In vitro characterization of biofilms formed by Kingella kingae

Abstract

The Gram-negative bacterium Kingella kingae is part of the normal oropharyngeal mucosal flora of children <4 years old. K. kingae can enter the submucosa and cause infections of the skeletal system in children, including septic arthritis and osteomyelitis. The organism is also associated with infective endocarditis in children and adults. Although biofilm formation has been coupled with pharyngeal colonization, osteoarticular infections, and infective endocarditis, no studies have investigated biofilm formation in K. kingae. In this study we measured biofilm formation by 79 K. kingae clinical isolates using a 96-well microtiter plate crystal violet binding assay. We found that 37 of 79 strains (47%) formed biofilms. All strains that formed biofilms produced corroding colonies on agar. Biofilm formation was inhibited by proteinase K and DNase I. DNase I also caused the detachment of pre-formed K. kingae biofilm colonies. A mutant strain carrying a deletion of the pilus gene cluster pilA1pilA2fimB did not produce corroding colonies on agar, autoaggregate in broth, or form biofilms. Biofilm forming strains have higher levels of pilA1 expression. The extracellular components of biofilms contained 490 μg cm^-2 of protein, 0.68 μg cm^-2 of DNA, and 0.4 μg cm^-2 of total carbohydrates. We concluded that biofilm formation is common among K. kingae clinical isolates, and that biofilm formation is dependent on the production of proteinaceous pili and extracellular DNA. Biofilm development may have relevance to the colonization, transmission, and pathogenesis of this bacterium. Extracellular DNA production by K. kingae may facilitate horizontal gene transfer within the oral microbial community.

Keywords: Kingella kingae; biofilms; oral microbiology.

Fig. 1

Biofilm formation by 58 corroding and 21 noncorroding K. kingae strains in 96-well microtiter plates. Biofilm was quantitated using a crystal violet binding assay. Absorbance at 595 nm is proportional to biofilm biomass.

Fig. 2

Colony morphologies on agar and biofilm phenotypes in broth of K. kingae corroding strain PYKK109 (top panels) and noncorroding strain ATCC 23330 (bottom panels). From left to right: bacterial colonies on agar; the agar surface beneath the colony after the colony was removed by rinsing with water; biofilm phenotype in broth (side view); biofilm phenotype in broth (bottom view).

Fig. 3

Confocal images of K. kingae bacterial cultures. The bacteria were grown in ibiTreat 60 µ-dishes plates for 24 h and then bacterial cultures were stained with 5 µM Sytox for 5 min at room temperature. The mages demonstrate extensive extracellular production of DNA by strain PYKK109 (Left panel) in contrast to strain ATCC 23330 (Right panel). Representative images are shown and are the results of three independent experiments. Scale bar =5µm.

Fig. 4

Effects of proteinase K and DNase I on K. kingae biofilm formation. In the left-hand graph, the enzymes were added to the broth at the time of inoculation and biofilm was quantitated after 24 h. In the right-hand graph, biofilms were cultured for 24 h and then treated with enzymes for 1 h. A₅₉₅ values are proportional to biofilm biomass. Asterisks indicate values significantly different from no-enzyme controls (P < 0.05).

Fig. 5

Autoaggregation, biofilm formation, and agar corrosion by K. kingae wild-type strain PYKK109 and pilus mutant strain VS1001. (A) Autoaggregation of cells in microcentrifuge tubes. (B) Biofilm formation on glass slides. In panel B, biofilms were visualized by staining with the green fluorescent nucleic acid stain SYTO9. (C) AFM topographic image (top) and cross-sectional profile (bottom) of agar surface after the bacteria were washed from the agar surfaces. For the pitted sample, the free amplitude and setpoint were 156 nm and 83 nm, respectively. For the control sample, those values were 208 nm and 145 nm. Areas of agar depression are designated with white circles.

Fig. 6

Transcript levels of genes pilA1, pilA2, fimB for type IV pili production in biofilm-forming strains PYKK081, PYKK109, 0211+12480 and non-biofilm forming strains ATCC 23330, 0604+15110. Transcripts levels are expressed relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SEM≥5%.