Studies on the enteropathogenic mechanism of non-O1 Vibrio cholerae. I. Enteropathogenicity of clinical and environmental isolates

Enteropathogenicity and plasmid DNA of clinical and environmental isolates of non-O1 V. cholerae were examined. Results were as follows: 1). The frequencies of enteropathogenic strains judged by the results from both ligated rabbit ileal loop (RIL) and suckling mouse tests were 36/38 (95%) for isolates from overseas travellers, 15/15 (100%) for isolates from food poisoning, 33/44 (75%) for isolates from fish and sea water, and 1/10 (10%) for isolates from river water. 2). Plasmid DNA was detected in eight of the 40 isolates examined, but the presence of plasmid did not correlate with enteropathogenicity. These results indicate that approximately three fourths of the strains isolated from fish and sea water are enteropathogenic, and that the genes controlling the enteropathogenicity of this organism probably exist in chromosomal DNA.     

Comparative genomics of clinical and environmental Vibrio mimicus

Whether Vibrio mimicus is a variant of Vibrio cholerae or a separate species has been the subject of taxonomic controversy. A genomic analysis was undertaken to resolve the issue. The genomes of V. mimicus MB451, a clinical isolate, and VM223, an environmental isolate, comprise ca. 4,347,971 and 4,313,453 bp and encode 3,802 and 3,290 ORFs, respectively. As in other vibrios, chromosome I (C-I) predominantly contains genes necessary for growth and viability, whereas chromosome II (C-II) bears genes for adaptation to environmental change. C-I harbors many virulence genes, including some not previously reported in V. mimicus, such as mannose-sensitive hemagglutinin (MSHA), and enterotoxigenic hemolysin (HlyA); C-II encodes a variant of Vibrio pathogenicity island 2 (VPI-2), and Vibrio seventh pandemic island II (VSP-II) cluster of genes. Extensive genomic rearrangement in C-II indicates it is a hot spot for evolution and genesis of speciation for the genus Vibrio. The number of virulence regions discovered in this study (VSP-II, MSHA, HlyA, type IV pilin, PilE, and integron integrase, IntI4) with no notable difference in potential virulence genes between clinical and environmental strains suggests these genes also may play a role in the environment and that pathogenic strains may arise in the environment. Significant genome synteny with prototypic pre-seventh pandemic strains of V. cholerae was observed, and the results of phylogenetic analysis support the hypothesis that, in the course of evolution, V. mimicus and V. cholerae diverged from a common ancestor with a prototypic sixth pandemic genomic backbone.     

Surveys on the contamination of marine fish with non-O1 Vibrio cholerae and Vibrio mimicus and food poisoning cases by these organisms

The present paper describes the relationship between the contamination with non-O1 Vibrio cholerae and Vibrio mimicus of marine fish, with special reference to the seasonal variation and the concentration of contamination, and the actual cases of domestic food poisoning by these organisms. A 10 year survey revealed that non-O1 Vibrio cholerae (non-O1 V. cholerae) strains were frequently isolated from fish during the summer season with some variations from one year to another, and isolates from fish showed similar biological properties to those of isolates from diarrhea cases of over-sea travellers. Experimentally enteropathogenic strains were included among these isolates. Vibrio mimicus (V. mimicus) strains were also isolated from fish, the frequency being not so high as in the case of non-O1 V. cholerae Strains of serovar O-41 which was most predominant among strains from diarrhea cases were also detected among the isolates from fish. The viable cell counts, however, were very small with regard to both non-O1 V. cholerae and V. mimicus From these observations, factors causing food poisoning by non-O1 V. cholerae or V. mimicus seemed to be essentially similar to those by Vibrio parahaemolyticus (V. parahaemolyticus); that is, the food poisoning by non-O1 V. cholerae or V. mimicus is apt to occur in the summer season and is caused by the consumption of raw fish, although the frequency might be significantly low in comparison to that of V. parahaemolyticus. The actual cases of the domestic food poisoning by non-O1 V. cholerae or V. mimicus were retrospectively surveyed by the literature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the enteropathogenic mechanism of non-O1 Vibrio cholerae. II. Lethality, adhesion, colonization and cytopathogenicity of enteropathogenic strains

Lethality, adhesion, colonization, hemagglutinable activity, invasiveness and cytopathogenicity of non-O1 V. cholerae were compared between enteropathogenic and non-enteropathogenic strains. The following results were obtained. 1) Minimum lethal doses (MLD) of enteropathogenic strains were significantly lower than those of non-enteropathogenic strains. 2) There were no differences in adhesive and hemagglutinating activities between enteropathogenic and non-enteropathogenic strains. 3) A greater majority of enteropathogenic strains showed cytopathogenic effect on HEp 2 cells, but non-enteropathogenic strains did not. 4) Regardless of enteropathogenicity of viable cells, none of the 13 strains examined were found to be invasive to HEp 2 cells. These results suggest that adhesion and colonization do not draw a clear distinction between enteropathogenic and non-enteropathogenic strains, and that both lethal and cytopathogenic activities of these organisms are correlated with enteropathogenicity.     

Enteropathogenicity and antimicrobial susceptibility of new Escherichia spp

To determine the mechanism of enteropathogenicity of the newly described Escherichia species, a total of 50 clinical isolates of Escherichia spp. from diarrhoeal stools were studied. Twelve isolates (24%) were found to be E. vulneris, 6 (12%) E. fergusonii, 2 (4%) E. hermannii, and the rest 30 (60%) were E. coli. Most isolates of the new species were resistant to ampicillin, tetracycline, and co-trimoxazole, but were susceptible to cephalosporins and aminoglycosides. The representative strains of all the new species produced significant fluid accumulation in the rat ileal loops both by live cells and their culture filtrates. E. vulneris, isolated from stools, showed maximum fluid accumulation. Thus, it can be inferred that these species are diarrhoeagenic, but their roles on extra-intestinal infections remain to be determined.     

Studies on the enteropathogenic mechanism of non-O1 Vibrio cholerae. IV. Role of hemolysin

The role of hemolysin in the enteropathogenic mechanism of non-O1 V. cholerae was experimentally investigated, in vitro and in vivo. Results are summarized as follows. 1). A greater majority of enteropathogenic strains produced hemolysin in Eagle MEM medium supplemented with 10% calf serum and in the rabbit ileal loop, while most non-enteropathogenic strains did not under the same conditions. 2). Non-enteropathogenic mutants derived from enteropathogenic parent strains produced much less hemolysin than that of parent strains. 3). A significant inhibition of the fluid accumulation in the ligated rabbit ileal loop test with viable cells was noted in rabbit immunized with purified hemolysin. These results indicate that hemolysin is the most important toxin in the enteropathogenic mechanism of non-O1 V. cholerae.     

O139霍乱弧菌肠毒素核苷酸序列分析

目的 探讨Ｏ１３９霍乱弧菌肠毒素（ＣＴＸ）核苷酸与Ｏ１群霍乱ＣＴＸ毒素核苷酸序列异同。方法 用聚合酶链反应、ＤＮＡ序列分析测定２株Ｏ１３９群、２株Ｏ１群古典型、２株Ｏ１群Ｅ１Ｔｏｒ型霍乱弧菌ＣＴＸＡ２－Ｂ亚单位核苷酸。结果 ２株Ｏ１３９群霍乱弧菌均含有ＣＴＸＡ２－Ｂ亚单位基因,Ｏ１３９群与Ｏ１群ＣＴＸＡ２－Ｂ核苷酸同源性为９７．１％～９８．９％结论 Ｏ１３９群与Ｏ１群霍乱弧菌ＣＴＸ核苷酸序列一致。     

Studies on the enteropathogenic mechanism of non-O1 Vibrio cholerae. III. Production of enteroreactive toxins

Cholera toxin gene and production of enteroreactive toxins were examined in 134 strains of non-O1 V. cholerae. Results obtained were summarized as follows. Frequencies of cholera-toxin-gene-positive strains were 2/58 (3.4%) from human sources and 2/76 (2.6%) from fish and environment. While, frequencies of production of hemolysin, fluid accumulating factor (FAF) related with protease, fluid accumulating factor in the suckling mouse, NAG-rTDH, NAG-ST and Vero toxin were 100, 72, 31, 2, 0 and 0%, respectively, for 58 strains from human sources, and 100, 57, 24, 0, 1.3 and 0%, respectively, for 76 strains from fish and environment. Among the 31 strains used for the injection of viable cells to the ligated rabbit ileal loop, detection frequencies of these enteroreactive toxins in the accumulated fluids were 100% for hemolysin, 3.2% for both FAF and NAG-rTDH and 0% for cholera toxin, Vero toxin or NAG-ST. Hemolysin and the fluid accumulating factor in the suckling mouse seemed to be identical in most strains. These results suggest that cholera toxin, NAG-ST, NAG-rTDH and Vero toxin may not be very important in the enteropathogenic mechanism of a great majority of non-O1 V. cholerae strains, whereas hemolysin may play an important role in the enteropathogenicity.     

Evidence for enterotoxin production by a classic enteropathogenic serotype of Escherichia coli.

Isolates of classic enteropathogenic Escherichia coli O:128 that had been implicated in an outbreak of diarrhea in a hospital nursery were found to produce heat-stable enterotoxin after storage for six years. This finding indicated that enteropathogenicity and the ability to produce enterotoxin may coincide in E. coli, and further study of enteropathogenic strains that produce enterotoxin may help in elucidation of the relationship between enteropathogenic and enterotoxigenic E. coli.     

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae. Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin in vitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans. Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum. We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.

Vibrio cholerae is known as the cause of cholera, a disease that can lead to fatal dehydration (1). The disease is caused by a few serogroups, and the main factor behind the symptoms is the cholera toxin (CT) encoded by genes located on a prophage mobile genetic element (CTX-φ) that induce severe disruption of intestinal cell function, leading to watery, secretory diarrhea (2). Most serogroups do not cause cholera, as they do not possess the genes for CT, but they cause other diseases [e.g., skin, wound, and gastrointestinal infections as well as bacteremia (3)]. The natural reservoirs of V. cholerae are aquatic sources such as rivers, brackish waters, and estuaries and are often associated with copepods, aquatic plants, and shellfish (4). The factors and mechanisms allowing V. cholerae and other Vibrionaceae to survive and thrive in harsh natural environments are of major interest to researchers (5).V. cholerae is motile by virtue of a single polar flagellum. The flagellum export machinery and the virulence-associated type-III secretion system (fT3SS and vT3SS, respectively) are suggested to share a common ancestor (6), explaining their similar structure and molecular organization. The vT3SS allows the delivery of effector proteins through a hollow channel directly to the eukaryotic host cell (7), and flagellar proteins are delivered via the fT3SS channel during flagellum assembly. In the bacterial cytoplasm, effectors secreted by the vT3SS are stabilized by chaperones to prevent aggregation. These chaperones are often encoded by genes adjacent to those encoding the effectors (8). Flagellar proteins are similarly protected by chaperones before they are transported to the growing distal end of the flagellum (9).We use Caenorhabditis elegans as a predatory organism model for identifying and assessing V. cholerae factors, other than CT, that may contribute to bacterial survival and persistence (10). With this model, we discovered a cytotoxin, MakA (motility-associated killing factor A), which we demonstrated to be an essential factor for the cytotoxic activity of V. cholerae in both C. elegans and Danio rerio (zebrafish) (11). We also demonstrated that secretion of MakA occurs via the flagellum in a manner that is undocumented in V. cholerae. Our crystal structure of MakA revealed similarities to ClyA (11), the pore-forming toxin first identified in nonpathogenic Escherichia coli (12, 13) and, subsequently, also in Salmonella enterica (14). ClyA from E. coli is expressed from a monocistronic operon and oligomerizes into a dodecameric pore upon release via membrane vesicles (13, 15, 16). MakA is also structurally related to two proteins from Bacillus cereus, the hemolysin BL binding component B (HBL-B) and the NheA component of the Nhe nonhemolytic enterotoxin. Both of these are considered components of tripartite toxins (17). Recently, a tripartite toxin, AhlABC, was identified and structurally characterized as a pore-forming toxin in Aeromonas hydrophila, and the structure of soluble AhlB shares the general structure described for MakA (18). A similar toxin complex of three proteins, SmhABC from Serratia marcescens, was also reported (19). However, if and how the Ahl and Smh proteins are released during normal growth, or if there is a dedicated secretion system, remain unclear.Here, we identify the proteins from the five V. cholerae genes, vca0880 through vca0884, that are coexpressed from the operon makDCBAE and analyze the crystal structures of MakA, MakB, and MakE. Our in vitro studies revealed that an equimolar combination of the MakA/B/E proteins acted as a tripartite cytotoxin causing lysis of red blood cells and cytotoxicity to epithelial cells. Examination of a large number of bacterial genomes revealed that the mak operon is present in many V. cholerae and other Vibrionaceae strains. These include Vibrio (Listonella) anguillarum, an inhabitant of estuarine and marine coastal ecosystems worldwide and the etiological agent of vibriosis in warm- and cold-water fish (20). The identification and structural characterization of the Mak proteins in V. cholerae presented here reveals a hitherto-unrecognized potential of many pathogenic Vibrionaceae strains to produce the tripartite Mak cytolytic toxin.     

Risk factors for Clostridium difficile colonisation and toxin production

OBJECTIVES: to examine risk factors for patients becoming Clostridium difficile culture and toxin positive. DESIGN: prospective cohort study. SETTING: two medicine for the elderly wards. PARTICIPANTS: patients admitted to the wards over a 17-month period. MEASUREMENT: presence of Clostridium difficile on culture of stool specimens and toxins A and/or B. Patient's age, sex, source of admission, antibiotic, laxative, antacid and steroid use, presence/absence of colonic disease, neoplasia, leukaemia and nasogastric or percutaneous endoscopic gastrostomy tube feeding. RESULTS: 390 of 865 patients admitted provided a total of 1003 faecal specimens. Age (OR 1.04, 95% CI 1.001-1.07 per year), admission from another hospital (OR 2.13, 95% CI 1.29-3.50), non-cephalosporin antibiotics (OR 2.08, 95% CI 1.25-3.46) and cephalosporin use (OR 8.45, 95% CI 2.99-23.9) increased risk of becoming Clostridium difficile culture positive. Becoming toxin-positive was associated with antibiotic use only (OR 3.02, 95% CI 1.15-7.92), specifically amoxycillin (OR 8.72, 95% CI 1.66-45.9) and cephalosporins other than ceftriaxone (OR 7.28, 95% CI 1.34-39.6). CONCLUSION: different risk factors are important for the two stages leading to Clostridium difficile diarrhoea. Age, source of admission and third generation cephalosporins increase risk of becoming culture positive, whilst only antibiotic use is associated with the step of becoming toxin-positive. Understanding these differential risks may aid infection control strategies.     

Vibriocidal antibody and antibodies to Vibrio cholerae lipopolysaccharide, cell-bound haemagglutinin and toxin in Thai population

Vibriocidal antibody and antibodies to Vibrio cholerae lipopolysaccharide (anti-LPS), cell-bound haemagglutinin (anti-CHA) and toxin (anti-CT) were determined in Thai individuals of various age groups who lived in areas with high (H) and low (L) cholera endemicity. The enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of class specific anti-LPS, anti-CHA and anti-CT. It was found that Thai individuals acquired the vibriocidal antibody early in life. Fifty percent of individuals aged 5 to 15 years old had detectable titre while more than 80% of adults had titres ranged from 1:5 to 1:125 or higher. Thai adults who lived in area with high cholera endemicity had significantly higher vibriocidal antibody levels than their counterparts who lived in area with low cholera endemicity. Lipopolysaccharide was not the only antigen responsible for stimulating the vibriocidal antibody production. Adult levels of all classes of anti-CHA from L were higher than those of H while the anti-LPS in the forms of total immunoglobulins, IgG and IgA were similar but IgM of L was higher than that of H. The levels of all classes of anti-CT from H seemed to increase with age except at the school age (5 years to 15 years old) when there were marked decreases of all antibody classes. Total immunoglobulin and IgM anti-CT at adult age of H and L were not different, although IgG anti-CT of L was higher than that of H and IgA anti-CT of H was higher than that of L.     

影响镰刀菌生长与产毒的基本因素的研究

通过对镰刀菌生长、产毒条件的观察，发现镰刀菌生长、产毒与环境条件关系密切：适宜温度（２０℃～３０℃）、湿度（４０％）条件下，真菌生长旺盛，孢子数量多；碱性（ｐＨ＞７．０）条件则明显抑制其生长，孢子数量也减少；无机元素对生长无影响；低温尤其是变温条件下，镰刀菌易产生Ｔ－２毒素；碱性条件可明显抑制真菌产毒；温度、湿度和ｐＨ之间存在明显的交互作用