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.     

Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure.     

Identification of the syntrophic partners in a coculture coupling anaerobic methanol oxidation to Fe(III) reduction

The repeating sequences of the toxin A gene from toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains of Clostridium difficile which were isolated in geographically separated facilities in Japan and Indonesia were determined. All six strains tested had identical repeating sequences with two deletions (1548 and 273 nucleotides in size) in the toxin A gene. A PCR method was designed to detect the deletions and the deletions were confirmed in all 50 toxin A-, toxin B+ strains examined by this method. Western immunoblot analysis revealed that polyclonal antiserum against native toxin A did not react with the concentrated culture filtrates of the toxin A-, toxin B+ strains. These results may suggest that toxin A-, toxin B+ strains have deletions of the two thirds of the repeating regions of the toxin A gene, which encodes the epitopes fully responsible for the reaction with the polyclonal antiserum.     

Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker

One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.     

Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.     

Zooplankton interactions with toxic phytoplankton: Some implications for food web studies and algal defence strategies of feeding selectivity behaviour, toxin dilution and phytoplankton population diversity

This study focuses on the interactions between toxic phytoplankton and zooplankton grazers. The experimental conditions used are an attempt to simulate situations that have, so far, received little attention. We presume the phytoplankton community to be a set of species where a population of a toxic species is intrinsically diverse by the presence of coexisting strains with different toxic properties. The other species in the community may not always be high-quality food for herbivorous zooplankton. Zooplankton populations may have developed adaptive responses to sympatric toxic phytoplankton species. Zooplankton grazers may perform a specific feeding behaviour and its consequences on fitness will depend on the species ingested, the effect of toxins, and the presence of mechanisms of toxin dilution and compensatory feeding. Our target species are a strain of the dinoflagellate Alexandrium minutum and a sympatric population of the copepod Acartia clausi. Mixed diets were used with two kinds of A. minutum cells: non-toxic and toxic. The flagellate Rhodomonas baltica and the non-toxic dinoflagellate Alexandrium tamarense were added as accompanying species. The effect of each alga was studied in separate diets. The toxic A. minutum cells were shown to have negative effects on egg production, hatching success and total reproductive output, while, in terms of its effect on fitness, the non-toxic A. minutum was the best quality food offered. R. baltica and A. tamarense were in intermediate positions. In the mixed diets, copepods showed a strong preference for toxic A. minutum cells and a weaker one for A. tamarense cells, while non-toxic A. minutum was slightly negatively selected and R. baltica strongly negatively selected. Although the level of toxins accumulated by copepods was very similar, in both the diet with only toxic A. minutum cells and in the mixed diet, the negative effects on fitness in the mixed diet could be offset by toxin dilution mechanisms. The implications of these findings are the fact that mesozooplankton may not play an important role in phytoplankton blooms development. Phytoplankton endotoxin production does not seem to be an evolutionary stable strategy as a defence against some herbivores.     

Effect of the endoparasite Amoebophrya sp. on toxin content and composition in the paralytic shellfish poisoning dinoflagellate Alexandrium fundyense (Dinophyceae)

Members of the Amoebophrya ceratii complex are endoparasitic dinoflagellates that parasitize a number of their dinoflagellate relatives, including toxic and/or harmful algal bloom-forming species. Despite many studies on the occurrence, prevalence, biology and molecular phylogeny of Amoebophrya spp., little attention has been given to toxin dynamics of host population following parasitism. Using Amoebophrya sp. infecting the paralytic shellfish toxin (PSP)-producing dinoflagellate Alexandrium fundyense, we addressed the following questions: (1) does parasitism by Amoebophrya sp. alter toxin content and toxin profiles of the dinoflagellate A. fundyense over the infection cycle? and (2) do parasite dinospores produced at the end of the infection cycle retain host toxins and thus potentially act as a vector to convey PSP toxin through the marine microbial food-web? Toxin time-course experiments showed that the PSP toxin contents did not vary significantly over the infection cycle, but mean toxin content for infected cultures was significantly higher than that for uninfected cultures. Host toxins were not detected in the free-living, dinospore stage of the parasite. Therefore, our results indicate that Amoebophrya sp. does not function as a vector for transferring PSP toxins to higher trophic levels. Rather, Amoebophrya infections appear to play an important role in maintaining healthy ecosystems by transforming potent toxins-producing dinoflagellates into non-toxic dinospores, representing “edible food” for consumers of the marine microbial food-web during toxic algal bloom event.     

Biocide gene(s) and biocidal activity in different strains of Bacillus sphaericus. Expression of the gene(s) in E. coli maxicells

Summary The recently cloned biocidal determinant of the highly toxic strain of B. sphaericus 1593M (Ganesan et al. 1983) was used as probe to investigate homologous sequences in different toxic and non-toxic strains of B. sphaericus. It was found that the potent strains we have analysed are characterised by the presence of DNA sequences (6.6, 6.4, 5.8, 1.6, 1.3 and 0.6 Kb) not found in the non-toxic strains. These results further show that one of the two weakly toxic strains analysed presents a hybridisation pattern completely different from that observed with the highly potent strains of B. sphaericus. When the DNA of the two non-toxic strains was analysed, SSII-I failed to hybridise to the probe and Rem4 exhibited mainly one hybridisable sequence of 2.3 Kb not detectable in the toxic strains.No region of homology to the probe was found in the DNA of two strains of B. thuringiensis (var. berliner and var. israeliensis) analysed.By dot blot hybridisation experiments it was estimated that the larvicidal determinant might be present in about one to three copies per genome.With the use of E. coli maxicells we have shown further that the toxin gene(s) encoded four polypeptides with molecular weights of 21, 19, 15, and 12 Kd. The significance of these findings is discussed.     

Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis

Summary The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 230 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.     

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.     

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.     

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.     

Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.     

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.     

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.     

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。     

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。     

Host- and tissue-specific pathogenic traits of Staphylococcus aureus

Comparative genomics were used to assess genetic differences between Staphylococcus aureus strains derived from infected animals versus colonized or infected humans. A total of 77 veterinary isolates were genetically characterized by high-throughput amplified fragment length polymorphism (AFLP). Bacterial genotypes were introduced in a large AFLP database containing similar information for 1,056 human S. aureus strains. All S. aureus strains isolated from animals in close contact with humans (e.g., pet animals) were predominantly classified in one of the five main clusters of the AFLP database (cluster I). In essence, mastitis-associated strains from animals were categorized separately (cluster IVa) and cosegregated with bacteremia-associated strains from humans. Distribution of only 2 out of 10 different virulence genes differed across the clusters. The gene encoding the toxic shock syndrome protein (tst) was more often encountered among veterinary strains (P < 0.0001) and even more in the mastitis-related strains (P<0.0001) compared to human isolate results. The gene encoding the collagen binding protein (cna) was rarely detected among invasive human strains. The virulence potential, as indicated by the number of virulence genes per strain, did not differ significantly between the human- and animal-related strains. Our data show that invasive infections in pets and humans are usually due to S. aureus strains with the same genetic background. Mastitis-associated S. aureus isolated in diverse farm animal species form a distinct genetic cluster, characterized by an overrepresentation of the toxic shock syndrome toxin superantigen-encoding gene.     

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.     

Toxic Bacillus pumilus from indoor air, recycled paper pulp, Norway spruce, food poisoning outbreaks and clinical samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 degrees C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1-10 microg ml(-1) (EC50) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.