Serological Studies of Clostridium botulinum Type E and Related Organisms II. Serology of Spores

Pure spore antigens for the immunization of rabbits were prepared by enzymic digestion of vegetative components and separation of the cleaned spores in polyethylene glycol. Spore antisera were prepared to strains representative of toxigenic Clostridium botulinum type E; nontoxigenic boticin E-producing variants; nontoxigenic nonproducers of boticin E; nontoxigenic "atypical" strains, which differ somewhat from C. botulinum type E in their physiology; C. botulinum types A and B; and C. bifermentans. They were tested against these and additional strains representative of the above groups, other types of C. botulinum, and other Clostridium species. There was no evidence of agglutination of flagellar or somatic antigens of vegetative cells by these antisera. Agglutination and agglutinin absorption tests showed common antigens among toxigenic type E strains and nontoxigenic variants, both producers and nonproducers of boticin E. Some nontoxigenic "atypical" strains varied in their ability to be agglutinated by type E antisera, and others did not agglutinate at all. Of those atypical strains that were not agglutinated, one was agglutinated by C. bifermentans antiserum. Antisera prepared against C. botulinum types A and B and C. bifermentans did not agglutinate the spores of type E or its variants nor share antigens common to each other. Similarly, antisera to type E, its nontoxigenic variants, and nontoxigenic atypical strains did not agglutinate other C. botulinum types or any other Clostridium species investigated.     

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)     

Production of toxin by Clostridium botulinum type A strains cured by plasmids

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid.     

Production of toxin by Clostridium botulinum type A strains cured by plasmids.

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one 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.     

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 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.     

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.     

Proteolytic mutants obtained from Clostridium botulinum type E.

Proteolytic mutants were isolated from toxigenic strains of Clostridium botulinum type E after several transfers. When these cultures were plated on blood agar, almost all of the colonies obtained were proteolytic, and there were fewer toxigenic colonies than nontoxigenic colonies. The proteolytic mutants and nonproteolytic original strains were different in their biological properties.     

Apparent Involvement of a Plasmid in Phaseotoxin Production by Pseudomonas phaseolicola

Three naturally occurring toxigenic strains (HB-36, G-50, and HB-33), one nontoxigenic strain (HB-20), and one ultraviolet light-induced toxinless mutant (G-50 Tox⁻) of Pseudomonas phaseolicola were examined by dye-buoyant density equilibrium centrifugation for the presence of plasmid deoxyribonucleic acid. All strains contained plasmid deoxyribonucleic acid. Comparison of the plasmid deoxyribonucleic acid of different strains by agarose gel electrophoresis showed that strain G-50 harbored three plasmids, whereas the rest of the strains contained two plasmids each. Irrespective of their toxigenicity, all strains shared the large-sized first plasmid band, but differed with respect to other plasmids. Restriction endonuclease analyses of the plasmids indicated that a 22.50-megadalton plasmid was common to two of the toxigenic strains (HB-36 and G-50). However, strain HB-33, which is also toxigenic, contained a much smaller plasmid (4.23 megadaltons). It is hypothesized that this small plasmid may have arisen by a recombination event from a larger plasmid.     

Immunodiffusion method for detection of type A Clostridium botulinum

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Effect of various sodium taurocholate preparations on the recovery of Clostridium difficile spores

The effect of four sodium taurocholate preparations, which are easily available in Japan, on recovery of Clostridium difficile spores was examined. All preparations, except for one, enabled the recovery of nearly all spores counted microscopically. Moreover, by using 69 toxigenic and 34 nontoxigenic C. difficile strains, the relationship between the recovery of spores in the medium with sodium taurocholate and toxigenicity of C. difficile was analyzed. It was noted that the number of strains with recovery rate of more than 70% was greater in toxigenic strains than in nontoxigenic strains, suggesting a more abundant recovery of toxigenic C. difficile strains in the presence of sodium taurocholate.     

Immunodiffusion method for detection of type A Clostridium botulinum.

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

兰州市婴幼儿艰难梭菌带菌状况

在兰州市随机采集的２１６名健康婴幼儿粪标本经分离并对其培养特性、菌落菌体形态特征、生化反应特性以及毒素原性等进行一系列检查,检出了３７人的粪便含有艰难梭菌（１０４─１０８／ｇ）,检出总阳性率为１７．１％,新生儿、婴儿及幼儿各年龄组的检出阳性率分别为１３．５％（１９／４１）、３３．３％（１３／３９）、１３．９％（５／３６）。３７株分离菌中毒素原性阳性者仅有８株（２１．６％）,均分布于婴儿与幼儿两组。新生儿１４１人中２７人的粪标本为胎粪,只有１人（３．７％）含艰难梭菌（２×１０６／ｇ）,但不产毒素。所有婴幼儿中有２２人因各种原因曾用过抗菌药物,但仅有２人的粪便含艰难梭菌,而且均为非产毒株,表明艰难梭菌检出率与药物服用之间似无显著的相关性。     

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.     

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.     

Rapid extraction of plasmids from Clostridium perfringens

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.     

Rapid extraction of plasmids from Clostridium perfringens.

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.