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J Med Microbiol 57 (2008), 225-231; DOI: 10.1099/jmm.0.47598-0
© 2008 Society for General Microbiology
ISSN 1473-5644

Human Fusobacterium necrophorum strains have a leukotoxin gene and exhibit leukotoxic activity

Sambasivarao Tadepalli1,{dagger}, George C. Stewart2, T. G. Nagaraja1 and Sanjeev K. Narayanan1

1 Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506-5606, USA

2 Department of Veterinary Pathobiology and Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA

Correspondence
Sanjeev K. Narayanan
sanjeev{at}vet.k-state.edu

Received 29 August 2007
Accepted 19 October 2007

Fusobacterium necrophorum, a Gram-negative anaerobe, causes a variety of necrotic infections in humans and animals. There are two subspecies: subsp. necrophorum and subsp. funduliforme. In cattle, subsp. necrophorum is more prevalent and production of leukotoxin is a major virulence factor. The leukotoxin operon (lktBAC) consists of three genes, lktB, lktA and lktC, of which lktA is the structural toxin gene. The subspecies identity of human F. necrophorum is less certain and it is not known whether human strains possess the leukotoxin gene or leukotoxin activity. Therefore, the objective of this study was to identify the subspecies status of four human clinical strains of F. necrophorum and determine whether they have the leukotoxin gene or leukotoxin activity. Phenotypic and genotypic characteristics suggested that the four strains belonged to subsp. funduliforme, which was confirmed by sequencing the 16S rRNA gene. Analysis of the four strains by PCR revealed the presence of the leukotoxin operon. Partial DNA sequencing identified one human strain with full-length lktA, whereas the others exhibited considerable heterogeneity in size. All strains had a leukotoxin operon promoter-containing intergenic region similar to that of bovine subsp. funduliforme strains, which was confirmed by DNA sequencing and Southern blotting. Despite variations in the lktA gene, all strains secreted leukotoxin as demonstrated by Western blotting. Flow cytometry assays revealed that the leukotoxin was toxic to human white blood cells. In conclusion, the human strains examined contained a leukotoxin gene whose gene product was biologically active. The importance of leukotoxin as a virulence factor in human fusobacterial infections needs further evaluation.

Abbreviations: PMN, polymorphonuclear leukocyte; PRAS, pre-reduced anaerobically sterilized.

{dagger}Present address: Novartis Animal Health US, Inc., Larchwood, IA 51241, USA.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the human Fusobacterium necrophorum strains RMA10682 and RMA16505 reported in this paper are EF447426 and EF447427, respectively.






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