Staphylococcal Enterotoxin C Subtypes Are Differentially Associated with Human Infections and Immunobiological Activities.

Staphylococcus aureus causes significant infections, responsible for toxic shock syndrome (TSS), hemorrhagic pneumonia, and many other infections. S. aureus secretes virulence factors, which include superantigens such as staphylococcal enterotoxins (SEs). We examined differences in immunobiological activities and disease associations among the four human SEC subtypes. We sequenced the sec gene from 35 human isolates to determine SEC subtypes. Upon finding differences in disease association, we used a [3H]thymidine uptake assay to examine SEC-induced superantigenicity. We also employed a rabbit model of SEC-induced TSS. SEC-2 and SEC-3 were associated with menstrual TSS and vaginal isolates from healthy women, whereas SEC-4 was produced by USA400 isolates causing purpura fulminans and hemorrhagic pneumonia. SEC subtypes differed in potency in a TSS rabbit model and in superantigenicity. There was no difference in superantigenicity when tested on human peripheral blood mononuclear cells. Despite differences, all SECs reacted with polyclonal antibodies raised against the other SEC subtypes. The associations of SEC subtypes with different infections suggest that S. aureus produces virulence factors according to host niches.IMPORTANCE Staphylococcal enterotoxin C has four subtypes that cause human diseases, designated SEC-1 to -4. This study shows that SEC-2 and SEC-3 are the most toxic subtypes in a rabbit model and are associated with human vaginal infections or colonization in association with another superantigen, toxic shock syndrome toxin 1. SEC-4 is associated with purpura fulminans and hemorrhagic pneumonia. SEC-1 is uncommon. The data suggest that there is some selective pressure for the SEC subtypes to be associated with certain human niches.

Rapid Serotyping of Salmonella Isolates Based on Single Nucleotide Polymorphism-Like Sequence Profiles of a Salmonella-Specific Gene.

Although serotyping is the most important method of identification of taxonomy in Salmonella, conventional serotype determination with a complete set of antisera is time consuming and laborious. Recently, rapid serotyping procedures with polymerase chain reaction (PCR) have been developed. In this study, we established a novel PCR-based rapid serotyping method that employs a unique target gene. Alignment study of Salmonella-specific gene (Salmonella enterotoxin [stn]) revealed a correlation between the stn gene sequence and the serotype of the organism. In 750 bp of stn gene, 55 nucleotides indicated single nucleotide polymorphism (SNP)-like polymorphism, and the correlation between the SNP-like polymorphism and the serotype of the organism suggests that SNP-like sequences in stn gene can serve as an index for serotyping. To develop a rapid serotyping method based on the SNP-like polymorphism, we selected serotype-associated 12 SNP-like sites in the stn gene and established a method based on high-resolution melting (HRM) and PCR, which identifies nucleotides at SNP-like sites within 1.5 h. This newly established rapid serotyping procedure (stn-HRM) could identify nine serotypes, including the frequently isolated serovar Enteritidis. These nine serotypes cover 64.3% of cases of Salmonella, as reported by the World Health Organization/Global Foodborne Infection Network (WHO/GFN) Country Databank from 2001 to 2010. In this study, we employed a unique target gene, stn, which is completely independent of the genes that were targeted in previously reported rapid serotyping procedures. Therefore, the results obtained by our newly developed stn-HRM procedure are independent of the results obtained by other procedures. Besides, stn-HRM can ensure accurate identification of the bacterial species as stn is a Salmonella-specific gene. It is expected that the combination of newly constructed stn-HRM and previously reported procedures could further improve the credibility of Salmonella isolate serotyping.

Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin.

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

Ant-infecting Ophiocordyceps genomes reveal a high diversity of potential behavioral manipulation genes and a possible major role for enterotoxins.

Much can be gained from revealing the mechanisms fungal entomopathogens employ. Especially intriguing are fungal parasites that manipulate insect behavior because, presumably, they secrete a wealth of bioactive compounds. To gain more insight into their strategies, we compared the genomes of five ant-infecting Ophiocordyceps species from three species complexes. These species were collected across three continents, from five different ant species in which they induce different levels of manipulation. A considerable number of (small) secreted and pathogenicity-related proteins were only found in these ant-manipulating Ophiocordyceps species, and not in other ascomycetes. However, few of those proteins were conserved among them, suggesting that several different methods of behavior modification have evolved. This is further supported by a relatively fast evolution of previously reported candidate manipulation genes associated with biting behavior. Moreover, secondary metabolite clusters, activated during biting behavior, appeared conserved within a species complex, but not beyond. The independent co-evolution between these manipulating parasites and their respective hosts might thus have led to rather diverse strategies to alter behavior. Our data indicate that specialized, secreted enterotoxins may play a major role in one of these strategies.

Multi-Typing of Enterobacteria Harboring LT and ST Enterotoxin Genes Isolated from Mexican Children.

Enterotoxigenic Escherichia coli is the most common cause of diarrhea in children younger than 5 years in the developing world. We used 16S rRNA gene sequencing, the Biolog® system, and an Amplified Ribosomal DNA Restriction Analysis (ARDRA) to identify 69 enterobacteria isolated from the feces of healthy children up to 12 years old and 54 enterobacteria isolated from stool samples obtained from children up to 5 years old with diarrhea from Morelia, Michoacán, Mexico. In the diarrheic group, 18 isolates belonged to the enterotoxigenic pathotype, 1 isolate had both LT (heat labile toxin) gene and ST (heat stable toxin) gene, and 17 had the ST gene. The identity of most of the strains harboring the ST gene was E. coli, and 3 of the strains were identified as Morganella morganii. The ST toxin gene of one of the strains identified as M. morganii showed 100% identity with an ST toxin gene of E. coli. The ARDRA was a very useful tool to differentiate between E. coli and M. morganii. The phenotypic and genetic analyses of the isolates using the Biolog® system and Random Amplified Polymorphic DNA, respectively, showed physiological variation among the studied strains and genetic differences between subgroups.

Animal Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors: adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17, and F18 fimbriae. Once established in the animal small intestine, ETEC produce enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes: heat-labile toxins that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This review describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics, and the identification of potential new targets by genomics are presented in the context of animal ETEC.

Differentiation of entC1 from entC2/entC3 with a single primer pair using simple and rapid SYBR Green-based RT-PCR melt curve analysis.

In spite of their involvement in foodborne illness, the epidemiological relevance of staphylococcal enterotoxin C (SEC) subtypes is poorly documented may be due to high sequence similarity. Among subtypes, SEC1, SEC2, and SEC3 exhibit more than 97 % homology because of which specific detection tools are seldom available to identify and differentiate them. In this study, a SYBR Green-based RT-PCR followed by melt curve analysis was developed for differentiation of entC1 from entC2/entC3 using a single primer pair. Nucleotide sequences of all three subtypes were analyzed using Clustal Omega program and the region with significant sequence variation/heterogeneity (where utmost SNPs were closely located and accessible for RT-PCR) was selected for amplification by designing a single primer pair that could amplify all three subtypes. In spite of same amplicon size, entC1 showed distinct melt peak at 76 °C. However, due to high similarity between entC2 and entC3, the developed format was deficient to discriminate between them and both showed melt peak at 82 °C. Reliability of developed RT-PCR was evaluated using various naturally contaminated samples and 91 food and clinical Staphylococcus aureus isolates where satisfactory results were obtained in comparison with commercial immunoassay kit and conventional PCRs using validated primers. To the best of our knowledge, this is the first method being reported to differentiate entC1 from entC2/entC3 using single primer pair which is unachievable by conventional PCR due to same amplicon size. As benefits, the method is sensitive, rapid, and inexpensive with no requirement of fluorescent probes, multiple primers, and post-PCR procedures. Thus, the assay might find its utility as a detection tool in epidemiological survey of foodborne outbreaks for simultaneous identification and differentiation of entC1 from entC2/entC3.

Generation of scFv phages specific to Staphylococcus enterotoxin C1 by panning on related antigens.

A method for generation of highly specific miniantibodies within the phage particle has been developed, and used to produce antibodies against Staphylococcus enterotoxin type C1. Under successive panning of the non-immune phage miniantibody (scFv) library with enterotoxins SE (types A, B, C1, D, E, G, and I) adsorbed on the plate surface, we generated 11 individual phage clones to Staphylococcus enterotoxin type C1. Five of them interacted specifically only with SEC1 and had no cross-reactions with the other enterotoxins.

LT-IIc, a new member of the type II heat-labile enterotoxin family encoded by an Escherichia coli strain obtained from a nonmammalian host.

Two families of bacterial heat-labile enterotoxins (HLTs) have been described: the type I HLTs are comprised of cholera toxin (CT) of Vibrio cholerae, LT-I of Escherichia coli, and several related HLTs; the type II HLTs are comprised of LT-IIa and LT-IIb. Herein, we report LT-IIc, a new type II HLT encoded from an enterotoxigenic E. coli (ETEC) strain isolated from an avian host. Using a mouse Y1 adrenal cell bioassay, LT-IIc was shown to be less cytotoxic than CT, LT-IIa, or LT-IIb. Cytotoxicity of LT-IIc was partially neutralized by antisera recognizing LT-IIa or LT-IIb but not by anti-CT antiserum. Genes encoding putative A polypeptide and B polypeptides of LT-IIc were arranged in an operon which was flanked by potential prophage sequences. Analysis of the nucleotide and predicted amino acid sequences demonstrated that the A polypeptide of LT-IIc has moderate homology to the A polypeptides of CT and LT-I and high homology to the A polypeptides of LT-IIa and LT-IIb. The B polypeptide of LT-IIc exhibited no significant homology to the B polypeptides of CT and LT-I and only moderate homology to the B polypeptides of LT-IIa and LT-IIb. The binding pattern of LT-IIc for gangliosides was distinctive from that of either LT-IIa or LT-IIb. The data suggest that other types of the type II HLT subfamily are circulating in the environment and that host specificity of type II HLT is likely governed by changes in the B polypeptide which mediate binding to receptors.

Toxinotype V Clostridium difficile in humans and food animals.

Clostridium difficile is a recognized pathogen in neonatal pigs and may contribute to enteritis in calves. Toxinotype V strains have been rare causes of human C. difficile-associated disease (CDAD). We examined toxinotype V in human disease, the genetic relationship of animal and human toxinotype V strains, and in vitro toxin production of these strains. From 2001 through 2006, 8 (1.3%) of 620 patient isolates were identified as toxinotype V; before 2001, 7 (<0.02%) of approximately 6,000 isolates were identified as toxinotype V. Six (46.2%) of 13 case-patients for whom information was available had community-associated CDAD. Molecular characterization showed a high degree of similarity between human and animal toxinotype V isolates; all contained a 39-bp tcdC deletion and most produced binary toxin. Further study is needed to understand the epidemiology of CDAD caused by toxinotype V C. difficile, including the potential of foodborne transmission to humans.

Heterogeneity of Escherichia coli STb enterotoxin isolated from diseased pigs.

To investigate the presence and frequency of estB variant(s), a collection of 100 STb-positive enterotoxigenic Escherichia coli (ETEC) strains isolated from 1980 to 2007 inclusively and randomly selected from diseased pigs in Québec, Canada, was analysed. A wide diversity of virulence gene profiles (virotypes) was detected in the strain collection. The estB gene was amplified by PCR using primers designed from the signal sequence and the C-terminal end, and the amplified fragment was sequenced using the forward primer. The translated DNA sequence revealed a His(12)-->Asn change in 23 of the 100 ETEC isolates tested. The STb-variant strains were observed throughout the sampling period covered in the study. No other STb-variant type was found in this study. All 23 variant strains were also positive for the STa enterotoxin and were resistant to tetracycline, as for strain 2173. The STb variant was associated with Stx2-positive strains (5/6) and STa : STb strains that did not harbour any of the tested porcine fimbrial adhesins (13/17). The remaining variant strains were associated with fimbriae F4 (1/40), F5 (1/6), F6 (1/1) and F18 (2/7; excluding F18 : Stx2 strains).

Genotype analysis of enterotoxin H-positive Staphylococcus aureus strains isolated from food samples in the Czech Republic.

Twenty-eight enterotoxin H-positive Staphylococcus aureus strains isolated from food samples collected in eleven districts of the Czech Republic between 2000 and 2005 were genotypically characterized by pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting and prophage carriage detection. These strains accounted for about 21% of the food-derived, staphylococcal enterotoxin (SE)-positive isolates. One strain, detected in feta cheese, was implicated in a case of enterotoxinosis. Sixteen of the twenty-eight isolates carried the seh gene alone. The remaining twelve strains harbored the seh gene in combination with other enterotoxin genes, most often the seg and sei genes, followed by the sea, seb, sec and sed genes. Comparison of various genomic profiles resulted in the determination of twenty genotypes designated G-1 to G-20. Two new, to date not defined, spa types (t2000 and t2002) were identified in one strain isolated from raw meat and two strains obtained from prepacked pizza. Evidence has been given that the seh-positive S. aureus isolates from foodstuffs did not originate from a single source or a common ancestor.

Genetic diversity of Clostridium perfringens isolated from healthy broiler chickens at a commercial farm.

Clostridium perfringens is an important commensal and bacterial pathogen of many animal species. It has particular significance in poultry, where it may cause necrotic enteritis. Our objective was to characterize the population diversity of C. perfringens colonizing healthy birds, and to observe how diversity changed over time. Isolates were obtained from broiler chicken cecal samples in two barns on a single farm, on days 7, 14, 22, 27, 30 and 34 of a single 42-day rearing cycle. Bacitracin was used as a feed additive in one of the barns and withdrawn from the second barn for the duration of the experiment. Each isolate was typed using pulsed-field gel electrophoresis (PFGE) using SmaI restriction endonuclease. A total of 205 cecal isolates from 49 birds were typed, as well as 93 isolates from the barn environment (bedding, drinking water and feces). Eight major PFGE types and 17 subtypes were found in the 298 total isolates. The results show that an optimal sampling strategy would involve a large number of birds, with only a few isolates sampled per bird. The diversity of C. perfringens in this study appears to be low within a single bird, and increases as the bird matures. There was no significant difference in genetic diversity between the two barns. In addition, isolates from fresh fecal samples appear to represent the cecal C. perfringens population accurately, although this was not proven statistically. Antimicrobial susceptibility testing was performed on selected isolates (n=41) representing a cross-section of PFGE types. Based on minimum inhibitory concentration distributions, 95% of the isolates tested were deemed resistant to bacitracin, with a 16 microg/mL breakpoint. Three new cpb2 (beta2 toxin gene) variants were found in the study.

Incidence and characterization of diarrheal enterotoxins of fecal Bacillus cereus isolates associated with diarrhea.

A total of 490 stool specimens were collected from patients with diarrhea and healthy controls without diarrhea to investigate the incidence of Bacillus cereus and its enterotoxins. B. cereus was found more significant in stools of persons with diarrhea than without diarrhea (9.5% versus 1.8%, P < 0.05), and was also detected more frequent but not significant in individuals aged > or =1 year and in adults than in children aged <1 year (11% and 8% versus 7.8%, P > 0.05). The hemolytic enterotoxin HBL genes of B. cereus isolates (hblA, hblC, hblD) were detected in 58%, 58%, and 68%, respectively, whereas the nonhemolytic enterotoxin NHE genes (nheA, nheB, nheC) were detected more frequent in 71.%, 84%, and 90% of the isolates, respectively. This study suggests that B. cereus isolates harboring 1 or more enterotoxin gene(s) can be a potential cause of diarrhea in Jordanian population.

Evolution of slime production by coagulase-negative staphylococci and enterotoxigenic characteristics of Staphylococcus aureus strains isolated from various human clinical specimens.

The present study was designed to determine the slime production of coagulase-negative staphylococci (CoNS) and the enterotoxigenic properties of Staphylococcus aureus strains, and to evaluate the clinical importance of slime-producing CoNS and enterotoxigenic S. aureus strains isolated from various human clinical specimens. For this purpose, a total of 120 Staphylococcus strains were isolated and identified, and further characterized for their slime production and enterotoxigenicity. Of the clinical isolates, 55 (45.8 %) were found to be S. aureus, and the others (54.2 %) were identified as CoNS. Of the CoNS, 20 (16.7 %) were further identified as Staphylococcus hominis, 18 (15 %) as Staphylococcus epidermidis, six (5 %) as Staphylococcus xylosus, six (5 %) as Staphylococcus warneri, five (4.2 %) as Staphylococcus sciuri, four (3.3 %) as Staphylococcus haemolyticus, and two each (1.7 %) as Staphylococcus simulans, Staphylococcus capitis and Staphylococcus saprophyticus, respectively. Thirty-nine (60 %) of 65 CoNS were found to be slime producers. Slime production was observed in all CoNS, except S. capitis, mostly from blood (38.5 %), tracheal aspiration (20.5 %) and urine (12.8 %) specimens. In addition, of the 55 S. aureus isolates, 46 (83.6 %) were found to be enterotoxigenic, and of these S. aureus strains, 39 (84.7 %) were positive for staphylococcal enterotoxin (SE)A. The results of this study showed that the slime-producing CoNS were mostly found in clinical specimens of blood, tracheal aspirate and urine. SEA was the predominant enterotoxin type detected in S. aureus strains from human clinical specimens.

Binary toxin-producing, large clostridial toxin-negative Clostridium difficile strains are enterotoxic but do not cause disease in hamsters.

Binary toxin CDT or its genes have been identified in some strains of Clostridium difficile that also produce the large clostridial toxins, toxins A and B (A+B+CDT+), including a newly recognized epidemic strain in the United States and Canada. To study the effects of binary toxin alone, we characterized 4 binary toxin CDT-positive only (A-B-CDT+) C. difficile strains. Unlike other clostridial binary toxins, binary toxin CDT required exogenous trypsin for activation. Supernatants from all A-B-CDT+ strains caused marked fluid accumulation in the rabbit ileal loop assay after concentration and trypsinization. In addition, the ileal loop response was neutralized by antisera raised against other binary toxin-producing clostridia. Challenge of clindamycin-treated hamsters with these strains resulted in colonization but not diarrhea or death. Binary toxin CDT may play an adjunctive role to toxins A and B in the pathogenesis of C. difficile-associated disease but by itself may not be sufficient to cause disease.

[Enterotoxin genes occurance among S. aureus strains isolated from inpatients and carriers]. / Wystkpowanie enterotoksyn u szczepów Staphylococcus aureus izolowanych od chorych hospitalizowanych i nosicieli.

We examined 44 inpatients and 66 carriers Staphylococcus aureus strains, isolated in years 2002-2005, for the presence of 18 enterotoxin genes (se/sel) (by PCR), the ability for A-D enterotoxin production (by SET-RPLA) and antibiotic resistance distribution (by disc diffusion method). se/sel genes were detected in 90,9% of all strains, sea (70,5%) and selk and selq (52,3%) - among inpatients strains and egc (65,2%) - among carriers strains were the most frequently se/sel genes found. Positive results of SET-RPLA were consistent with PCR results. There was no correlation observed between antibiotic resistance and se/sel genes distribution among tested S. aureus strains.

Detection of enterotoxic Bacillus cereus producing hemolytic and non hemolytic enterotoxins by PCR test.

Nine strains belonging to Bacillus cereus group has been isolated from food and environmental samples. Their taxonomic position was confirmed by RFLP analysis of 16S rRNA gene digested with TaqI. The detection of DNA sequences encoding the hemolysin BL complex and enterotoxin NHE, was studied in Bacillus sp. isolates. Set of primers was used to amplify fragment of hblD gene by PCR. For the detection of nheB gene a new primer set was developed which allowed to amplify 273 bp fragment from wide number of strains belonging to B. cereus group. The hblD gene was present in 7 out of 9 isolates whereas nheB gene occurred in all of them. Reference strains of B. cereus LOCK 0807, and B. thuringiensis NCAIM 01262 contained both genes. Strains of B. subtilis ATCC 6633 and B. pumilus LOCK 0814 do not contain both genes. Obtained results showed that B. thuringiensis NCAIM 01262 contains both genes and therefore may be harmful for human beings.

Humans as reservoir for enterotoxin gene--carrying Clostridium perfringens type A.

We found a prevalence of 18% for enterotoxin gene-carrying (cpe+) Clostridium perfringens in the feces of healthy food handlers by PCR and isolated the organism from 11 of 23 PCR-positive persons by using hydrophobic grid membrane filter-colony hybridization. Several different cpe genotypes were recovered. The prevalence was 3.7% for plasmidial IS1151-cpe, 2.9% for plasmidial IS1470-like-cpe, 0.7% for chromosomal IS1470-cpe, and 1.5% for unknown cpe genotype. Lateral spread of cpe between C. perfringens strains was evident because strains from the same person carried IS1470-like cpe but shared no genetic relatedness according to pulsed-field gel electrophoresis analysis. Our findings suggest that healthy humans serve as a rich reservoir for cpe+ C. perfringens type A and may play a role in the etiology of gastrointestinal diseases caused by this organism. The results also indicate that humans should be considered a risk factor for spread of C. perfringens type A food poisoning and that they are a possible source of contamination for C. perfringens type A food poisoning.