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 Voges-Proskauer 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.

FIG. 1

Dendrogram showing the levels of similarity between P. alvei isolates based on their API 50CHB biochemical reactions.

FIG. 2

Restriction endonuclease profiles of amplified regions of the 16S rRNA and alveolysin genes of P. alvei. 16S rRNA gene fragments (length, 1,416 bp) were separately amplified by PCR and digested with CfoI, RsaI, FokI, HinfI, DraI, and AluI. Representative profiles generated by using CfoI (lane 2), RsaI (lane 3), FokI (lane 4), HinfI (lanes 5 and 6), DraI (lane 7), and AluI (lane 8) are shown. The profiles were generated after digestion of the 16S rRNA gene fragment derived from isolate 17 (except in lane 6, which shows the HinfI profile of the 16S rRNA gene fragment derived from isolate 18). CfoI profiles of the alveolysin gene fragment derived from P. alvei isolates 23, 14, and 19 are shown in lanes 9 through 11, respectively. RsaI profiles of the alveolysin gene fragment derived from P. alvei isolates 30, 29, and 17 are shown in lanes 12 through 14, respectively. The molecular size markers used included a 100-bp ladder (lanes 1 and 15) and lambda DNA digested with HindIII (lane 16). Restriction fragments were separated by using 1% (wt/vol) agarose and were stained with ethidium bromide.

FIG. 3

Silver-stained 3.5% polyacrylamide gel of CfoI-generated REFPs of geographically diverse Australian isolates of P. alvei. The gel was electrophoresed for 15 h and shows DNA fragments between 0.45 and 8.5 kb long. The lanes show the DNA profiles for New South Wales isolate 5, Tasmania isolates 9 and 12, South Australia isolates 25 and 27, Victoria isolates 16 through 18, South Australia isolates 29 and 28, Tasmania isolate 15, Victoria isolates 19 and 20, and Queensland isolate 21; isolate numbers are indicated at the top. Lane M contained molecular size markers (lambda DNA digested with HindIII).

FIG. 4

CfoI-generated REFPs of P. alvei DNA recovered from geographically diverse Australian isolates. Gels (3.5% polyacrylamide) were electrophoresed for 21 h to separate large molecular fragments and were stained with silver. (A) REFPs for isolates 30 and 31 (origin unknown), New South Wales isolates 4 and 6, and Victoria isolates 19, 17, 18, and 20. Isolate numbers are indicated at the top. The positions of molecular size markers (lambda DNA digested with HindIII) are indicated on the left. (B) REFPs for Queensland isolates 22 and 21, Tasmania isolates 12 and 13, South Australia isolates 29 and 24, New South Wales isolate 2, Tasmania isolate 15, and South Australia isolate 28. Isolate numbers are indicated at the top.

FIG. 5

CfoI profiles of P. alvei DNA isolated from eastern and southern states of Australia. The lanes show the profiles for Victoria isolates 18 and 20, South Australia isolates 25 and 27, New South Wales isolates 3 and 5, Tasmania isolate 15, and South Australia isolates 28 and 23. Isolate numbers are indicated at the top. Lane M contained molecular size markers (phage SPPI digested with EcoRI).

FIG. 6

CfoI profiles of a subset of genetically related P. alvei isolates. The lanes show the profiles for New South Wales isolate 14, Tasmania isolate 11, New South Wales isolates 1, 8, and 7, South Australia isolates 24, 28, 27, and 26, Victoria isolate 16, Tasmania isolate 9, and New South Wales isolate 5. Isolate numbers are indicated at the top. Lane M contained a 1-kb ladder (Bio-Rad).

FIG. 7

Whole-cell DNA profiles of P. alvei isolates on 1.0% (wt/vol) agarose gels. The lane designations correspond to isolate numbers. Extrachromosomal elements (arrowheads) were observed in the whole-cell DNA profiles of 17 isolates. Lane M contained a 1-kb ladder.