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)     

Interconversion of Type C and D Strains of Clostridium botulinum by Specific Bacteriophages

These studies show that Clostridium botulinum types C and D cultures can be cured of their prophages and converted to either type C or D depending on the specific phage used. Strains of types C and D were cured of their prophages and simultaneously ceased to produce their dominant toxins designated as C(1) and D, respectively. Cured nontoxigenic cultures derived from type C strain 162 were sensitive to the phages from the toxigenic type C strain 162 and type D strain South African. When cured nontoxigenic cultures derived from strain 162 were infected with the tox(+) phages from the 162 strain of type C and the South African strain of type D, they then produced toxin neutralized by types C and D antisera, respectively. Cured nontoxigenic cultures isolated from the type D South African strain were only sensitive to the parent phage, and, when reinfected with the tox(+) phage, they produced toxin neutralized by type D antiserum. Type C strain 153 and type D strain 1873, when cured of their respective prophages, also ceased to produce toxins C(1) and D, but, unlike strain 162 and the South African strain, they continued to produce a toxin designated as C(2). When the cured cultures from strains 153 and 1873 were infected with the tox(+) phage from type D strain 1873, the cultures simultaneously produced toxin that was neutralized by type D antiserum. When these cured cultures were infected with the tox(+) phage from type C strain 153, the cultures produced toxin that was neutralized by type C antiserum. These studies with the four strains of C. botulinum confirm that the toxigenicity of types C and D strains requires the continued participation of tox(+) phages. Evidence is presented that types C and D cultures may arise from a common nontoxigenic strain.     

Evaluation of different methods for detection of Clostridium difficile toxins in Poland

The aim of this study was to compare different methods for C. difficile toxins detection. Fifty three stool samples taken from patients with antibiotic-associated diarrhoea were studied. TCD toxin A EIA (Becton Dickinson, USA), Tox A/B ELISA test (TechLab, USA), cytotoxicity and neutralization assay on McCoy cells and PCR for detection of both toxin A and B genes were performed in vivo (in stool samples) and in vitro (in isolated strains). Reference toxigenic and nontoxigenic and two Japanese toxin A-negative and toxin B-positive C. difficile strains were used as a controls. TCD toxin A EIA detected in vivo only 19 positive samples. Tox A/B test detected 52 positive samples out of 53 studied. All 53 stool samples were C. difficile culture positive (53 strains were cultured). Toxin B was detected in 52 strain-supernatants and in all controls (except the nontoxigenic one). Both toxin A and B genes were detected by PCR in all 53 isolated strains, Japanese and reference strain (except the nontoxigenic one). In vitro toxin A was detected by TCD toxin A EIA in 42 strains. These results were compared with those obtained in Tox A/B ELISA test. We observed 52 positive strains. Toxigenic reference strain and two Japanese toxA(-)/toxB(+) strains were also positive. Only 2 negative results were obtained with the nontoxigenic reference strain and unique nontoxigenic isolated strain. Tox A/B ELISA test seems to be the best for detection of C. difficile toxins in vivo and in vitro. Test avoids the false-negative results in the case of presence of toxin A-negative and toxin B-positive strain.     

Isolation of Nontoxigenic Variants Associated with Enhanced Sporulation and Alteration in the Cell Wall from Clostridium botulinum Type A 190L by Treatment with Detergents

Nontoxigenic variants were isolated from Clostridium botulinum type A strain 190L after treatment with detergents such as deoxycholate, sodium dodecyl sulfate, Tween 80 and Brij-58. Deoxycholate was most effective for obtaining the variants. The variants exhibited a markedly increased frequency of sporulation compared with the oligosporogenic parent strain. The cell wall of the parent strain was composed of an outer layer and an inner layer, whereas that of the variants lost the outer layer. After treatment with mitomycin C the parent strain was subjected to lysis and produced bacteriophages with a hexagonal head and a contractible tail, while the nontoxigenic variants did not yield bacteriophages or phage-like structures. There appears to be a close relationship among the toxigenic and sporogenic properties, formation of the outer cell wall layer and lysogeny.     

Effect of lysogenic conversion of Corynebacterium diphtheriae strain on its iron assimilation and spectrum of cellular proteins

Growth of the isogenic pair of C. diphtheriae strains, the nontoxigenic strain C7 (-) and the toxigenic strain C7 (beta), was studied under conditions of limited availability of the iron source. The growth of the toxigenic strain was shown to depend on the concentration of iron in the medium to a lesser degree than the nontoxigenic one. Lysogenic conversion results in the synthesis of additional iron-dependent proteins, absent in C. diphtheriae initial nontoxigenic strain C7 (-). Special attention was paid to proteins with a mol. wt. 66 kD, synthesized by the toxigenic strain irrespective of the concentration of iron in the medium, while in the toxigenic strain these proteins were detected only under conditions of iron deficiency.     

Observations on bacteriophages of Clostridium botulinum type C isolates from different sources and the role of certain phages in toxigenicity.

Twenty strains of Clostridium botulinum type C, including 12 isolates from avian sources with varying toxigenic properties, were examined by electron microscope for the presence of bacteriophages. All toxigenic strains were infected with one or two types of phages. Three types of phages designated large, small, and intermediate were observed. Most of the strains carried the large and small phage, with the large phage being present in much greater numbers. Since there is evidence that highly toxigenic strains of C. botulinum type C are responsible for large outbreaks of botulism in wild birds, the phenomenon of toxigenic variation among the type C strains was investigated. Experiments were carried out employing a broth medium on a phagefree nontoxigenic strain for elucidating the role of bacteriophages in toxigenicity. All phage suspensions contained large phages, with the exception of one that caused conversion. The exception was a preparation containing an intermediate type of phage. Phages from different strains produced cultures of varying toxigenic characteristics. By employing a tube-lytic test and an agar-overlay-phage assay technique, it was determined that whenever the phage-bacterium relationship resulted in an initial high degree of lysis, the potency of toxin in the culture was weak. It appeared that in highly toxigenic strains, the phage-bacterium relationship is characterized by a stable lysogenic type of association. It was also found that in a highly toxigenic converted culture the percentage of toxigenic cells was 100, whereas in hypotoxigenic culture the percentage was only 20.     

Observations on bacteriophages of Clostridium botulinum type C isolates from different sources and the role of certain phages in toxigenicity.

Twenty strains of Clostridium botulinum type C, including 12 isolates from avian sources with varying toxigenic properties, were examined by electron microscope for the presence of bacteriophages. All toxigenic strains were infected with one or two types of phages. Three types of phages designated large, small, and intermediate were observed. Most of the strains carried the large and small phage, with the large phage being present in much greater numbers. Since there is evidence that highly toxigenic strains of C. botulinum type C are responsible for large outbreaks of botulism in wild birds, the phenomenon of toxigenic variation among the type C strains was investigated. Experiments were carried out employing a broth medium on a phagefree nontoxigenic strain for elucidating the role of bacteriophages in toxigenicity. All phage suspensions contained large phages, with the exception of one that caused conversion. The exception was a preparation containing an intermediate type of phage. Phages from different strains produced cultures of varying toxigenic characteristics. By employing a tube-lytic test and an agar-overlay-phage assay technique, it was determined that whenever the phage-bacterium relationship resulted in an initial high degree of lysis, the potency of toxin in the culture was weak. It appeared that in highly toxigenic strains, the phage-bacterium relationship is characterized by a stable lysogenic type of association. It was also found that in a highly toxigenic converted culture the percentage of toxigenic cells was 100, whereas in hypotoxigenic culture the percentage was only 20.     

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G.

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.     

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.     

Observations on Nonconverting Phage,c-n71, Obtained from a Nontoxigenic Strain of Clostridium botulinum Type C

A nontoxigenic mutant (C-N71) obtained from a toxigenic strain of Clostridium botulinum type C, Stockholm, with nitrosoguanidine treatment was found to be lysogenic by the lysis test. Although the filtrate of a passaged lysate of this nontoxigenic but lysogenic strain, C-N71, lysed cells of the nontoxigenic strain C-AO2 equally as well as the converting phage c-st obtained from the strain C-Stockholm, it did not convert C-AO2 to the toxigenic state. The lysis spectrum of this filtrate was the same as that of the c-st phage. The ability of the filtrate to lyse the indicator cells, C-AO2, was destroyed neither by trypsin nor DNase but was inactivated by heat treatment at 80 C for 10 min. This suggested that the agent which caused lysis was not boticin but probably a phage. An electron micrograph of the complete phage, c-n71, which was similar in morphology to that of the c-st phage was obtained from the filtrate of strain C-N71. Anti-c-n71 phage rabbit serum neutralized both the lytic and the converting activities of the c-st phage. These findings strongly suggest that the c-n71 phage is a mutant of the c-st phage which lacks the gene controlling production of botulinum type C toxin.     

Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus

The relevance of free radical generation and oxidative stress with regard to aflatoxin production was examined by comparing the oxygen requirement and antioxidant status of a toxigenic strain of Aspergillus parasiticus with that of a nontoxigenic strain at early (trophophase) and late logarithmic (idiophase) growth phases. In comparison to the nontoxigenic strain, wherein the oxygen requirements were relatively unaltered at various growth phases, the toxigenic strain exhibited greater oxygen requirements at trophophase coinciding with onset of aflatoxin production. The activities of antioxidant enzymes such as xanthine oxidase, superoxide dismutase, and glutathione peroxidase and the mycelial contents of thiobarbituric acid-reactive substances as well as of reduced glutathione were all enhanced during the progression of toxigenic strain from trophophase to idiophase. The combined results suggest that aflatoxin production by the toxigenic strain may be a consequence of increased oxidative stress leading to enhanced lipid peroxidation and free radical generation.     

Evidence that the regulation of diphtheria toxin production is directed at the level of transcription.

It has been known for several decades that iron inhibits the production of diphtheria toxin by Corynebacterium diphtheriae by preventing expression at maximal levels. We examined the inhibition kinetics of toxin production after the addition of either iron or rifampin to iron-limited cultures of C7 (betatox+). Iron-mediated inhibition of toxin production was found to be linear within the range of 16 nM to 16 micron. The inhibition kinetics following the addition of iron or rifampin was almost identical. [3H]RNA extracted from iron-limited toxigenic C. diphtheriae was found to hybridize to a greater extent to corynephage beta DNA than either [3H]RNA extracted from toxigenic C. diphtheriae before the onset of toxin production or [3H]RNA extracted from nonlysogenic, nontoxigenic C. diphtheriae.     

The use of polymerase chain reaction in the diagnosis of diphtheria infection

624 Corynebacterium diphtheriae strains, newly isolated from patients and carriers, were studied with the use of the methods of gel immunodiffusion (Elek's test) and polymerase chain reaction (PCR). In the evaluation of 388 C. diptheriae strains, found to be toxigenic in PCR, the results of Elek's test coincided with those of PCR on 98% of cases. In 38 out of 143 strains (26.5%), nontoxigenic according to the results of Elek's test, the presence of the A-fragment of the tox-gene was established. Subculturing in nutrient media made it possible to determine the presence of toxin in 19 out of 38 of these strains; the remaining strains, isolated mainly from carriers, were found to have the "silent" gene. The advantage of using PCR for the detection of toxigenic and nontoxigenic C. diphtheriae strains of different origin was shown.     

Corynebacterium diphtheriae nontoxigenic strain carrying the gene of diphtheria toxin

Among 828 C. diphtheriae nontoxigenic cultures isolated in different region of Russia in 1994-2002, 114 cultures (13.8%) had the gene of diphtheria toxin (gene tox) and were thus called nontoxigenic tox-carrying (NTTC) strains. All NTTC strains were found to belong to biovar mitis and formed neither normal, nor "defective" diphtheria toxin. The most of NTTC strains (94%) belonged to ribotype "Moskva", not occurring among C. diphtheriae toxigenic strains. The incapacity of NNTC strains of forming diphtheria toxin was caused by mutation: the deletion of one nucleotide which led to the shift of the open reading frame and to the formation of the stop codon. The results of these studies are indicative of the fact that a sufficiently homogeneous and isolated group of C. diphtheriae nontoxigenic strains is spread in Russia. These strains carry the nonexpressing gene of diphtheria toxin and are of no epidemic importance in diphtheria infection.     

Characterization of two inducible bacteriophages, alpha 1 and alpha 2, isolated from Clostridium botulinum type A 190L and their deoxyribonucleic acids

Two inducible bacteriophages, alpha 1 and alpha 2, isolated from Clostridium botulinum type A strain 190L and their deoxyribonucleic acids (DNAs) were purified and characterized. Phage alpha 1, which is unable to form plaques on any strain of C. botulinum, was produced in large quantities after treatment with mitomycin C (MC), whereas phage alpha 2, which was induced in much lower quantities than phage alpha 1, propagated in cultures of type A strain Hall. The phage DNAs were exclusively synthesized after induction with MC. Alpha 1 and alpha 2 DNAs had sedimentation coefficients of 34.0 and 30.6 S, corresponding to molecular weights of 31.9 x 10(6) and 23.5 x 10(6), respectively. The buoyant density in CsC1 was 1.682 g/cm3 for alpha 1 DNA and 1.680 g/cm3 for alpha 2 DNA. Based on thermal denaturation characteristics, the genomes of both phages were shown to be double-stranded DNAs. Agarose gel electrophoretic profiles of the phage DNAs digested with restriction endonuclease EcoRI revealed nine fragments for alpha 1 DNA and six fragments for alpha 2 DNA. The molecular weights of the phage DNAs as determined by restriction enzyme analysis were 30.55 x 10(6) for alpha 1 DNA and 25.83 x 10(6) for alpha 2 DNA. Nontoxigenic mutants obtained from strain 190L could, like the toxigenic parent strain, produce the two phages after treatment with MC. Lysogenic conversion to toxigenicity by phage alpha 2 was not observed with the nontoxigenic mutants. It seems likely that there is no relationship between either phage genome and the toxigenicity of C. botulinum type A.     

Serological Studies of Clostridium botulinum Type E and Related Organisms

Clostridium botulinum type E antigens prepared from washed cells by either Formalin treatment or heating at 100 C were used for immunizing rabbits. Agglutination tests showed that high levels of antibody were produced by both types of preparations. Flagellar antigens were highly strain-specific, whereas the somatic antigens were sufficiently similar to produce complete cross-agglutination. One toxigenic strain produced toxigenic and nontoxigenic progeny which were physiologically and antigenically identical in all other respects. Other nontoxigenic strains whose growth, physiological, and morphological characters were identical to type E and strains which had some physiological differences completely cross-agglutinated with type E strains via the somatic antigen. Neither type of antiserum agglutinated other clostridia against which they were tested except for C. acetobutylicum. This reaction seems to be due to a nonspecific anamnestic response and does not appear to be related to the immunizing strains. The nontoxigenic strains studied seem to have no greater antigenic differences from type E strains than the type E strains have from each other.     

Whole genome PCR scanning reveals the syntenic genome structure of toxigenic Vibrio cholerae strains in the O1/O139 population

Vibrio cholerae is commonly found in estuarine water systems. Toxigenic O1 and O139 V. cholerae strains have caused cholera epidemics and pandemics, whereas the nontoxigenic strains within these serogroups only occasionally lead to disease. To understand the differences in the genome and clonality between the toxigenic and nontoxigenic strains of V. cholerae serogroups O1 and O139, we employed a whole genome PCR scanning (WGPScanning) method, an rrn operon-mediated fragment rearrangement analysis and comparative genomic hybridization (CGH) to analyze the genome structure of different strains. WGPScanning in conjunction with CGH revealed that the genomic contents of the toxigenic strains were conservative, except for a few indels located mainly in mobile elements. Minor nucleotide variation in orthologous genes appeared to be the major difference between the toxigenic strains. rrn operon-mediated rearrangements were infrequent in El Tor toxigenic strains tested using I-CeuI digested pulsed-field gel electrophoresis (PFGE) analysis and PCR analysis based on flanking sequence of rrn operons. Using these methods, we found that the genomic structures of toxigenic El Tor and O139 strains were syntenic. The nontoxigenic strains exhibited more extensive sequence variations, but toxin coregulated pilus positive (TCP+) strains had a similar structure. TCP+ nontoxigenic strains could be subdivided into multiple lineages according to the TCP type, suggesting the existence of complex intermediates in the evolution of toxigenic strains. The data indicate that toxigenic O1 El Tor and O139 strains were derived from a single lineage of intermediates from complex clones in the environment. The nontoxigenic strains with non-El Tor type TCP may yet evolve into new epidemic clones after attaining toxigenic attributes.     

Epiedmiologic significance of the distribution of corynephages in different collectives]

Corynephage distribution was studied in the nasopharyngeal washings of 252 persons infected with C. diphtheriae of gravis type, toxigenic (21 patients and 147 carriers) and non-toxigenic ones (84 carriers), and in 468 uninfected persons in collective bodies under different epidemic conditions. Corynephages were isolated from the nasopharyngeal washings only in persons infected with toxigenic C. diphtheriae--in 4 (of 21) patients, and in 21% (of 147) carriers. Phages tox+ (4--6.2%) were revealed only in carriers of toxigenic C. diphtheriae with numerous bacteria in the nasopharynx and in diphtheria patients. Carriers of nontoxigenic diphtheria bacilli can become infected with phage tox+ only together with the toxigenic strains (reinfection). The data obtained indicated that toxigenic and nontoxigenic C. diphtheriae strains were individual variants.     

Bacteriophage and the Toxigenicity of <Emphasis Type="Italic">Clostridium botulinum</Emphasis> Type D

THE data of Inoue and Iida^1,2 strongly suggest that bacteriophage is involved in the toxigenicity of Clostridium botulinum types C and D. They were able to recover toxigenic isolates from nontoxigenic cultures incubated in broth containing filtrates of the toxigenic strains (Stockholm strain of type C and strain 1873 of type D). Lysis was observed in the cultures, but plaques were not demonstrated on solid medium. The change from hontoxigenicity to toxigenicity in C. botulinum type C strain 468C has been shown³ to require the active and continued participation of a specific bacteriophage designated CEβ.