Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes

Highlights

• Genotypic relationships of Clostridium perfringens were evaluated by MLST.

• netB gene was found in human's strains, and tpeL gene in humans and chicken strains.

• Some C. perfringens from humans act as reservoirs of virulence genes.

• Human and animal strains were genetically distinct and human strains were more diverse.

Abstract

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains.

Introduction

Clostridium perfringens is an anaerobic, endospore-forming, gram-positive rod ubiquitous in various environments, especially in soils and the gastrointestinal tract of healthy humans and animals [1]. This species produces at least 17 different toxin and according to the production of major toxins alpha, beta, epsilon and iota it has been classified into five toxinotypes (A, B, C, D and E) [2]. C. perfringens type A causes gangrene and food poisoning in humans, diarrhea in foals and pigs, and necrotic enteritis (NE) in chickens [3], [4].

The α-toxin produced by C. perfringens type A is phospholipase C which hydrolyzes phospholipids causing the release of inflammatory mediators and acute cell death [5]. Strains of C. perfringens produce low, intermediate or high levels of α-toxin which depends on the growth conditions, such as the medium type and pH [6].

Large clostridial cytotoxins produced by Clostridium spp. are important virulence factors in pathogenesis of myonecrosis and intestinal diseases. They include Clostridium difficile toxins A (TcdA) and B (TcdB), C. sordellii lethal toxin (TcsL) and hemorrhagic toxin (TcsH), C. novyi alpha toxin (TcnA), and more recently discovered C. perfringens large cytotoxin (TpeL) [7], [8].

The TpeL toxin was initially detected in culture supernatants of C. perfringens type C isolated from swine. It is lethal to mice and cytotoxic to Vero cells, resulting in enlarge, rounded cells forming aggregates, which eventually detach from the plate [8], [9]. This toxin has been detected in C. perfringens Type A isolated from avian hosts; and it may potentiate or contribute to the pathogenesis of NE [2].

The necrotic enteritis toxin B like (NetB) of C. perfringens type A was first discovered in a chicken with NE from Australia [10]. These toxins form pores, which disrupt the phospholipid membrane bilayer of both human and animal cells, causing ion influx that may lead to osmotic cell lyses [11], [12]. The TpeL and NetB toxins have not yet been reported in humans.

Certain molecular typing techniques are used to verify bacterial diversity of C. perfringens. The methods include pulsed-field gel electrophoresis [13], multilocus variable number of tandem repeats analysis [14], and multilocus sequence typing (MLST) [15]. Such phylogenetic studies have elucidated distinct lineages of C. perfringens strains associated with specific diseases [16], [17].

MLST is an excellent method for bacterial characterization based on sequencing of several housekeeping genes [18]. Genes are amplified by polymerase chain reaction [PCR] and sequenced, and the differences in DNA sequences define the allelic profile for each isolate, known as the sequence type (ST). Genetic events such as point mutations or recombinations result in different alleles and subsequently, in different allele combinations [19]. Most bacterial species have sufficient variation within housekeeping genes to provide many alleles per locus, allowing multiple STs to be identified using a limited number of loci [20]. This global approach would be of great value for the study of the diseases-causing C. perfringens.

In this study, the phylogenetic relationships and niche partitioning were verified in a diverse collection of C. perfringens isolated from humans and broiler chickens using the MLST protocol described by Jost et al. [15].