Genetic characterization of gut-associated Clostridium perfringens isolates from Austrian animals using a combination of established and novel methods

Abstract

Clostridium perfringens ( C. perfringens ) is a pathobiont of the intestinal microbiota that can cause severe systemic and enteric diseases. C. perfringens colonizes a variety of hosts and environmental niches, which is the theorized reason for its large genome, low guanine-cytosine content, and small stable core genome. Over 20 putative virulence factors have been described, but only six are included in the used typing scheme. Epidemiological analysis is limited, and to the authors’ knowledge, this is the first study for Austria. Furthermore, C. perfringens frequently harbors different and/or multiple genes associated with antibiotic resistance (AMR genes) targeting various antibiotic classes. A novel DNA microarray – targeting 15 C. perfringens -associated genes – was evaluated against a published multiplex PCR ( n  = 143) and in silico analysis of Whole Genome Sequences (WGS) ( n  = 20). Multi Locus Sequence Typing (MLST) was performed ( n  = 124), the results were analyzed using the goeBURST algorithm, and a maximum likelihood tree was constructed. Genomes of this study and publicly available ( n  = 44) were analyzed in silico for AMR genes. The DNA microarray detected all target genes except becA and becB , which were absent across all tested strains and methods. The most frequent virulence factors were cpa ( n  = 143), tpi ( n  = 132), and cpb2 ( n  = 104). MLST revealed 77 Sequence Types (STs) – including 42 novel STs – clustered into six Clonal Complexes (CCs). CC39 was the most abundant and exclusively associated with pork/swine. At least one AMR gene was present in 58.7% of genomes and multiple AMR genes in 34.8%. AMR genes associated with resistance to tetracycline, aminoglycosides, pleuromutilin, lincosamides, macrolides, and streptogramins but non to beta-lactams were detected. Notably, one isolate carried lsa (E), which was not previously described for C. perfringens . The DNA microarray showed detection rates comparable to those of multiplex PCR and WGS analysis while being less labor-intensive and more cost-effective. This is the first epidemiological study on C. perfringens for Austria. We described 42 novel STs and six CCs of which CC39 appears to be porcine-associated. The lsa (E) gene was detected in a C. perfringens isolate.