Biogeography of Paenibacillus larvae, the causative agent of American foulbrood, using a new multilocus sequence typing scheme

Abstract

American foulbrood is the most destructive brood disease of honeybees (Apis mellifera) globally. The absence of a repeatable, universal typing scheme for the causative bacterium Paenibacillus larvae has restricted our understanding of disease epidemiology. We have created the first multilocus sequence typing scheme (MLST) for P. larvae, which largely confirms the previous enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR)-based typing scheme's divisions while providing added resolution and improved repeatability. We have used the new scheme to determine the distribution and biogeography of 294 samples of P. larvae from across six continents. We found that of the two most epidemiologically important ERIC types, ERIC I was more diverse than ERIC II. Analysis of the fixation index (FST ) by distance suggested a significant relationship between genetic and geographic distance, suggesting that population structure exists in populations of P. larvae. Interestingly, this effect was only observed within the native range of the host and was absent in areas where international trade has moved honeybees and their disease. Correspondence analysis demonstrated similar sequence type (ST) distributions between native and non-native countries and that ERIC I and II STs mainly have differing distributions. The new typing scheme facilitates epidemiological study of this costly disease of a key pollinator.

Fig 1

eburst diagram of P. larvae MLST scheme. The numbers in the circles represent the ST of the isolates (Table 2). The size of the circle represents the number of isolates with that type (Table 2). STs with variation at more than three loci are not connected. Clockwise from left, groups are: isolates typed as ERIC I, isolates typed as ERIC II and isolates typed as ERIC III and IV.

Fig 2

Neighbour-joining tree of all sequence types. The evolutionary history was inferred using the neighbour-joining method. The optimal tree with the sum of branch length = 0.01386545 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.

Fig 3

Chaol estimates of ERIC I and ERIC II ST richness as a function of sample size. Dotted lines are 95% CIs and were calculated with the variance formula derived by Chao (1987). The lower solid line represents ERIC II and the upper solid line represents ERIC I.

Fig 4

F_ST by distance.A. Describes the F_ST by distance of P. larvae populations within the native range of the host (Apis mellifera).B. Describes the F_ST by distance of P. larvae populations both within (dark grey dots) and outside of the native range of the host (light grey dots).

Fig 5

Correspondence analysis ordination graph. The CA ordination graph illustrates the associations among countries of isolate origins and MLST STs. Filled circles represent countries (dark grey represents native host range, light grey represents introduced host range) Diamonds represent STs.