Fig 1.
India ink staining of K. pneumoniae cells from overnight co-culturing at serial density.
(A). Acanthamoeba cells at a density of 4 × 104, 8 × 104, and 1.6 × 105 were overnight co-cultured with K. pneumoniae adjusted at OD600 value of 0.2. (B). K. pneumoniae cells at OD600 of 0.2, 0.4, 0.8, and 1.6 were overnight co-cultured with Acanthamoeba cells at a density of 5 × 104 for India ink staining. Scale bar = 10 μm.
Fig 2.
Measurement of K. pneumoniae cell size using India ink staining after co-culturing with Acanthamoeba.
(A). India ink staining images of K. pneumoniae at a magnification of 1000× after co-culturing with Acanthamoeba. Bacterial cells were screened after co-culturing with Acanthamoeba at 24, 72, and 168 h. Scale bar = 10 μm. (B). Comparison of average cell size between co-cultured and cultured alone K. pneumoniae at 24, 72, and 168 h. The average cell size was calculated for 50 bacterial cells observed under a magnification of 1000×. Statistical significance was determined by Student’s t-test. ***P < 0.001.
Fig 3.
Images of K. pneumoniae cells passaged two times after culturing alone or co-culturing with Acanthamoeba.
Acanthamoeba co-cultured K. pneumoniae or K. pneumoniae cultured alone was passaged two times in Luria-Bertani broth. The third generation of K. pneumoniae cells were harvested using India ink staining. The images were captured from three independent experiments.
Fig 4.
Phenol-sulfuric acid assay for capsular polysaccharide of K. pneumoniae.
The cells from K. pneumoniae cultured alone or Acanthamoeba co-cultured K. pneumoniae were treated with sulfuric acid. Uronic acid from K. pneumoniae capsular polysaccharide was identified as an orange complex at a wavelength of 520 nm after phenol treatment. Statistical significance was determined by Student’s t-test. ***P < 0.001.
Fig 5.
K. pneumoniae capsular viscosity examination by sedimentation assay and string test.
(A). The cells from K. pneumoniae cultured alone or Acanthamoeba co-cultured K. pneumoniae were incubated overnight in Luria-Bertani broth. The broth was centrifuged at 3000 rpm for 10 min. The absorbance of the supernatants was measured at 600 nm. Statistical significance was determined by Student’s t-test. ***P < 0.001. (B). Comparison of stretched colonies between K. pneumoniae cultured alone and Acanthamoeba co-cultured K. pneumoniae. A positive string and hypermucoviscous phenotype were defined by strings 5 mm in length or longer. The black arrows indicate the top of the string; however, no stretched string was observed for K. pneumoniae cultured alone.
Fig 6.
India ink staining of various serotypes K. pneumoniae environmental isolates treated with Acanthamoeba.
The images of Acanthamoeba co-cultured K. pneumoniae environmental isolates or isolates cultured alone after staining with India ink at a magnification of 1000×. The serotypes of the environmental isolates are as follows: CMU-A belongs to serotype K1, CMU-B belongs to serotype K2, CMU-C belongs to serotype K5, and CMU-D belongs to serotype K20. Scale bar = 10 μm.