Mammalian serum stimulates <i>S. aureus</i> colony-spreading activity.

<p>(A) Stimulation of colony-spreading activity by calf serum. Overnight culture of <i>S. aureus</i> MRSA NI-15 was spotted on soft agar supplemented with serially diluted calf serum and incubated for 8 h at 37°C. Each plate contained 20 ml soft agar medium. (B) Stimulation of colony-spreading by porcine serum. Porcine serum or calf serum was serially diluted 2-fold and applied to 20 ml soft agar medium and its colony-spreading stimulatory activity was measured. Open circles indicate the halo diameters of giant colonies supplemented with porcine serum and filled circles indicate those supplemented with calf serum. Horizontal axis represents the volume of serum added to 20 ml soft agar medium in a plate. (C) Stimulation of colony-spreading activity by silkworm hemolymph. Hemolymph was collected from fifth instar larvae of silkworms and applied to the soft agar plates in 2-fold serial dilutions and its colony-spreading stimulatory activity was measured. Open circles indicate the halo diameters of giant colonies supplemented with silkworm hemolymph and filled circles indicate those supplemented with calf serum. (D) Growth curves of MRSA NI-15, MW2, or FRP3757 in tryptic soy broth supplemented with or without 1.25% (v/v) calf serum.</p

Stimulation of colony-spreading by bovine serum albumin.

<p>(A) Colony-spreading stimulation by purified bovine serum albumin. Purified bovine serum albumin of 96% grade (open triangles, Nacalai, cat. no. 01861-26) or 99% grade (open circles, Sigma, cat. no. A0281) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities against MRSA NI-15 strain were measured. (B) Elution profile of DEAE-cellulose column chromatography using bovine serum albumin (96% grade, Nacalai). Open circles indicate the colony-spreading stimulatory activity. Filled circles indicate absorbance at 280 nm. (C) SDS-PAGE analysis of DEAE-cellulose column chromatography fractions. The gel was stained with Coomassie Brilliant Blue. The 66-kDa protein coincided with colony-spreading stimulatory activity in fractions 10–20. (D) Bovine serum albumin (Nacalai), fatty acid-free albumin from bovine serum (Wako Chemicals, cat. no. 017-15146), casein from bovine milk (Sigma, cat. no. C4032), or fetuin from calf serum (Sigma, cat. no. F2379) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured.</p

Gel filtration column chromatography of calf serum.

<p>(A) Elution profile of gel filtration column chromatography using a Superdex 200. Open circles indicate diameters of colonies. Filled circles indicate absorbance at 280 nm. Molecular weight markers were eluted in the fraction described below. Catalase (250-kDa) was eluted in fraction 21, bovine serum albumin (66-kDa) in fractions 25–26, and cyanocobalamin (1.3-kDa) in fraction 38. 250-µl aliquots of each sample were applied to soft agar medium and their colony-spreading stimulatory activity was measured. (B) SDS-PAGE analysis of gel filtration fractions. The gel was stained with Coomassie Brilliant Blue. A 66-kDa protein coincided with the colony-spreading stimulatory activity in fractions 24–29.</p

Therapeutic effects of antibiotics against bacterial infection in honeybees.

<p>A, Effect of co-injection of vancomycin or gentamycin on survival of in-hive worker bees infected with <i>S. aureus</i> (n = 20). B, Dose-dependency of therapeutic effects of antibiotics on <i>S. aureus</i> infection in in-hive worker bees. Survival numbers were determined 24 h after infection (n = 5).</p

Comparison of susceptibilities to bacterial infection between honeybees in different labor divisions.

<p>LD₅₀ values of <i>S. aureus</i> were determined 24 h after infection in either in-hive workers, out-hive workers, or drones (n = 5–8). Data represent mean ± SDs of three experiments. Statistical analysis was performed by one-way ANOVA, and differences compared with the in-hive workers group were analyzed by Dunnett’s multiple comparison tests (*, p<0.05).</p

Establishment of a bacterial infection model in the honeybee.

<p>A, Images of sample injection into the body cavities of bees (left) and oral administration (right). In the left panel, bees anesthetized on ice were injected into the abdomen with 25 µl of each sample using a 1-ml syringe. In the right panel, anesthetized bees were placed in 1.5-ml tubes and fixed with paper tape. Ten microliters of sample containing sucrose was directly administered to bees extending the proboscis. B–C, Survival of in-hive worker bees (n = 20) after infection with Gram-negative (left) and Gram-positive (right) bacteria. Overnight culture of each bacterium was diluted 100-fold with saline. Survival numbers of bees were monitored at 25°C (B) or 37°C (C). The data shown are representative of three experiments with similar results. D, Effect of incubation temperature on survival of in-hive worker bees after <i>S. aureus</i> infection. LD₅₀ values were determined 24 h after injection with a live bacterial suspension of <i>S. aureus</i> (n = 5).</p

ED₅₀ values of antibiotics in the honeybee <i>S. aureus</i> infection model.

<p>ED₅₀ values of tetracycline, vancomycin, and kanamycin against <i>S. aureus</i> infection in in-hive worker bees (n = 5) were determined 24 h after infection. Antibiotics were injected into the cavities (i.c.) or orally administered (p.o.), and live bacterial suspension of <i>S. aureus</i> was injected immediately after antibiotic treatment.</p>a<p>ED₅₀ values of <i>B. mori</i> were previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089917#pone.0089917-Hamamoto1" target="_blank">[16]</a>.</p>b<p>µg/insect.</p

Purification of colony-spreading stimulatory factor in calf serum.

<p>Calf serum (19 ml) was used as the starting medium. To measure the colony-spreading stimulatory activity, samples were serially diluted 2-fold and applied to 20 ml of soft agar and incubated for 8 h at 37°C. The diameters of giant colonies were measured from each dish, and stimulatory activities were calculated.</p

Diverse colony-spreading response to calf serum among HA-MRSA and CA-MRSA strains.

<p>(A) Representative images of the colony-spreading activity of MRSA NI strains stimulated by calf serum. Overnight cultures of MRSA NI strains were spotted onto soft agar plates supplemented with or without calf serum (250 µl/plate) and incubated for 8 h. (B) Colony-spreading response of CA-MRSA strains to calf serum. Overnight cultures of MW2 (USA400, open circles) or FRP3757 (USA300, filled circles) were spotted onto soft agar plates supplemented with 2-fold serially diluted calf serum and incubated for 8 h. The halo diameter was measured. (C) Amount of PSMα3 in MRSA strains cultured in the presence or absence of calf serum. MRSA strains with high colony-spreading response against calf serum (NI-15, MW2, FRP3757, NI-5, NI-7, NI-27, NI-29, NI-36, and NI-38) were cultured in the presence or absence of 1.25% (v/v) calf serum and the culture supernatants were analyzed by HPLC as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097670#pone.0097670-Kaito4" target="_blank">[6]</a>. Asterisks means not detected.</p

Lipoprotein and albumin-depleted serum decreases colony-spreading stimulatory activity.

<p>(A) Stimulatory activities of calf serum, lipoprotein-depleted serum, lipoprotein- and albumin-depleted serum were examined. Fractionation was performed from a 50-ml volume of calf serum containing 3850 units of stimulatory activity. Each fraction was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. Relative total activities of each fraction compared with that of calf serum are presented in graph. (B) SDS-PAGE analysis of the lipoprotein-depleted serum and Affi-Gel blue gel column effluent. 10 µg protein of the lipoprotein-depleted serum and the Affi-Gel blue gel column effluent was electrophoresed in 12.5% SDS-polyacrylamide gel.</p