Abstract
Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P) and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human pathogen.
Highlights
Clostridium butyricum is a butyric acid-producing anaerobic and spore-forming bacterium widely distributed in the environment and commonly present in the gut microflora of healthy humans and animals
Neurotoxigenic C. butyricum type E has been implicated in further cases of infant botulism, as well as in several episodes of intestinal toxaemia in adults and of foodborne botulism [11,12,13,14,15,16,17]
This result was apparently in contrast with the result previously obtained by Wang et al [29], who detected by Pulsed-field gel electrophoresis (PFGE) a single DNA chromosomal band in several neurotoxigenic C. butyricum type E strains, some of which were analyzed in the present study (Table 1)
Summary
Clostridium butyricum is a butyric acid-producing anaerobic and spore-forming bacterium widely distributed in the environment and commonly present in the gut microflora of healthy humans and animals. It is generally considered a harmless saprophyte and it may be used for bioproduction of butyric acid, a widely applied food preservation and flavour-enhancing additive [1]. Neurotoxigenic C. butyricum type E strains were first isolated in Italy in 1984 from two distinct cases of infant botulism [6], an intestinal toxaemia by botulinum toxin-producing clostridia that affects babies under 1 year of age. Neurotoxigenic C. butyricum type E has been rarely isolated up to now, it seems to be spread throughout the world, and in Italy it has PLoS ONE | www.plosone.org ß-Lactamase-Encoding Linear Megaplasmids been implicated in botulism cases more frequently than C. botulinum type E [19]
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