High Toxin Production Research Articles

Benthic cyanobacterial metabolites interact to reduce coral larval survival and settlement

Benthic cyanobacterial mats (BCMs) are becoming increasingly abundant on coral reefs worldwide. High growth rates and prolific toxin production give them the potential to cause widespread coral recruitment failure through allelopathic effects, but few studies have made the link between their toxicity for coral larvae and in situ toxin concentrations. Here we investigated the allelopathic effects of the benthic cyanobacterium Anabaena sp.1 on larvae of the coral Pocillopora acuta. This cyanobacterium produces several non-ribosomal cyclic lipopeptides of the laxaphycin family with cytotoxic properties. Therefore, we measured the concentration of laxaphycins A and B in Anabaena mats and in the water column and tested their effects on coral larvae. We found that Anabaena crude extract reduces both larval survivorship and settlement and that laxaphycin B reduces settlement. When larvae were exposed to both laxaphycins, there was a reduction in both larval survival and settlement. In the natural reef environment, laxaphycin A and B concentrations increased with increasing proximity to Anabaena mats, with concentrations being consistently above LC50 and EC50 thresholds within a 1 cm distance of the mats. This study demonstrates that laxaphycins reduce the survival and inhibit the settlement of coral larvae at concentrations found near Anabaena mats in situ. It further shows a combined effect between two cyanobacterial metabolites. As BCMs become more common, more of their secondary metabolites might be released in the water column. Their occurrence will lead to a reduction in coral recruitment rates, contributing to the continuing decline of coral reefs and shift in community structure.

Assessment of the Appearance and Toxin Production Potential of Invasive Nostocalean Cyanobacteria Using Quantitative Gene Analysis in Nakdong River, Korea.

Invasive nostocalean cyanobacteria (INC) were first reported in tropical regions and are now globally spreading rapidly due to climate change, appearing in temperate regions. INC require continuous monitoring for water resource management because of their high toxin production potential. However, it is difficult to analyze INC under a microscope because of their morphological similarity to nostocalean cyanobacteria such as the genus Aphanizomenon. This study calculates the gene copy number per cell for each target gene through quantitative gene analysis on the basis of genus-specific primers of genera Cylindrospermopsis, Sphaerospermopsis, and Cuspidothrix, and the toxin primers of anatoxin-a, saxitoxin, and cylindrospermopsin. In addition, quantitative gene analysis was performed at eight sites in the Nakdong River to assess the appearance of INC and their toxin production potential. Genera Cylindrospermopsis and Sphaerospermopsis did not exceed 100 cells mL−1 at the maximum, with a low likelihood of related toxin occurrence. The genus Cuspidothrix showed the highest cell density (1759 cells mL−1) among the INC. Nakdong River has potential for the occurrence of anatoxin-a through biosynthesis by genus Cuspidothrix because the appearance of this genus coincided with that of the anatoxin-a synthesis gene (anaF) and the detection of the toxin by ELISA.

Updating the Methodology of Identifying Maize Hybrids Resistant to Ear Rot Pathogens and Their Toxins—Artificial Inoculation Tests for Kernel Resistance to Fusarium graminearum, F. verticillioides, and Aspergillus flavus

Resistance to toxigenic fungi and their toxins in maize is a highly important research topic, as mean global losses are estimated at about 10% of the yield. Resistance and toxin data of the hybrids are mostly not given, so farmers are not informed about the food safety risks of their grown hybrids. According to the findings aflatoxin regularly occurs at preharvest in Hungary and possibly other countries in the region can be jeopardized. We tested, with an improved methodology (two isolates, three pathogens, and a toxin control), 18 commercial hybrids (2017–2020) for kernel resistance (%), and for toxin contamination separately by two–two isolates of F. graminearum, F. verticillioides (mg/kg), and A. flavus (μg/kg). The preharvest toxin contamination was measured in the controls. Highly significant kernel resistance and toxin content differences were identified between hybrids to the different fungi. Extreme high toxin production was found for each toxic species. Only about 10–15% of the hybrids showed higher resistance to the fungal species tested and lower contamination level of their toxins. The lacking correlations between resistance to different fungi and toxins suggest that resistance to different fungi and response to toxin contamination inherits independently, so a toxin analysis is necessary. For safety risk estimation, separated artificial and natural kernel infection and toxin data are needed against all pathogens. Higher resistance to A. flavus and F. verticillioides stabilizes or improves feed safety in hot and dry summers, balancing the harmful effect of climate changes. Resistance and toxin tests during variety registration is an utmost necessity. The exclusion of susceptible or highly susceptible hybrids from commercial production results in reduced toxin contamination.

Effect of in-package gas composition on growth and aflatoxin production of Aspergillus flavus in culture medium and red pepper

In this study, the effect of in-package gas composition on growth and aflatoxin production of toxigenic Aspergillus flavus was investigated. For this purpose, semi-dried red pepper samples and potato dextrose agar media with different pH values (3.5, 5.6, 7.0 and 9.0) were inoculated with A. flavus and packaged under different gas atmospheres (air, 100% N2, 70% N2 + 30% CO2 and 100% CO2). The growth of the fungus was monitored during storage at 25 °C for 8 days and the aflatoxin levels produced were determined at the end of storage. The highest growth rate was observed in the medium at pH 9.0 and the highest toxin production was in the medium at pH 5.6. Slowdowns up to 55.6% and 28.0% in the growth of A. flavus in the culture medium and red pepper were observed when packaging under 100% N2 was employed. It was found that 70% N2 + 30% CO2 atmosphere was more effective than 100% N2 in inhibiting the growth of A. flavus in red peppers; however, the difference between the aflatoxin levels produced in the samples packaged under these gas compositions was not significant (p > 0.05). CO2 concentrations higher than 90% in the package were quite effective against A. flavus resulting in 60% and 99% reductions in the growth and aflatoxin production of the fungus in red pepper.

Transcriptional and physiological responses to inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: Implications for nutrient uptakes and assimilation

The marine dinoflagellate Alexandrium minutum is known to produce saxitoxins that cause paralytic shellfish poisoning in human worldwide through consumption of the contaminated shellfish mollusks. Despite numerous studies on the growth physiology and saxitoxin production of this species, the knowledge on the molecular basis of nutrient uptakes in relation to toxin production in this species is limited. In this study, relative expressions of the high-affinity transporter genes of nitrate, ammonium, and phosphate (AmNrt2, AmAmt1 and AmPiPT1) and the assimilation genes, nitrate reductase (AmNas), glutamine synthase (AmGSIII) and carbamoyl phosphate synthase (AmCPSII) from A. minutum were studied in batch clonal culture condition with two nitrogen sources (nitrate: NO3− or ammonium: NH4+) under different N:P ratios (high-P: N:P of 14 and 16, and low-P: N:P of 155). The expression of AmAmt1 was suppressed in excess NH4+-grown condition but was not observed in AmNrt2 and AmNas. Expressions of AmAmt1, AmNrt2, AmNas, AmGSIII, AmCPSII, and AmPiPT1 were high in P-deficient condition, showing that A. minutum is likely to take up nutrients for growth under P-stress condition. Conversely, relative expression of AmCPSII was incongruent with cell growth, but was well correlated with toxin quota, suggesting that the gene might involve in arginine metabolism and related toxin production pathway. The expression of AmGSIII is found coincided with higher toxin production and is believed to involve in mechanism to detoxify the cells from excess ammonium stress. The gene regulation observed in this study has provided better insights into the ecophysiology of A. minutum in relation to its adaptive strategies in unfavorable environments.

Induction of domoic acid production in diatoms—Types of grazers and diatoms are important

Grazers can induce toxin (domoic acid, DA) production in diatoms. The toxic response has been observed in two species of Pseudo-nitzschia and was induced by Calanus copepods. In this study, interactions between diatoms and copepods were further explored using different species of diatoms and copepods. All herbivorous copepods induced toxin production, whereas exposure to carnivorous copepods did not. In line with this, increasing the number of herbivorous copepods resulted in even higher toxin production. The induced response is thus only elicited by copepods that pose a real threat to the responding cells, which supports that the induced toxin production in diatoms evolved as an inducible defense.The cellular toxin content in Pseudo-nitzschia was positively correlated to the concentration of a group of specific polar lipids called copepodamides that are excreted by the copepods. This suggests that copepodamides are the chemical cues responsible for triggering the toxin production. Carnivorous copepods were found to produce less or no copepodamides.Among the diatoms exposed to grazing herbivorous copepods, only two of six species of Pseudo-nitzschia and none of the Nitzschia or Fragilariopsis strains responded by producing DA, indicating that not all Pseudo-nitzschia species/strains are able to produce DA, and that different diatom species might have different strategies for coping with grazing pressure. Growth rate was negatively correlated to cellular domoic acid content indicating an allocation cost associated with toxin production. Long-term grazing experiments showed higher mortality rates of grazers fed toxic diatoms, supporting the hypothesis that DA production is an induced defense mechanism.

Comparison of Fusarium graminearum Transcriptomes on Living or Dead Wheat Differentiates Substrate-Responsive and Defense-Responsive Genes.

Fusarium graminearum is an opportunistic pathogen of cereals where it causes severe yield losses and concomitant mycotoxin contamination of the grains. The pathogen has mixed biotrophic and necrotrophic (saprophytic) growth phases during infection and the regulatory networks associated with these phases have so far always been analyzed together. In this study we compared the transcriptomes of fungal cells infecting a living, actively defending plant representing the mixed live style (pathogenic growth on living flowering wheat heads) to the response of the fungus infecting identical, but dead plant tissues (cold-killed flowering wheat heads) representing strictly saprophytic conditions. We found that the living plant actively suppressed fungal growth and promoted much higher toxin production in comparison to the identical plant tissue without metabolism suggesting that molecules signaling secondary metabolite induction are not pre-existing or not stable in the plant in sufficient amounts before infection. Differential gene expression analysis was used to define gene sets responding to the active or the passive plant as main impact factor and driver for gene expression. We correlated our results to the published F. graminearum transcriptomes, proteomes, and secretomes and found that only a limited number of in planta- expressed genes require the living plant for induction but the majority uses simply the plant tissue as signal. Many secondary metabolite (SM) gene clusters show a heterogeneous expression pattern within the cluster indicating that different genetic or epigenetic signals govern the expression of individual genes within a physically linked cluster. Our bioinformatic approach also identified fungal genes which were actively repressed by signals derived from the active plant and may thus represent direct targets of the plant defense against the invading pathogen.

Screening of Bifidobacteria and Lactobacilli Able to Antagonize the Cytotoxic Effect of Clostridium difficile upon Intestinal Epithelial HT29 Monolayer.

Clostridium difficile is an opportunistic pathogen inhabiting the human gut, often being the aetiological agent of infections after a microbiota dysbiosis following, for example, an antibiotic treatment. C. difficile infections (CDI) constitute a growing health problem with increasing rates of morbidity and mortality at groups of risk, such as elderly and hospitalized patients, but also in populations traditionally considered low-risk. This could be related to the occurrence of virulent strains which, among other factors, have high-level of resistance to fluoroquinolones, more efficient sporulation and markedly high toxin production. Several novel intervention strategies against CDI are currently under study, such as the use of probiotics to counteract the growth and/or toxigenic activity of C. difficile. In this work, we have analyzed the capability of twenty Bifidobacterium and Lactobacillus strains, from human intestinal origin, to counteract the toxic effect of C. difficile LMG21717 upon the human intestinal epithelial cell line HT29. For this purpose, we incubated the bacteria together with toxigenic supernatants obtained from C. difficile. After this co-incubation new supernatants were collected in order to quantify the remnant A and B toxins, as well as to determine their residual toxic effect upon HT29 monolayers. To this end, the real time cell analyser (RTCA) model, recently developed in our group to monitor C. difficile toxic effect, was used. Results obtained showed that strains of Bifidobacterium longum and B. breve were able to reduce the toxic effect of the pathogen upon HT29, the RTCA normalized cell-index values being inversely correlated with the amount of remnant toxin in the supernatant. The strain B. longum IPLA20022 showed the highest ability to counteract the cytotoxic effect of C. difficile acting directly against the toxin, also having the highest capability for removing the toxins from the clostridial toxigenic supernatant. Image analysis showed that this strain prevents HT29 cell rounding; this was achieved by preserving the F-actin microstructure and tight-junctions between adjacent cells, thus keeping the typical epithelium-like morphology. Besides, preliminary evidence showed that the viability of B. longum IPLA20022 is needed to exert the protective effect and that secreted factors seems to have anti-toxin activity.

Influence of the Mississippi River on Pseudo-nitzschia spp. Abundance and Toxicity in Louisiana Coastal Waters

The presence of domoic acid (DA) toxin from multiple species of Pseudo-nitzschia is a concern in the highly productive food webs of the northern Gulf of Mexico. We documented the Pseudo-nitzschia presence, abundance, blooms, and toxicity over three years along a transect ∼100 km west of the Mississippi River Delta on the continental shelf. Pseudo-nitzschia were present throughout the year and occurred in high abundances (>104 cells l−1) in the early spring months during high Mississippi River (MSR) flow (∼20,000 m3 s−1) but were most abundant (>106 cells l−1) when MSR discharge was relatively lower among the spring months. A high particulate toxin production (maximum reaching 13 μg DA l−1) was associated with the high cell abundances and exceeded, by an order of magnitude, prior reports of particulate DA concentrations in Louisiana coastal waters. Differences in Pseudo-nitzschia peak times and its toxicity were correlated mainly with the timing and magnitude of MSR discharge and changes in associated parameters such as nutrient stoichiometry and salinity. A negative relationship between high MSR discharge and Pseudo-nitzschia and particulate DA concentrations was documented. These riverine dynamics have the potential to influence DA contamination in pelagic and benthic food webs in the coastal waters of the northern Gulf of Mexico.

Genetic features of human and bovine Escherichia coli O157:H7 strains isolated in Argentina

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens associated with human diseases. In Argentina, O157:H7 is the dominant serotype in hemolytic uremic syndrome (HUS) cases. Previously, we have described the almost exclusive circulation of human E. coli O157 strains belonging to the hypervirulent clade 8 in Neuquén Province. The aim of the present study was to investigate, by a broad molecular characterization, if this particular distribution of E. coli O157 clades in Neuquén is similar to the situation in other regions of the country and if it may be originated in a similar profile in cattle, its main reservoir. Two-hundred and eighty O157 strains (54 bovine and 226 human) isolated between 2006 and 2008 in different regions of Argentina were studied. All strains harbored rfbO157, fliCH7, eae, and ehxA genes. The predominant genotype was stx2a/stx2c in human (76.1%) and bovine (55.5%) strains. All human isolates tested by Lineage-Specific Polymorphism Assay (LSPA-6), were lineage I/II; among bovine strains, 94.1% belonged to lineage I/II and 5.9% to lineage I. No LSPA-6 lineage II isolates were detected. Single nucleotide polymorphism (SNP) analysis has revealed the existence of nine clade phylogenetic groups. In our clinical strains collection, 87.6% belonged to the hypervirulent clade 8, and 12.4% were classified as clade 4/5. In bovine isolates, 59.3% strains were clade 8, 33.3% clade 4/5 and 7.4% clade 3. More than 80% of human strains showed the presence of 6 of the 7 virulence determinants described in the TW14359 O157 strain associated with the raw spinach outbreak in the U.S. in 2006. More than 80% of bovine strains showed the presence of 3 of these factors. The q933 allele, which has been related to high toxin production, was present in 98.2% of clinical strains and 75.9% of the bovine isolates. The molecular characterization of human STEC O157 strains allows us to conclude that the particular situation previously described for Neuquén Province, may actually be a characteristic of the whole country. These genetic features are quite similar to those observed in the bovine reservoir and may be derived from it. This data confirms that, unlike the rest of the world, in Argentina most of the STEC O157 strains present in cattle may cause human infections of varying severity and the marked virulence described for these strains may be related to the high incidence of HUS in our country.

Genotypic characterization of Escherichia coli O157:H7 strains that cause diarrhea and hemolytic uremic syndrome in Neuquén, Argentina

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens associated with cases of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). E. coli O157:H7 is the dominant serotype in Argentina and also in Neuquén Province, in which HUS incidence is above the national average, with a maximum of 28.6 cases per 100,000 children less than 5 years old reported in 1998. The aim of this study was to characterize a collection of 70 STEC O157 strains isolated from patients with diarrhea and HUS treated in the province of Neuquén, Argentina, between 1998 and 2011. All strains harbored eae, ehxA, rfbO157, and fliCH7 genes, and stx2a/stx2c (78.7%) was the predominant genotype. A total of 64 (91.4%) STEC O157 strains belonged to the hypervirulent clade 8 tested using both 4 and 32 SNP typing schemes. The strains showed the highest values reported in the literature for 6 of the 7 virulence determinants described in the TW14359 O157 strain associated with the raw spinach outbreak in the U.S. in 2006. Clade 8 strains were strongly associated with two of them: ECSP_3286, factor encoding an outer membrane protein that facilitates the transport of the heme complex (P=0.001), and in particular extracellular factor ECSP_2870/2872, coding proteins related to adaptation to plant hosts (P=0.000004). The q933 allele, which has been related to high toxin production, was present in 97.1% of the strains studied for the anti-terminator Q gene. In summary, this study describes, for the first time in Argentina, the almost exclusive circulation of strains belonging to the hypervirulent clade 8, and also the presence of putative virulence factors in higher frequencies than those reported worldwide. These data may help to understand the causes of the particular epidemiological situation related to HUS in Neuquén Province.

Co-amoxiclav induces proliferation and cytotoxin production of Clostridium difficile ribotype 027 in a human gut model

Co-amoxiclav is widely prescribed in hospitals. Although reports have suggested it may be linked to onset of Clostridium difficile infection (CDI), data on the risk of CDI associated with specific antibiotics is difficult to obtain, due to confounding clinical factors. We have examined the propensity of co-amoxiclav to induce CDI using a human gut model. We used a triple-stage chemostat human gut model to study the effects of co-amoxiclav on indigenous gut microorganisms and C. difficile PCR ribotype 027. C. difficile viable counts and spores were evaluated, and cytotoxin titres were assayed. Co-amoxiclav concentrations were measured using a large plate bioassay. Co-amoxiclav induced rapid C. difficile germination and high toxin production in the gut model, from 5 days after commencement of instillation. Cell proliferation and toxin production were prolonged and continued throughout the duration of the experiment. Only very low levels of co-amoxiclav antimicrobial activity could be detected within the gut model, despite having a marked effect on gut flora microorganisms. Co-amoxiclav induced CDI within the gut model, supporting clinical observations linking co-amoxiclav treatment with CDI onset. This reinforces the value of the gut model as a clinically relevant means of studying CDI. Caution should be exercised in the prescription of co-amoxiclav to patients in high CDI risk settings.

Lack of Association between Clinical Outcome of Clostridium difficile Infections, Strain Type, and Virulence-Associated Phenotypes

Clostridium difficile strain NAP1/027 (North American pulsed-field gel electrophoresis [PFGE] type 1 and PCR ribotype 027 [R027]) has been associated with recent outbreaks in North America and Europe. It has been associated with more severe disease symptoms, higher mortality rates, and greater risk of relapse. This strain is thought to produce more toxins and sporulate to higher levels. However, recent studies suggest that this may not always be the case. The objective of our study was to assess, in a nonoutbreak situation, whether specific strains, such as NAP1/027, were associated with more severe disease symptoms, higher toxin production, and/or greater sporulation in vitro. We isolated and characterized C. difficile strains from 21 patients with mild to moderate, severe, or complicated symptoms of C. difficile infection (CDI). The isolates were characterized by different molecular typing methods, including PCR ribotyping, tandem repeat sequence typing (TRST), and sequencing of the tcdC gene. Fourteen isolates were of PCR ribotype 027 with deletions in tcdC, but no association with severity or clinical outcome was found. We show by immunodot blot detection of toxins with monoclonal antibodies that all R027 isolates produced more TcdA and TcdB than other strains. On the other hand, they consistently produced fewer spores than non-R027 isolates. Taken together, our data suggest that NAP1/027 isolates are not always associated with more severe disease, even though they may produce larger amounts of toxins. Our study also suggests that current assertions regarding the NAP1/027 may not apply to all isolates and that other factors may come into play.

Increased production of Coleoptera-specific delta-endotoxin by homologous expression of cry3Aa11 in Bacillus thuringiensis Mm2

The cry3Aa11 gene of Bacillus thuringiensis strain Mm2 (B. thuringiensis Mm2) was PCR-amplified and cloned into E. coli/B. thuringiensis shuttle vector pHT315. The recombinant plasmid carrying the cry3Aa11 gene was introduced into the parental B. thuringiensis Mm2. The toxin production capacities of parental and recombinant strains grown in DSM, containing either 50 mM or 200 mM inorganic phosphate (Pi), were compared by SDS-PAGE and western blot analyses. In comparison to the parental strain, recombinant B. thuringiensis Mm2 produced 4-fold more Cry3Aa11 toxin after 12 h of growth in the presence of 50 mM Pi. However, the 200 mM Pi concentration did not cause a drastic increase in toxin production by the recombinant strain. This study shows that higher toxin production could be obtained from the cultures during the early hours of growth by introducing the multicopy cry3Aa11 gene into the source organism.

Evaluation of the Anti-Terminator Q933 Gene as a Marker for Escherichia coli O157:H7 with High Shiga Toxin Production

The anti-terminator Q933 gene of the bacteriophage 933W was evaluated as a marker for Escherichia coli O157:H7 strains with high Shiga toxin production. In total, 262 environmental strains of E. coli O157:H7 isolated from feces of beef cattle and the digestive tract of houseflies were screened for the Q933 and Q21 (anti-terminator Q21 of bacteriophage 21) genes by polymerase chain reaction. Nine (3.4%) isolates tested positive for Q933 alone, 161 (61.5%) were positive for the Q21 gene alone, and 92 (35.1%) isolates carried both Q alleles. Results from the enzyme-linked immunosorbent assay show that the isolates with Q933 alone produced significantly more Shiga toxin than the remaining isolates. The difference was even greater after the induction of the toxin production by a short exposure of cells to ultraviolet light. These data suggest that Q933 is a promising indicator for environmental E. coli O157:H7 with high production of Shiga toxins and, therefore, for potentially clinically relevant strains.

Optimization of casein-based semisynthetic medium for growing of toxigenicCorinebacterium diphtheriaein a fermenter

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.

Molecular composition of diphtheria toxoid produced using semi-synthetic and meat extract-based broths

The use of semi-synthetic broths for cultivation of Corynebacterium diphtheriae instead of a meat extract-based broth avoids the presence of highly undesirable bovine meat antigens in the diphtheria toxoid. As information on the composition of casein digest-based broths used for the production of diphtheria toxoid is scarce, we have now developed one. The composition of a casein-based medium that supports vigorous bacterial growth as well as high toxin production is described below. The comparative analysis of the toxoids, produced using the meat-based Pope–Lingood and the casein digest-based broths, showed considerable differences in their molecular composition. The variance of weight distribution of toxoid-containing molecular complexes was smaller when the semi-synthetic broth was used. Normal human therapeutic IgG recognizes some of the proteins in the meat-based medium but does not react with any components of the semi-synthetic medium. While precipitation at the isoelectric point of the diphtheria toxoid produced by culturing the C. diphtheriae strain in the semi-synthetic medium resulted in a preparation meeting the requirement for purity (more than 1500 limit floculation Lf/mg protein nitrogen PN), the toxoid produced in the Pope–Lingood broth failed to meet this requirement in some cases, even after a second purification step using ultrafiltration.

Serotypes and virulence factors of Shiga toxin-producing Escherichia coli isolated from healthy Norwegian sheep.

To characterize a number of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep and to discuss the potential of these isolates as human pathogens. Twelve different O-groups and seven different H-types were identified by standard serotyping methods. The most common serotypes were O5:NM, O6:H10, O91:NM and O128:NM. Polymerase chain reaction (PCR) was used for the detection of virulence factor genes. Of 102 isolates, 86.3% carried stx1 and 83% of these were also positive in the stx1OX3-specific PCR. stx2 was carried by 55.9% of the isolates and 77.2% of these were also positive in the stx2d-specific PCR. The Vero cell assay showed high toxin production in 70.6% of the isolates. None of the isolates carried eae. The study supports the animal-host relationship suggested in other studies with STEC serogroups O5, O91 and O128 strongly associated with sheep. Most sheep STEC carry stx1OX3 (except O91) and the dominating stx2 variant is stx2d. One stx profile clearly dominates within a serotype. In spite of the predominance of certain sheep-associated STEC, sheep cannot be excluded as carriers of human pathogenic STEC.

Proteins released during high toxin production in Clostridium difficile.

The mechanism by which toxins A and B are released by Clostridium difficile is unknown and information about the other extracellular proteins of this bacterium is limited. The authors identified exported proteins from C. difficile strain VPI 10463 during conditions promoting high toxin production. Toxins A and B were released in a 1:1 ratio and the proportion of toxin in the extracellular fraction reached 50% during the stationary phase as compared to a proportion of <1% for typical cytoplasmic proteins, showing that toxin export was not due to bacterial lysis. A 47 kDa protein, released with similar kinetics to the toxins, was processed and showed weak similarity to the channel-forming protein TolC. Another protein released during high toxin production was unprocessed and showed similarity to XkdK encoded by the prophage PBSX in Bacillus subtilis, a protein supposedly exported via phage-specific holins. The two most abundant extracellular C. difficile proteins, found during both high and low toxin production, were processed and identified as shed S-layer proteins. As shown by N-terminal sequencing and PCR-based methods, there was a considerable sequence variation of the S-layer gene slpA in different serogroup reference strains. To conclude, C. difficile uses the classical Sec-dependent and probably also holin-like pathways to secrete a comparatively small repertoire of proteins.

Toxins, butyric acid, and other short-chain fatty acids are coordinately expressed and down-regulated by cysteine in Clostridium difficile.

It was recently found that a mixture of nine amino acids down-regulate Clostridium difficile toxin production when added to peptone yeast extract (PY) cultures of strain VPI 10463 (S. Karlsson, L. G. Burman, and T. Akerlund, Microbiology 145:1683-1693, 1999). In the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (n = 6) and clinical isolates (n = 28) of C. difficile (>99% suppression by cysteine in the highest toxin-producing strain). Also, cysteine derivatives such as acetylcysteine, glutathione, and cystine effectively down-regulated toxin expression. An impact of both cysteine and cystine but not of thioglycolate on toxin yield indicated that toxin expression was not regulated by the oxidation-reduction potential. Several metabolic pathways, including butyric acid and butanol production, were coinduced with the toxins in PY and down-regulated by cysteine. The enzyme 3-hydroxybutyryl coenzyme A dehydrogenase, a key enzyme in solventogenesis in Clostridium acetobutylicum, was among the most up-regulated proteins during high toxin production. The addition of butyric acid to various growth media induced toxin production, whereas the addition of butanol had the opposite effect. The results indicate a coupling between specific metabolic processes and toxin expression in C. difficile and that certain amino acids can alter these pathways coordinately. We speculate that down-regulation of toxin production by the administration of such amino acids to the colon may become a novel approach to prophylaxis and therapy for C. difficile-associated diarrhea.