Genetic and Biochemical Diversity among Isolates of Paenibacillus alvei Cultured from Australian Honeybee ( Apis mellifera ) Colonies

Abstract

Twenty-five unique CfoI-generated whole-cell DNA profiles were identified in a study of 30 Paenibacillus alvei isolates cultured from honey and diseased larvae collected from honeybee (Apis mellifera) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with DraI, HinfI, CfoI, AluI, FokI, and RsaI. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in the HinfI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using DraI, FokI, and HinfI differentiated P. alvei from the phylogenetically closely related species Paenibacillus macerans and Paenibacillus macquariensis. Alveolysin gene fragments (length, 1,555 bp) were enzymatically amplified from some of the P. alvei isolates (19 of 30 isolates), and RFLP were detected by using the enzymes CfoI, Sau3AI, and RsaI. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%) P. alvei whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28 P. alvei isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and VogesProskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity in P. alvei isolates may be a reflection of adaptation to the special habitats in which they originated.Paenibacillus alvei is a spore-forming bacterium that swarms vigorously on routine culture media. P. alvei, Enterococcus faecalis, Enterococcus faecium, and Achromobacter eurydice are often recovered from diseased larvae obtained from honeybee (Apis mellifera) colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB) (3). In Australia, P. alvei is the third-most common bacterium detected in honeybee colonies, and E. faecalis, E. faecium, and A. eurydice are rarely recovered from EFB-affected colonies (17, 18). P. alvei can produce signs in larvae that are similar to the signs produced by Paenibacillus larvae subsp. larvae, which causes American foulbrood, the other major bacterial disease of honeybees.Several studies based on comparative analyses of 16S rRNA gene sequences of different Bacillus species revealed five phylogenetically distinct clusters (groups 1 through 5), which confirmed that this genus is genetically heterogeneous and in need of extensive taxonomic revision (1, 27). Furthermore, the authors sug...

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