Identification of Novel Linear Megaplasmids Carrying a ß-Lactamase Gene in Neurotoxigenic Clostridium butyricum Type E Strains

Aureli P (2011) Identification of Novel Linear Megaplasmids Carrying a -Lactamase Gene in Neurotoxigenic Clostridium butyricum Type E Strains. PLoS ONE 6(6): e21706. doi:10.1371/journal.pone.0021706 Identification of Novel Linear Megaplasmids Carrying a -Lactamase Gene in Neurotoxigenic Clostridium butyricum Type E Strains Giovanna Franciosa 0 Concetta Scalfaro 0 Paola Di Bonito 0 Marco Vitale 0 Paolo Aureli 0 Holger Bruggemann, Max Planck Institute for Infection Biology, Germany 0 1 Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanita` , Rome , Italy , 2 Department of Infectious, Parasytic and Immunomediated Diseases, Istituto Superiore di Sanita` , Rome , Italy 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 -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 -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. - Funding: Funding was provided by the Istituto Superiore di Sanita`, Rome, Italy (www.iss.it). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. In addition, in certain countries, C. butyricum strains with proven absence of toxicological effects are allowed for use as probiotics in humans and animals, based on the beneficial effects that butyric acid and the bacteriocin(s) produced by those strains are believed to exert on the host intestine [2,3]. On the other hand, an in vitro cytotoxic effect of butyric acid produced by C. butyricum has been demonstrated in various cells. This effect has been hypothesized to be responsible for the initiation of the intestinal lesions that lead to the mucosal necrosis of the ileum and colon observed in neonatal necrotizing enterocolitis, although conclusive evidence is still lacking [4,5]. Most notably, some strains of C. butyricum have been described that are capable of synthesizing the type E botulinum neurotoxin (BoNT/E), which is one of the seven (A to G) antigenically distinct protein toxins that cause the flaccid paralysis of botulism. 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. Except for the ability to produce botulinum toxin, which is conventionally considered a distinctive characteristic of members of the C. botulinum species, these particular strains had all the phenotypic characteristics of the C. butyricum species [7]: their taxonomic identity was later genetically confirmed by DNA hybridization experiments and 16S rRNA gene sequencing [810]. Since then, 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 (namely the classic intoxication caused by the ingestion of preformed BoNT in improperly preserved foods) [11 17]. Moreover, C. butyricum type E has been isolated from numerous lake sediments in China [18], where an outbreak of foodborne botulism had previously occurred [11]. Although 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 been implicated in botulism cases more frequently than C. botulinum type E [19]. It is assumed that neurotoxigenic C. butyricum type E strains acquired the bont/E gene from a progenitor strain through mobile genetic elements [20]. Recently, the bont/A,/B and/F genes of certain C. botulinum strains were localized within circular plasmids [2024], whereas the bont/G gene of C. argentinense has long been known to be plasmid-encoded [25], although whether the bont/Gencoding plasmid has a circular or linear structure remains unknown. Remarkably, some of the bont-encoding plasmids have been shown to be conjugative [26]. Previous studies have investigated the bont/E gene chromosomal or plasmid location in the genomes of neurotoxigenic C. butyricum type E. Although earlier experiments yielded discordant results, more recent studies indicated a chromosomal location for the bont/ E gene in the neurotoxigenic C. butyricum type E isolates that had been analyzed [20,2729]. Here, we present evidence for the existence of previously unrecognized extremely large linear plasmids in ten neurotoxigenic C. butyricum type E strains isolated from clinical, food, and environmental sources in two widely separated geographical areas, Italy and China (Table 1). Given these unexpected findings, and because the genes encoding other BoNT types have been demonstrated to be chromosomally or plasmid located, depending on the clostridia strains being examined (...truncated)