Virulence Differences among Melissococcus plutonius Strains with Different Genetic Backgrounds in Apis mellifera Larvae under an Improved Experimental Condition

European foulbrood (EFB) caused by Melissococcus plutonius is an important bacterial disease of honeybee larvae. M. plutonius strains can be grouped into three genetically distinct groups (CC3, CC12 and CC13). Because EFB could not be reproduced in artificially reared honeybee larvae by fastidious strains of CC3 and CC13 previously, we investigated a method to improve experimental conditions using a CC3 strain and found that infection with a potassium-rich diet enhanced proliferation of the fastidious strain in larvae at the early stage of infection, leading to the appearance of clear clinical symptoms. Further comparison of M. plutonius virulence under the conditions revealed that the representative strain of CC12 was extremely virulent and killed all tested bees before pupation, whereas the CC3 strain was less virulent than the CC12 strain, and a part of the infected larvae pupated. In contrast, the tested CC13 strain was avirulent, and as with the non-infected control group, most of the infected brood became adult bees, suggesting differences in the insect-level virulence among M. plutonius strains with different genetic backgrounds. These strains and the improved experimental infection method to evaluate their virulence will be useful tools for further elucidation of the pathogenic mechanisms of EFB.


Supplementary Methods
Survival of M. plutonius in water and saline. In this study, for preparation of inocula for experimental infections, M. plutonius was suspended in sterile water or saline at room temperature and then mixed with an artificial diet within 10 min. In order to know the influence of the suspension media (in particular, hypoosmotic shock in water) on the M. plutonius survival, we investigated the survival of DAT606(CC3) in water and saline. DAT606(CC3) cultured on KSBHI agar [Brain Heart Infusion (BHI) agar with 1% soluble starch and 150 mM KH 2 PO 4 ] plates at 37ºC for five days under anaerobic conditions was suspended in KSBHI broth (BHI broth with 1% soluble starch and 150 mM KH 2 PO 4 ) (optical density at 600 nm of 1.0). Bacterial cells in 500 µl of the suspension were collected by centrifugation (15,000 rpm, 5 min), and the supernatant was removed completely. Collected bacterial cells were then suspended with 500 µl of sterile water or saline in microtubes.
Immediately after the suspension and after incubation for 10, 30 and 60 min at room temperature (25ºC), 10 µl of the suspension was collected from each tube, and serial dilutions of the suspensions were made with KSBHI broth. Bacterial survival in the suspension was investigated by plating the serial dilutions onto KSBHI agar plates and counting colonies on the plates after incubation of the plates at 35-37ºC for four days under anaerobic conditions. Data were collected from five independent suspensions. The differences in the concentration of viable M. plutonius were analysed by repeated ANOVA.

Growth of M. plutonius in liquid culture media with known concentrations of Na and K.
In order to investigate the influence of the Na:K ratio in liquid culture media on the growth of M. plutonius, we cultured DAT606(CC3) in kSBHI broth (BHI broth with 1% soluble starch and 13.5 mM KH 2 PO 4 ) and KSBHI broth (BHI broth with 1% soluble starch and 150 mM KH 2 PO 4 ). Na and K concentrations in BHI broth determined by flame atomic absorption spectrometry using Shimadzu Atomic Absorption Spectrophotometer AA-6800 (the combustion gases: air and acetylene, the wavelengths: Na 589.0 nm and K 766.5 nm) in Prophoenix Division, Food and Life-Science Laboratory, Idea Consultants, Inc. (Osaka, Japan) were 2,700 mg/L and 860 mg/L, respectively. Therefore, Na and K concentrations in kSBHI broth were considered to be 117.44 mM and 35.50 mM, respectively, and the Na:K ratio (Na/K = 3.31, Na > K condition) was comparable to those of the Day 0 diet for experimental condition nos. 1, 2, 3 and 5. On the other hand, Na and K concentrations in KSBHI broth were considered to be 117.44 mM and 172.00 mM, respectively (Na/K = 0.68, K > Na condition).
DAT606(CC3) cultured on KSBHI agar plates at 37ºC for five days under anaerobic conditions was suspended in KSBHI broth (optical density at 600 nm of 0.8). Four ml of kSBHI or KSBHI broth was inoculated with 40 µl of the suspension. Immediately after the inoculation (0 h) and after incubation for 24 h at 34ºC ± 0.5ºC under anaerobic conditions, 200 µl of the culture was collected from each tube, and serial dilutions of the samples were made with KSBHI broth.
Bacterial growth in the culture media was investigated by plating the serial dilutions onto KSBHI agar plates and counting colonies on the plates after incubation of the plates at 37ºC for four days under anaerobic conditions. Data were collected from five independent cultures, and the results were expressed as fold-increase relative to the concentration of viable bacteria at 0 h (mean ± SEM). The difference in the increase of DAT606(CC3) concentration between the two culture media was compared by two-tailed Welch's t-test.  Table 3.
Supplementary Figure S1. Survival of M. plutonius DAT606(CC3) in water (red) and saline (blue). DAT606(CC3) was suspended in sterile water or saline and incubated for 10, 30 and 60 min at room temperature (25ºC). The survival of bacteria in the suspensions was investigated as described in Supplementary Methods. Data were collected from five independent suspensions and expressed as CFU/ml (mean ± SEM). There was no significant difference in the concentration of viable bacteria by the sampling period of 0, 10, 30 and 60 min (repeated ANOVA, P=0.17 and 0.47 for water and saline, respectively).  Figure S2. Growth of M. plutonius DAT606(CC3) in Na-rich and K-rich liquid media. DAT606(CC3) was cultured in kSBHI broth (BHI broth with 1% soluble starch and 13.5 mM KH 2 PO 4 , Na/K = 3.31) and KSBHI broth (BHI broth with 1% soluble starch and 150 mM KH 2 PO 4 , Na/K = 0.68) for 24 h at 34ºC ± 0.5ºC under anaerobic conditions. The growth of bacteria was investigated as described in Supplementary Methods. Data were collected from five independent cultures, and the results were expressed as fold-increase relative to the concentration of viable bacteria at 0 h (mean ± SEM). The difference in the increase of DAT606(CC3) concentration between the two culture media was compared by the two-tailed Welch's t-test.  Threshold cycle Threshold cycle 10 3 10 4 10 5 10 6 10 7 10 8 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 2