An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells

1. HSV Strains and Handling

NOTE: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ^– with beta-galactosidase reporter activity is used for the assay.

1. Use virus from a single lot and store at -80 °C at a 1:1 ratio of Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS) and skim milk until use. Before viral lot storage, determine virus concentration by o-nitrophenyl-β-D-galactopyranoside (ONPG) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) assay.
2. Determine virus viability and multiplicity of infection (MOI) by X-Gal staining for each experimental assay run using reporter virus entry assay, as previously described (Figure 1).
3. Dilute virus (Opt-MEM) to desired MOI. Fix monolayers (paraformaldehyde; 0.5 ml/well) before staining. Place virus viability controls in a separate microtiter plate in parallel with polymicrobial assay plates.

2. HeLa 299 Cell Handling

1. Grow at 37 °C, 5% CO₂ in DMEM with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum (FBS), gentamicin (50 µg/ml) and L-glutamine. Passage cells at 80% confluence with Trypsin solution (ethylenediaminetetraacetic acid, 0.53 M EDTA; 0.05% Trypsin; 5 ml/flask).

3. C. albicans Handling

NOTE: C. albicans obtained from a clinical laboratory source is stored at -80 °C in Remmel skim milk 2x medium.

1. Culture frozen stock onto Sabouraud Dextrose Medium (37 °C). After 24 hr subculture the C. albicans onto Fungisel medium (37 °C; 48 hr) for use.
2. Generate germ tube (GT) forms (pick representative colonies; 3 ml FBS; 3 hr; 37 °C; absorbance₆₀₀ (Abs₆₀₀), 0.3). After incubation, wash GT (Hanks' Balanced Salt Solution, HBSS; 2x; 4,000 x g). Add washed GT to warmed HBSS (37 °C; 0.32 Abs₆₀₀). GT forms should be 99% of cells observed as determined by hemocytometer count.
3. Make yeast form (YF) stock suspensions by picking representative Fungisel colonies (HBSS, 3 ml; 0.32 Abs₆₀₀). Count the number of YF forms/ml microscopically using a hemocytometer. YF forms should be 99% of cells observed as determined by hemocytometer count.
4. Make working fungal stock (250 µl of GT or YF stock in 25 ml HBSS; 37 °C; 10⁵ colony forming unit, (CFU/ml)

4. S. aureus Handling

1. Store S. aureus American Type Culture Collection, ATCC 25923 (-80 °C; Remmel skim milk 2x). Culture onto sheep blood agar (5%; 37 °C; 24 hr). Pick representative colonies and transfer to mannitol salts medium within 2 days for stock (37 °C; 18 hr).
2. Make S. aureus stock suspension (3 ml HBSS; 1.32 Abs_{600 ;}10⁸ CFU/ml)
   1. Make working S. aureus stock (100 µl of the stock in 25 ml HBSS; 10⁵ CFU/ml).

5. Candida and S. aureus Suspensions

1. Make mixed C. albicans and S. aureus suspension (250 µl YF or GT stock and 100 µl S. aureus stock in 25 ml HBSS).

6. Polymicrobial Biofilm Assay

1. Seed 96 well plates with 200 µl of 2 x 10⁵ HeLa cells/ml (85% confluence level). Rock plates (30 – 45 min; 37 °C) before incubation (37 °C; 5% CO₂ incubator; 18 hr). Wash monolayers (1x; Opt-MEM) then seed with HSV (HSV-1 (KOS) gL86 or HSV-2 (KOS) 33 gJ^– in 100 µl Opt-MEM ; MOI 50 and 10). Incubate plates (3 hr; 37 °C; 5% CO₂). Use only one viral strain per day.
2. Wash infected monolayers (1x; phosphate-buffered saline (PBS) with Mg⁺² and Ca⁺²; 100 µl). Replace PBS with warm HBSS leaving 25 µl in each well.
   1. Add YF, GT and/or S. aureus working suspensions (100 µl; target to cell ratio =5:1; n=16) as indicated in Table 1. Incubate plates (static; 30 min; 37 °C; 5% CO₂).
3. After incubation, aspirate one column at a time immediately refilling with 300 µl PBS with Mg⁺² and Ca⁺². Repeat this step twice then add radio-immunoprecipitation assay lysis buffer (RIPA; filter sterilized; 200 µl of a 1:50 dilution).
4. Rapidly triturate the HeLa cell lysate then place 50 µl onto mannitol salts (MS) and/or Fungisel (F) media (Figure 2). Spread the lysate using a glass rod bent at a 90° angle. Incubate the plates (18 hr at 37 °C). Manually count the number of colonies per plate. Controls consist of S. aureus and/or C. albicans adherence to HSV-uninfected HeLa cells.

Table 1. General Template Determination of Microbial Adherence in Polymicrobial Interactions. GT= Candida albicans germ tube phenotype; YF= C. albicans yeast form phenotype; MSSA= methicillin-sensitive Staphylococcus aureus; MS= mannitol salts medium; F= Fungisel medium.