Multilocus Sequence Typing Subtypes of Poultry Clostridium perfringens Isolates Demonstrate Disease Niche Partitioning

Abstract

Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance.

Identification of Accessory Genome Regions in Poultry Clostridium perfringens Isolates Carrying the netB Plasmid

“…A recent MLST study examining NE and healthy bird C. perfringens isolates found that the majority of those associated with NE belonged to two phylogenetically distinct clonal groups: clonal cluster 4 (CC-4) and sequence type 31 (ST-31) (13). Based on in silico MLST analysis, CP4 and JGS4143 belong to CC-4 and ST-31, respectively (data not shown).…”

“…Previous pulsed-field gel electrophoresis (PFGE) analyses of NE-associated C. perfringens isolates failed to provide evidence for a relationship between subtypes from different flocks (11,12), indicating that there were numerous clonal origins of virulent strains that resulted from horizontal transfer of virulence genes. However, two recent multilocus sequence typing (MLST) studies identified two prevalent clonal groups among NE-associated isolates, thus suggesting that specific core chromosomal genes might be important for NE pathogenesis (13,14).…”

“…Previous pulsed-field gel electrophoresis (PFGE) analyses of NE-associated C. perfringens isolates failed to provide evidence for a relationship between subtypes from different flocks (11,12), indicating that there were numerous clonal origins of virulent strains that resulted from horizontal transfer of virulence genes. However, two recent multilocus sequence typing (MLST) studies identified two prevalent clonal groups among NE-associated isolates, thus suggesting that specific core chromosomal genes might be important for NE pathogenesis (13,14).In this study, we used microarray comparative genomic hybridization (CGH) to examine the genome content of a set of 54 genetically diverse C. perfringens isolates derived from either healthy broiler chickens (intestinal tract or retail chicken meat) or from the intestine of broiler chickens with NE. We have determined the variable gene regions that represent, in part, the accessory genome among these isolates, and we have identified those regions that are significantly associated with the presence of the netB plasmid.…”

“…We have determined the variable gene regions that represent, in part, the accessory genome among these isolates, and we have identified those regions that are significantly associated with the presence of the netB plasmid. The genotypic relatedness of these isolates was also determined based on CGH data, providing insight into the evolutionary basis of the prevalence of certain NE-associated clones (13,14). DNA extraction and Cy labeling.…”

Necrotic enteritis in chickens: an important disease caused by Clostridium perfringens

“…However, recent studies have indicated that the C. perfringens strains that are commonly present in low numbers within the healthy gut are generally distinct from the strains that can go on to cause disease 3,4 . The origin of pathogenic strains has not yet been clarified and the mechanisms driving displacement of non-pathogenic strains by pathogenic strains are not clear but may be partly driven by bacteriocin expression by the pathogenic strains 5 .…”