be directly visualized as negative or positive in the isothermal amplification (IAM) tube, which saved time and prevented the possibility of cross-contamination. The detection limits of each assay were similar, and all three assays could directly detect the DNA of Escherichia coli in clinical samples successfully. This is the first report on the application of CPA and IMSA methods for the detection of LT-I. The findings suggest that the three assays may be important tools for the rapid detection of enterotoxigenic Escherichia coli (ETEC) in the clinic.

An electrochemical aptasensor for staphylococcal enterotoxin B detection based on reduced graphene oxide and gold nano-urchins.

Detection of staphylococcal enterotoxin B (SEB) as a bacterial toxin causing severe food poisoning is of great importance. Herein, we developed an electrochemical aptasensor for SEB detection using a screen printed electrode modified with reduced graphene oxide (rGO) and gold nano-urchins (AuNUs). Afterward, the single-stranded DNA probe was attached to the surface of AuNUs on the modified electrode and then the specific aptamer was attached to the probe. In the presence of SEB molecules, the aptamer detached from the electrode surface and after applying the electrochemical signal generator, hematoxylin and the peak current of differential pulse voltammetry (DPV) were recorded. Due to the intercalation mechanism of hematoxylin-DNA interaction, the detachment of aptamer from electrode surface decreased the DPV peak current and the calibration graph (peak current vs SEB concentration) can be used for quantification of SEB. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) and also field emission scanning electron microscope imaging were used for electrode characterization. Selectivity experiments of the developed aptasensor showed a very distinct difference between SEB and other nonspecific molecules. A wide linear range from 5.0 to 500.0 fM was achieved and the detection limit was calculated as 0.21 fM. The performance of the aptasensor was checked in spiked food samples as simulated real samples and the results showed no significant difference compared to the synthetic samples. Results of selectivity and repeatability of the aptasensor were satisfactory. In addition, better recovery percentages and also lower standard deviation of aptasensor compared to a commercial ELISA kit of SEB detection proved the superior performance of the proposed aptasensor.

An enhanced green fluorescence protein (EGFP)-based reporter assay for quantitative detection of sporulation in Clostridium perfringens SM101.

Clostridium perfringens type F is a spore-forming anaerobe that causes bacterial food-borne illness in humans. The disease develops when ingested vegetative cells reach the intestinal tract and begin to form spores that produce the diarrheagenic C. perfringens enterotoxin (CPE). Given that CPE production is regulated by the master regulator of sporulation (transcription factor Spo0A), the identification of sporulation-inducing factors in the intestine is relevant to better understanding of the disease. To examine these factors, we established assays to quantify C. perfringens sporulation stage under microscopy by using two fluorescent reporters, namely, Evoglow-Bs2 and CpEGFP. When the reporter genes were placed under control of the cpe promoter, both protein products were expressed specifically during sporulation. However, the intensity of the anaerobic reporter Evoglow-Bs2 was weak and rapidly photobleached during microscopic observation. Alternatively, CpEGFP, a canonical green fluorescence protein with optimized codon usage for Clostridium species, was readily detectable in the mother-cell compartment of most bacteria at early stages of sporulation. Additionally, CpEGFP expression predicted final spore yield and was quantifiable in 96-well plates using fluorescence plate reader. These results indicate that CpEGFP can be used to analyze the sporulation of C. perfringens and has a potential application in the large-scale screening of sporulation-regulating biomolecules.