Fig 1.
Biofilm-formation by E. coli on the surface of the PFs in the silkworms.
(A) Experimental scheme. Saline or E. coli cell suspension (2 x 106–2 x 108 cells/50 μL) was inoculated into PF-inserted silkworms, and biofilms on PFs isolated after 18 h of rearing at 27°C were stained with crystal violet. (B) Microscopic observation. (C) The biofilm mass was determined by measuring absorbance 590 nm (A590). n = 7/group. Statistically significant differences between groups were evaluated using Tukey’s test. * P < 0.05.
Fig 2.
Viable E. coli cells in the biofilm on the surface of the PFs in the silkworms.
Saline or E. coli cell suspension (2 x 106–2 x 108 cells/50 μL) was inoculated into PF-inserted silkworms, and the viable E. coli cells in the biofilms on PFs isolated after 18 h of rearing at 27°C were determined by the CFU method. n = 7/group. Statistically significant differences between groups were evaluated using Dunnett’s test. * P < 0.05.
Fig 3.
Effect of MEPM on viable E. coli cells forming a biofilm on the surface of the PFs in the silkworms.
(A) Experimental scheme. E. coli cell suspension (2 x 108 cells/50 μL) was inoculated into PF-inserted silkworms, and the infected silkworms were incubated at 27°C for 18 h. After incubation, saline or MEPM solution (3.1–1000 μg/50 μL) was administered, and the silkworms were incubated at 27°C for 1 h. (B, C) Viable E. coli cells on the surface of the PFs in the silkworms administered MEPM (3.1–50 μg/50 μL) (B) or MEPM (62.5–1000 μg/50 μL) (C) were measured. n = 6/group. Statistically significant differences between groups were evaluated using Dunnett’s test. * P < 0.05.
Fig 4.
Biofilm formation by E. coli and C. albicans on the surface of the PFs in the silkworms.
(A) Experimental scheme. Saline, E. coli cell suspension (2 x 108 cells/50 μL), C. albicans cell suspension (106 cells/50 μL), or mixed cell suspension (E. coli: 2 x 108 cells and C. albicans: 106 cells/50 μL) was inoculated into PF-inserted silkworms, and biofilms on PFs isolated after 18 h of rearing at 27°C were stained with crystal violet. (B) Microscopic observation. (C) The biofilm mass was determined by measuring absorbance 590 nm (A590). n = 7/group. Statistically significant differences between groups were evaluated using Tukey’s test. * P < 0.05.
Fig 5.
Viable E. coli and C. albicans cells in the dual-species biofilm on the surface of the PFs in the silkworms.
(A, B) E. coli cell suspension (2 x 108 cells/50 μL), C. albicans cell suspension (1 x 106 cells/50 μL), or a mixed cell suspension (E. coli: 2 x 108 cells and C. albicans: 1 x 106 cells/50 μL) were inoculated into PF-inserted silkworms, and the viable E. coli cells (A) and C. albicans cells (B) in the biofilms on PFs isolated after 18 h of rearing at 27°C were determined by the CFU method. n = 7/group. Statistically significant differences between groups were evaluated using Student’s t-test. *P < 0.05.
Fig 6.
MEPM tolerance of E. coli induced by C. albicans in a dual-species biofilm on the surface of the PFs in silkworms.
(A) Experimental scheme. (B) E. coli cell suspension (2 x 108 cells/50 μL) or a mixed cell suspension (E. coli: 2 x 108 cells and C. albicans: 1 x 106 cells/50 μL) was inoculated into PF-inserted silkworms, and the infected silkworms were incubated at 27°C for 18 h. After incubation, saline or MEPM solution (0 or 250 μg/50 μL) was administered, and the silkworms were incubated at 27°C for 1 h. Viable E. coli cells on the surface of the PFs in the silkworms were measured. n = 9/group. Statistically significant differences between groups were evaluated using Tukey’s test. * P < 0.05. (C) E. coli cell suspension (2 x 108 cells/50 μL) or mixed cell suspension (E. coli: 2 x 108 cells and C. albicans: 104–106 cells/50 μL) was inoculated into PF-inserted silkworms, and the infected silkworms were incubated at 27°C for 18 h. After incubation, saline or MEPM solution (250 μg/50 μL) was administered, and the silkworms were incubated at 27°C for 1 h. Viable E. coli cells on the surface of the PFs in the silkworms were measured. n = 9/group. Statistically significant differences between groups were evaluated using Dunnett’s test. *P < 0.05.
Fig 7.
Effect of MCFG on MEPM tolerance of E. coli induced by C. albicans in a dual-species biofilm on the surface of the PFs in the silkworms.
(A) Experimental scheme. (B, C) Mixed cell suspensions (E. coli: 2 x 108 cells and C. albicans: 106 cells/50 μL) were inoculated into PF-inserted silkworms, and the infected silkworms were incubated at 27°C for 18 h. After incubation, MEPM solution (250 μg/50 μL) or a mixed drug solution (MEPM: 250 μg and MCFG: 10 μg/50 μL) were administered, and the silkworms were incubated at 27°C for 1 h. Viable E. coli cells (B) and C. albicans cells (C) on the PF surface in silkworms were measured. n = 9/group. Statistically significant differences between groups were evaluated using Student’s t-test. *P < 0.05.