A type II restriction endonuclease of Streptococcus thermophilus ST117

Three separate sets of polymerase chain reaction primers were designed to specifically detect the presence of a toxin A gene fragment, a toxin B gene fragment, and the entire toxin B gene. In addition toxin gene fragments that were amplified from well characterized toxic strains were tagged fluorescently and used as hybridization probes to screen C. difficile strains. A survey of 37 toxic strains and 10 non-toxic strains demonstrated that toxic strains normally contain the genetic composition for toxin A and toxin B simultaneously; whereas, non-toxic strains typically did not contain detectable toxin determinants. The only exception found was strain 39, which had the genetic composition for toxins A and B, but was not cytotoxic under the conditions tested.     

Isolation and characterization of two toxins from the Mexican scorpion Centruroides limpidus limpidus Karsch

1. Several toxic polypeptides were found in the venom of the scorpion Centruroides limpidus limpidus. Comparative studies of the potency of the venom in different strains of mice were conducted. 2. A new type of toxin (component II.9), specific for crustaceans (crayfish and isopods), was isolated from this scorpion and was shown to have the following N-terminal amino acid sequence: Lys-Lys-Asp-Gly-Tyr-Leu-Val-Asn-Lys-Tyr-Thr-Gly-Cys-Lys-Val-Asn-Cys- Tyr-Lys-Leu-Gly-Glu-Asn-Lys-Phe-Cys-Asn-Arg-Glu-. 3. A polypeptide toxic to mice (component II.6) from this venom was shown to have the following N-terminal sequence: Lys-Glu-Gly-Tyr-Leu-Val-Asn-His-Ser-Thr-Gly-Cys-Lys-Tyr- Glu-Cys-Tyr-Lys-Leu-Gly-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-. 4. In cultured chick dorsal root ganglion cells, 1 microM of toxin II.6 was shown to reduce the size of sodium currents and to slow-down their activation-inactivation kinetics. The toxin had also a depressive action on the classical Ca2+ current activated at high membrane potentials (greater than 0 mV).     

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers

Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.     

Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from China

A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.     

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N₂ fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.     

Toxic shock syndrome toxin 1 (TSST-1) production by staphylococci isolated from goats and presence of specific antibodies to TSST-1 in serum and milk.

The ability of staphylococcal strains isolated from different anatomical sites in 133 healthy goats to produce toxic shock syndrome toxin 1 (TSST-1) and the presence of antibodies to this toxin in serum and milk were studied. The enzyme-linked immunosorbent assay method was used to detect both the toxin and the presence of antibodies. Of a total of 342 staphylococcal strains studied, 86 (25.2%) were found to produce TSST-1. Specific antibodies to TSST-1 were found in the serum of 57 (42.9%) of the animals studied and the milk of 63 (47.4%) of the animals. These results suggest that goats are frequently in contact with staphylococci that produce TSST-1, a toxin usually associated with Staphylococcus aureus strains isolated from cases of toxic shock syndrome in humans.     

Toxic shock syndrome toxin 1 (TSST-1) production by staphylococci isolated from goats and presence of specific antibodies to TSST-1 in serum and milk

The ability of staphylococcal strains isolated from different anatomical sites in 133 healthy goats to produce toxic shock syndrome toxin 1 (TSST-1) and the presence of antibodies to this toxin in serum and milk were studied. The enzyme-linked immunosorbent assay method was used to detect both the toxin and the presence of antibodies. Of a total of 342 staphylococcal strains studied, 86 (25.2%) were found to produce TSST-1. Specific antibodies to TSST-1 were found in the serum of 57 (42.9%) of the animals studied and the milk of 63 (47.4%) of the animals. These results suggest that goats are frequently in contact with staphylococci that produce TSST-1, a toxin usually associated with Staphylococcus aureus strains isolated from cases of toxic shock syndrome in humans.     

球形芽孢杆菌C3—41二元毒素基因在苏云金芽孢杆菌以色列亚种中的？…

球形芽孢杆菌Ｃ３－４１是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于２３６２菌株,Ｓｏｕｔｈｅｒｎ杂交证明Ｃ３－４１总ＤＮＡ中３．５ＫｂＨｉｎｄＩＩＩ片段上带有４１．９和５１．４ｋＤ二元毒素基因。     

球形芽孢杆菌C3-41二元毒素基因在苏云金芽孢杆菌以色列亚种中的克隆和表达

球形芽孢杆菌C3-41是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于2362菌株,Southern杂交证明C\-3\|41总DNA中35Kb HindIII片段上带有419和514kD二元毒素基因,该片段由3479个核苷酸组成,核苷酸序列同2362菌株的二元毒素基因序列完全相同。含二元毒素基因的重组质粒pCW\|1和pCW\|2能在大肠杆菌中表达产生二元毒蛋白,但表达量低,重组子杀蚊毒性低。无晶体型苏云金芽孢杆菌以色列亚种重组子在其芽孢形成中能产生以晶体形式存在的二元毒素蛋白,其全发酵液和纯化晶体蛋白的杀蚊活性与C\-3\|41相近。     

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.     

Erythrogenic toxins A, B and C: Occurrence of the genes and exotoxin formation from clinical Streptococcus pyogenes strains associated with streptococcal toxic shock-like syndrome

We report the study of 53 clinical isolates of group A streptococci, all from patients with streptococcal toxic shock-like syndrome. The strains were analysed for the occurrence of the genes of erythrogenic toxins (pyrogenic exotoxins) types A, B and C and in vitro production of these toxins. In contrast to reports indicating that 85% of the toxic shock-like syndrome-associated isolates contained the erythrogenic toxin A gene, only 58.5% of our strains harboured this gene. The erythrogenic toxin C gene was detected in 22.6% of the isolates. Erythrogenic toxin A and erythrogenic toxin B were produced by 68.7% and 58.3% of the strains containing either gene. For all group A streptococci, irrespective of clinical association, the erythrogenic toxin B gene was detected in all the isolates tested. Thus, it is difficult to define a specific role for erythrogenic toxin B in toxic shock-like syndrome as there was no clear correlation between this disease and the presence of toxin genes. Our results suggest the existence of other pathogenic factor(s) produced by group A streptococci which may stimulate human peripheral T lymphocytes in a manner similar to that of erythrogenic toxins, thus explaining different observations in previous epidemiological genetic studies.     

Increasing oxygen radicals and water temperature select for toxic Microcystis sp

Pronounced rises in frequency of toxic cyanobacterial blooms are recently observed worldwide, particularly when temperatures increase. Different strains of cyanobacterial species vary in their potential to produce toxins but driving forces are still obscure. Our study examines effects of hydrogen peroxide on toxic and non-toxic (including a non-toxic mutant) strains of M. aeruginosa. Here we show that hydrogen peroxide diminishes chlorophyll a content and growth of cyanobacteria and that this reduction is significantly lower for toxic than for non-toxic strains. This indicates that microcystins protect from detrimental effects of oxygen radicals. Incubation of toxic and non-toxic strains of M. aeruginosa with other bacteria or without (axenic) at three temperatures (20, 26 and 32°C) reveals a shift toward toxic strains at higher temperatures. In parallel to increases in abundance of toxic (i.e. toxin gene possessing) strains and their actual toxin expression, concentrations of microcystins rise with temperature, when amounts of radicals are expected to be enhanced. Field samples from three continents support the influence of radicals and temperature on toxic potential of M. aeruginosa. Our results imply that global warming will significantly increase toxic potential and toxicity of cyanobacterial blooms which has strong implications for socio-economical assessments of global change.     

Presence of enterotoxin C and toxic shock syndrome toxin--1 (TSST-1) genes in population of Staphylococcus aureus phage type 187

The aim of this study was to examine whether Staphylococcus aureus of phage type 187 possess the genes of enterotoxins and toxic shock syndrom toxin. Sixteen phage type 187 strains were isolated from the hospital patients (12) and the carriers (4) in twelve medical centres in Poland during 1991 and 2005. Biotyping, phage typing, antibiotic susceptibility, detection of the genes of enterotoxins (sea--sed) and toxic shock syndrome toxin (tst) was tested. The results of this study showed that all staphylococci of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Almost all of them (93.8%) had the enterotoxin C gene and TSST-1 gene. This fact allows to consider them the strains of potentially high virulence.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3-3.5 and temperature 16°C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.     

Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain.

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.     

Identification and characterization of a "biomarker of toxicity" from the proteome of the paralytic shellfish toxin-producing dinoflagellate Alexandrium tamarense (Dinophyceae)

The objective of this study was to identify and characterize a "biomarker of toxicity" from the proteome of Alexandrium tamarense, a paralytic shellfish toxin (PST)-producing dinoflagellate. A combination of 2-DE and MS approaches was employed to identify proteins of interest in the vegetative cells of several strains of A. tamarense with different toxin compositions and from different geographical locations. The electrophoretic analysis of the total water-soluble proteins from these toxic strains by 2-DE showed that several abundant proteins, namely AT-T1, AT-T2 and AT-T3, differing slightly in apparent Mr and pIs, were consistently present in all toxic strains of A. tamarense. Further analysis by MALDI-TOF MS and N-terminal amino acid sequencing revealed that they are isoforms of the same protein. Even more intriguing is that these proteins in A. tamarense have similar amino acid sequences and are closely related to a "biomarker of toxicity" previously reported in A. minutum. Unambiguous and highly species-specific identification was later achieved by comparing the PMFs of proteins in these two species. An initial attempt to characterize these proteins by generation of murine polyclonal antibodies against the AT-T1 protein was successful. Western blot analysis using the murine AT-T1-polycolonal antibodies identified all the toxic strains of A. tamarense and A. minutum, but not the nontoxic strain of A. tamarense. These results indicate that these protein characteristics for toxic strains are species-specific and that they are stable properties of the tested algae which are clearly distinguishable irrespective of geographical location and toxin composition. To our knowledge, this is the first study to demonstrate the use of polyclonal antibodies against marker proteins purified from 2-DE gels to distinguish different strains and species of the PST-producing dinoflagellate Alexandrium. It provides the basis for the production of monoclonal antibody probes against the "biomarkers of toxicity" for those dinoflagellates whose genome is incompletely characterized. Potentially, immunoassays could be developed to detect the presence of toxic algae in routine monitoring programs as well as to predict bloom development and movement.     

Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1.

A cosmid library was prepared from a partial BamHI digest of total DNA from Bacillus sphaericus SSII-1. Two hundred fifty Escherichia coli clones were screened for toxicity against larvae of the mosquito Culex quinquefasciatus. One toxic clone, designated pKF2, was chosen for further study. Two toxic subclones, designated pXP33 and pXP34, obtained by ligating PstI-derived fragments of pKF2 into pUC18, contained the same 3.8-kb fragment, but in opposite orientations. Sequence analysis revealed the presence of an open reading frame corresponding to a 100-kDa protein and the 3' end of a further open reading frame having significant homology to open reading frames of transposons Tn501 and Tn21. The sequence of the SSII-1 toxin was compared with those of known toxins and was found to show regional homology to those of ADP-ribosyltransferase toxins. The distribution of the toxin gene among other B. sphaericus strains was examined.     

VARIATIONS OF PSP TOXIN PROFILES DURING DIFFERENT GROWTH PHASES IN GYMNODINIUM CATENATUM (DINOPHYCEAE) STRAINS ISOLATED FROM THREE LOCATIONS IN THE GULF OF CALIFORNIA,MEXICO1

In vitro experiments were performed with Gymnodinium catenatum Graham strains isolated from three locations in the Gulf of California to determine the variability in toxicity and toxin profiles. Strains were cultivated in GSe at 20°C±1°C, 150 μmol photons·m^?2·s^?1 (12:12 light:dark cycle), and harvested during different growth phases. Growth rates were higher than in previous studies, varying between 0.70 and 0.82 day^?1. The highest cell yields were reached at 16 and 19 days, with maximum densities between 1090 and 3393 cells·mL^?1. Bahía de La Paz (BAPAZ) and Bahía de Mazatlán (BAMAZ) were the most toxic (101 pg STXeq·cell^?1), whereas strains from Bahía Concepción (BACO) were the least toxic (13 pg STXeq·cell^?1). A strain isolated from cyst germination was one of the least toxic strains. No significant changes in toxin content with culture age were observed (0.2 and 0.6 pmol paralytic shellfish poisoning·cell^?1). All strains contained neosaxitoxin (NEOSTX), decarbamoyl‐saxitoxin (dcSTX), decarbamoyl‐gonyautoxin‐2,‐3, (dcGTX2‐3), N‐sulfo‐carbamoylsaxitoxin (B1), N‐sulfo‐carbamoylneosaxitoxin (B2), and N‐sulfo‐carbamoylgonyautoxin‐2,‐3 (C1‐2). Bahía Concepción strains had the highest content of C1; BAPAZ and BAMAZ strains had a higher percentage of NEOSTX. Differences in toxin composition with culture age were observed only in BAMAZ and BAPAZ strains. Cultures with a higher percentage of long chains had more NEOSTX, while those with a higher proportion of short chains had a lower content of NEOSTX. Gulf of California strains are characterized by a high proportion of NEOSTX, and seem to have evolved particular physiological responses to their environment that are reflected in the toxin profile, suggesting different populations.