Prevalence of Staphylococcus argenteus among food handlers, kitchen utensils, and food samples in Japan.

Staphylococcus argenteus has received increased attention from an aspect of food safety since several food poisoning outbreaks caused by the bacterium were reported in Japan. However, S. argenteus prevalence among food handlers and utensils has not yet been investigated. In this study, we investigated S. argenteus prevalence among a collection of coagulase-positive staphylococci (CPS) that were isolated during food sanitary inspections in Japan. Out of a total of 191 CPS isolates, 14 were identified as S. argenteus. One was isolated from shelled shrimp, nine were isolated from food handlers' hand swabs, and four were isolated from kitchen utensils. Whole-genome sequencing revealed that transmission of S. argenteus from human hands to utensils was possible. Though all 14 isolates were negative for the pvl and tst-1 genes, 6 harbored the seb gene. Only 21.4% of S. argenteus isolates were resistant to antibiotics, while 62.1% of the S. aureus isolates from the same sources were confirmed to be resistant. To the best of our knowledge, this is the first report to demonstrate possible transmission of S. argenteus from food handlers to utensils in food-processing environments.

Phosphorothioation of foreign DNA influences the transformation efficiency in Clostridium perfringens NCTC8239.

Major advances in Clostridium perfringens genetics have been achieved through the development of electroporation-induced transformation; however, highly transformable strains are still limited. SM101 is the only useful strain for genetic manipulation via transformation in C. perfringens causing foodborne illness (FBI). We focused on the FBI strain NCTC8239, which is transformed at a low frequency, because it has a gene cassette that is predicted to encode enzymes involved in DNA phosphorothioation (PT). The oxidant-dependent degradation of NCTC8239 genomic DNA suggested that the genome is PT-modified. When foreign DNA was PT-modified using a plasmid expressing Salmonella enterica PT modification enzymes, the transformation efficiency of NCTC8239 was significantly higher than that using an unmodified plasmid. We then attempted to establish a highly transformable derivative of NCTC8239, and focused on DptFGH, which are predicted to be PT restriction enzymes. A dptG-null mutant exhibited significantly higher transformation efficiency with unmodified foreign DNA than did the wild-type strain. Furthermore, the mutant was transformed with the unmodified plasmid as efficiently as with a PT-modified plasmid, implying that DptG is involved in PT-dependent restriction. Thus, the present results revealed that PT modifications of foreign DNA influence the transformation frequency of NCTC8239 and suggest that PT is a factor contributing to transformation efficiency in NCTC8239.

Effect of sublethal heat treatment on the later stage of germination-to-outgrowth of Clostridium perfringens spores.

Sublethal heating of spores has long been known to stimulate or activate germination; however, the underlying mechanisms are not yet fully understood. In this study, the entire germination-to-outgrowth process of spores from Clostridium perfringens, an anaerobic sporeformer, was visualized at single-cell resolution. Quantitative analysis revealed that sublethal heating significantly reduces the time from completion of germination to the beginning of the first cell division, indicating that sublethal heating of C. perfringens spores not only sensitizes the responsiveness of germinant receptors but also directly or indirectly facilitates multiple steps during the bacterial regrowth process.

Transcriptional Profile during Deoxycholate-Induced Sporulation in a Clostridium perfringens Isolate Causing Foodborne Illness.

UNLABELLED: Clostridium perfringens type A is a common source of foodborne illness (FBI) in humans. Vegetative cells sporulate in the small intestinal tract and produce the major pathogenic factor C. perfringens enterotoxin. Although sporulation plays a critical role in the pathogenesis of FBI, the mechanisms inducing sporulation remain unclear. Bile salts were shown previously to induce sporulation, and we confirmed deoxycholate (DCA)-induced sporulation in C. perfringens strain NCTC8239 cocultured with human intestinal epithelial Caco-2 cells. In the present study, we performed transcriptome analyses of strain NCTC8239 in order to elucidate the mechanism underlying DCA-induced sporulation. Of the 2,761 genes analyzed, 333 were up- or downregulated during DCA-induced sporulation and included genes for cell division, nutrient metabolism, signal transduction, and defense mechanisms. In contrast, the virulence-associated transcriptional regulators (the VirR/VirS system, the agr system, codY, and abrB) were not activated by DCA. DCA markedly increased the expression of signaling molecules controlled by Spo0A, the master regulator of the sporulation process, whereas the expression of spo0A itself was not altered in the presence or absence of DCA. The phosphorylation of Spo0A was enhanced in the presence of DCA. Collectively, these results demonstrated that DCA induced sporulation, at least partially, by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes in strain NCTC8239 while altering the expression of various genes. IMPORTANCE: Disease caused by Clostridium perfringens type A consistently ranks among the most common bacterial foodborne illnesses in humans in developed countries. The sporulation of C. perfringens in the small intestinal tract is a key event for its pathogenesis, but the factors and underlying mechanisms by which C. perfringens sporulates in vivo currently remain unclear. Bile salts, major components of bile, which is secreted from the liver for the emulsification of lipids, were shown to induce sporulation. However, the mechanisms underlying bile salt-induced sporulation have not yet been clarified. In the present study, we demonstrate that deoxycholate (one of the bile salts) induces sporulation by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes using a transcriptome analysis. Thus, this study enhances our understanding of the mechanisms underlying sporulation, particularly that of bile salt-induced sporulation, in C. perfringens.

Nitrate salts suppress sporulation and production of enterotoxin in Clostridium perfringens strain NCTC8239.

Clostridium perfringens type A is a common source of food-borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food-borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co-cultured with Caco-2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co-cultured in Roswell Park Memorial Institute-1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO3 ]2 ) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO3 )2 -deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO3 )2 down-regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down-regulating Spo0A-regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation.

Voltammetric detection and profiling of isoprenoid quinones hydrophobically transferred from bacterial cells.

We have developed a novel bacterial detection technique by desiccating a bacterial suspension deposited on an electrode. It was also found that the use of an indium-tin-oxide (ITO) electrode dramatically improved the resolution of the voltammogram, allowing us to observe two pairs of redox peaks, each assigned to the adsorption of isoprenoid ubiquinone (UQn) and menaquinone (MKn), which were present in the bacterial cell envelopes, giving midpeak potentials of -0.015 and -0.25 V versus Ag|AgCl|saturated KCl| at pH 7.0, respectively. Most of the microorganisms classified in both the Gram-negative and -positive bacteria gave well-defined redox peaks, demonstrating that this procedure made the detection of the quinones possible without solvent extraction. It has been demonstrated that the present technique can be used not only for the detection of bacteria, but also for profiling of the isoprenoid quinones, which play important roles in electron and proton transfer in microorganisms. In this respect, the present technique provides a much more straightforward way than the solvent extraction in that one sample can be prepared in 1 min by heat evaporation of a suspension containing the targeted bacteria, which has been applied on the ITO electrode.

In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity.

IFN-γ elicits macrophage autophagy via the p38 MAPK signaling pathway.

Autophagy is a major innate immune defense pathway in both plants and animals. In mammals, this cascade can be elicited by cytokines (IFN-γ) or pattern recognition receptors (TLRs and nucleotide-binding oligomerization domain-like receptors). Many signaling components in TLR- and nucleotide-binding oligomerization domain-like receptor-induced autophagy are now known; however, those involved in activating autophagy via IFN-γ remain to be elucidated. In this study, we engineered macrophages encoding a tandem fluorescently tagged LC3b (tfLC3) autophagosome reporter along with stably integrated short hairpin RNAs to demonstrate IFN-γ-induced autophagy required JAK 1/2, PI3K, and p38 MAPK but not STAT1. Moreover, the autophagy-related guanosine triphosphatase Irgm1 proved dispensable in both stable tfLC3-expressing RAW 264.7 and tfLC3-transduced Irgm1(-/-) primary macrophages, revealing a novel p38 MAPK-dependent, STAT1-independent autophagy pathway that bypasses Irgm1. These unexpected findings have implications for understanding how IFN-γ-induced autophagy is mobilized within macrophages for inflammation and host defense.

Prunin Laurate Derived from Natural Substances Shows Antibacterial Activity against the Periodontal Pathogen Porphyromonas gingivalis.

Periodontal disease is an inflammatory disease caused by infection with periodontopathogenic bacteria. Oral care is essential to prevent and control periodontal disease, which affects oral and systemic health. However, many oral hygiene products currently on the market were developed as disinfectants, and their intense irritation makes their use difficult for young children and older people. This study investigated the antibacterial effects of prunin laurate (Pru-C12) and its analogs on periodontopathogenic bacteria, Porphyromonas gingivalis (P. gingivalis). Pru-C12 and its analogs inhibited in vitro bacterial growth at more than 10 µM and biofilm formation at 50 µM. Among its analogs, only Pru-C12 showed no cytotoxicity at 100 µM. Three of the most potent inhibitors also inhibited the formation of biofilms. Furthermore, Pru-C12 inhibited alveolar bone resorption in a mouse experimental periodontitis model by P. gingivalis infection. These findings may be helpful in the development of oral hygiene products for the prevention and control of periodontal disease and related disorders.

Isolation and characterization of Staphylococcus argenteus strains from retail foods and slaughterhouses in Japan.

Staphylococcus argenteus has been recently established as a novel species of Staphylococcus aureus complex. It is known to cause various human diseases, such as skin and soft-tissue infections, sepsis, and staphylococcal food poisoning, although the source of infection has not been clearly described. In food poisoning cases, the source of bacterial contamination in food is unknown. This study examined the prevalence of S. argenteus among retail fresh food and poultry slaughterhouses in Japan. Among 642 food samples examined, successful isolation of S. argenteus was achieved in 21 of 151 (13.9%) chicken samples. No isolations from pork, beef, fish, or vegetables in retail markets were confirmed. Multiple-locus sequence typing revealed that the 21 isolates were classified into four sequence types (ST) that were divided into 14 subtypes using spa-typing. All food isolates were susceptible to methicillin and did not show positivity for the Panton-Valentine leukocidin gene. When bacteria were isolated from two poultry slaughterhouses in the same region, 14 S. argenteus strains were successfully isolated from only one slaughterhouse. Thirteen of 14 strains were isolated from a poultry carcass and slaughterhouse environments during a certain sampling period and were all classified as ST5961 with identical spa-type. Also, the number of single nucleotide variants (SNVs) detected on the core genomes of the same 13 strains were between 0 and 17, suggesting a long-term inhabitation of an S. argenteus strain inside the facility. Furthermore, one isolate from chicken meat was also genetically linked with the same lineage of slaughterhouse isolates, with ≤15 SNVs being detected. Additionally, one slaughterhouse isolate from chiller water and three chicken isolates were classified into the same cluster by phylogenetic analysis, although the number of pairwise SNVs ranged from 62 to 128. To the best of the authors' knowledge, this is the first study that demonstrated S. argenteus in a food processing facility and the possible bacterial contamination on food during food processing.

Clostridium perfringens enterotoxin interacts with claudins via electrostatic attraction.

Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr(306), Tyr(310), Tyr(312), and Leu(315), which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction.

Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G(q)- and G(12/13)-dependent pathways through the deamidation of a glutamine residue in the alpha-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575-1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590-670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT DeltaC1(4H)) neither localized to the plasma membrane nor stimulated the G(q/12/13)-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT DeltaC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.

Whole-genome analyses of extended-spectrum or AmpC ß-lactamase-producing Escherichia coli isolates from companion dogs in Japan.

The emergence and global spread of extended-spectrum or AmpC ß-lactamase (ESBL/AmpC)-producing Enterobacteriaceae in companion animals have led to the hypothesis that companion animals might be reservoirs for cross-species transmission because of their close contact with humans. However, current knowledge in this field is limited; therefore, the role of companion animals in cross-species transmission remains to be elucidated. Herein, we studied ESBL/AmpC-producing Enterobacteriaceae, Escherichia coli in particular, isolated from extraintestinal sites and feces of companion dogs. Whole-genome sequencing analysis revealed that (i) extraintestinal E. coli isolates were most closely related to those isolated from feces from the same dog, (ii) chromosomal sequences in the ST131/C1-M27 clade isolated from companion dogs were highly similar to those in the ST131/C1-M27 clade of human origin, (iii) certain plasmids, such as IncFII/pMLST F1:A2:B20/blaCTX-M-27, IncI1/pMLST16/blaCTX-M-15, or IncI1/blaCMY-2 from dog-derived E. coli isolates, shared high homology with those from several human-derived Enterobacteriaceae, (iv) chromosomal blaCTX-M-14 was identified in the ST38 isolate from a companion dog, and (v) eight out of 14 tested ESBL/AmpC-producing E. coli isolates (i.e., ST131, ST68, ST405, and ST998) belonged to the human extraintestinal pathogenic E. coli (ExPEC) group. All of the bla-coding plasmids that were sequenced genome-wide were capable of horizontal transfer. These results suggest that companion dogs can spread ESBL/AmpC-producing ExPEC via their feces. Furthermore, at least some ESBL/AmpC-producing ExPECs and bla-coding plasmids can be transmitted between humans and companion dogs. Thus, companion dogs can act as an important reservoir for ESBL/AmpC-producing E. coli in the community.

Association of Bordetella dermonecrotic toxin with the extracellular matrix.

BACKGROUND: Bordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown. RESULTS: Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active. CONCLUSIONS: Components in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

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.

The morphological changes in cultured cells caused by Bordetella pertussis adenylate cyclase toxin.

Bordetella pertussis is the causative agent for human whooping cough. It was found that Bordetella pertussis infection caused a change in shape from flat to round in L2 cells, which are derived from rat type 2 alveolar cells. This phenomenon was reproduced using the culture supernatant of B. pertussis, and bacterium-free adenylate cyclase toxin (CyaA) was identified as the factor responsible. A purified preparation of wild-type CyaA but not an enzyme-dead mutant caused the cell rounding. It was examined whether CyaA causes similar morphological changes in various cultured cell lines. L2, EBL, HEK293T, MC3T3-E1, NIH 3T3, and Vero cells were rounded by the toxin whereas Caco-2, Eph4, and MDCK cells were not, although all these cells showed a significant elevation of the intracellular cAMP level in response to CyaA treatment, which indicates that there is no quantitative correlation between the rounding phenotype and the intracellular cAMP level. CyaA has been believed to target various immunocompetent cells and support the establishment of the bacterial infection by subverting the host immune responses. The possibility that CyaA may also affect tissue cells such as respiratory epithelial cells and may be involved in the pathogenesis of the bacterial infection is also indicated.

A colorimetric assay for studying effector secretion through the bacterial type III secretion system.

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.

Evaluation of multinomial logistic regression models for predicting causative pathogens of food poisoning cases.

In cases of food poisoning, it is important for food sanitation inspectors to determine the causative pathogen as early as possible and take necessary measures to minimize outbreaks. Interviews are usually conducted to obtain epidemiological information to aid in the rapid determination of the cause. However, the current method of determining the causative pathogen has the disadvantage of being reliant upon the experience and knowledge of food sanitation inspectors. Here, we analyzed 529 infectious food poisoning incidents reported in five municipalities in the Kinki region to develop a tool for evaluation using a multinomial logistic regression model, which can predict the causative pathogen based on the patients' epidemiological information. This tool predicts the most probable cause of the incident by generating a list of pathogens with the highest probability. As a result of leave-one-out cross validation, the agreement ratio with the actual pathogen was 86.4%, and this ratio increased to 97.5% when the agreement was judged by including the true pathogen within the top three pathogens with the highest probability. In cases where the difference of probability between the first and second candidate pathogen was ≥50%, the agreement ratio increased to 94.2%. Using this tool, it is possible to accurately estimate the causative pathogen at an early stage based on patient information, and this will further help narrow the target of investigations to identify causative agent, thereby leading to a prompt identification, which can prevent the spread of food poisoning.

Adherence of Clostridium perfringens spores to human intestinal epithelial Caco-2 cells.

Clostridium perfringens is a gram-positive, spore-forming bacillus, and is a causative agent of foodborne infection, antibiotic-associated diarrhoea and sporadic diarrhoea in humans. In cases of antibiotic-associated and sporadic diarrhoea, C. perfringens colonises the intestine, proliferates and causes disease. However, bacterial colonisation of the intestine is not considered necessary in the pathogenesis of foodborne illness, because such pathogenesis can be explained by anchorage-independent production of diarrhoeic toxin by the bacterium in the intestine. In this study, we used an in vitro adherence assay to examine the adherence of C. perfringens spores to human intestinal Caco-2 cells. Adherence of spores from isolates of foodborne illness and nosocomial infection was observed within 15 min, and plateaued 60 min after inoculation. Electron microscopy revealed a tight association of spores with the surface of Caco-2 cells. The adherence of vegetative cells could not be confirmed by the same method, however. These results suggest that C. perfringens spores may adhere to intestinal epithelial cells in vivo, although its biological significance remains to be determined.

Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells.

Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.