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Immunology and Infection

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

doi: 10.3791/54162 Published: July 6, 2016


A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.


The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV's effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.


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The human microbiome includes diverse organisms from multiple taxonomic kingdoms that share geographic regions in the body. Adherence to cell surfaces is an essential first step in biofilm formation, which is part of the microbiome colonization process. Included in the microbiome can be viruses that cause chronic and persistent infections. The chronic cell infection by these viruses can cause an alteration in putative receptor availability.1,2 In addition, cell entry by intracellular pathogens could also affect host membrane fluidity/hydrophobicity which in turn may alter attachment of other microbiome members, including bacteria and fungi. In order to understand the interactions that can occur between these multiple pathogens that co-localize in the same geographic regions of the human host, we must be able to study the interaction of pathogens that represent the spectrum of taxonomic kingdoms present at the mucosal surface.

The Herpesviridae are a family of microbes present in 100% of humans as permanent members of the microbiome3,4. In addition they can also be persistently shed both in the presence and absence of symptoms. Specifically, herpes simplex virus-1 and herpes simplex virus-2 (HSV-1 and HSV-2, respectively) are permanent members of the microbiome in the oronasopharynx and genital tract. In immune-competent individuals, both HSV-1 and HSV-2 cause gingivostomatitis, as well as genital herpes5-8. At these sites, HSV causes a latent infection characterized by chronic persistent asymptomatic viral shedding9. Entry of HSV into cells results in alterations in surface expression of nectins, heparan sulfate, lipid rafts and herpesvirus entry mediator/tumor necrosis factor receptor (HVEM/TNFr)10-25. These potentially represent shared receptors for some bacteria and fungi, e.g. S. aureus and C. albicans,which while opportunistic pathogens, can also reside as members of the mucosal microbiome of the oronasopharynx 26,27. Within the oronasopharynx S. aureus and C. albicans occupy two distinct sites of colonization. In hosts with natural teeth, the oral mucosa is shared by HSV-1 and C. albicans, while the anterior nasal nares are occupied by S. aureus28. However, despite in vitro findings that S. aureusadheres to mouth epithelial cells, 29,30 S. aureus is infrequently isolated from oral specimens when normal tissue is present29,30. Little is known concerning genital tract co-colonization niches beyond the clinical findings that S. aureus is associated with aerobic vaginitis, characterized by genital inflammation, discharge and dyspareunia, while C. albicans produces mucosal lesions similar to that observed in the oral cavity31-35. Thus, although these members of the oral and genital microbiome cross taxonomic kingdoms little is known concerning their interaction as it impacts their ability to initiate biofilm formation through adherence to the host cell surface5. This protocol has been effectively applied to determine the functional consequences of co-colonization/infection.

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1. HSV Strains and Handling

Note: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ- with beta-galactosidase reporter activity used were provided by V. Twiari36,37.

  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)14.
  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; Abs600, 0.3). After incubation, wash GT (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 CFU/ml)

4. S. aureus Handling

  1. Store S. aureus 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 Abs600 ;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.

7. Imaging Studies

  1. Wash each round glass coverslip (12 mm; 50 ml acetone in 100 ml beaker). Dry and sterilize coverslips (Kimwipes; glass petri dishes). Place dry sterile coverslips into the wells of sterile 24 well plates with alcohol flame sterilized forceps.
  2. Add HeLa cells (1 ml; 5x volume used for 96 well plates) to the wells of 24 well plates containing the washed sterile round glass coverslips. Add the virus, bacteria and fungi according to the template (Table 2) at 5x the volume used for the 96 well plates, then incubate and process as described in steps 6.2.1 to 6.4 above.
  3. After the final wash, fix the cells for microscopy by flooding the slide with methanol and allowing it to evaporate. Store the plates at RT until staining.
  4. For bright field microscopy (1,000x final original magnification) fill the wells containing methanol-fixed coverslips with deionized water. Immediately aspirate water. Cover each coverslip with Grams crystal violet. Wash coverslips free of non-bound stain (deionized water). Dry coverslips in situ then adhere them with hard set mounting medium to a labeled slide (Figure 3).
  5. For fluorescent microscopy (100x objective; 1,000x final original magnification) wash coverslips free of methanol essentially as described in step 7.4. Dry the coverslips in the wells.
    1. After drying, remove the cover slips and place them on labeled slides. Then add a sufficient amount of 1:20 dilution of fluorescein isothiocyanate (FITC)-conjugated Herpes Simplex Virus Type 1 + 2 gD antibody to cover the coverslip (1 - 5 µl).
    2. Incubate the slides in a moisture chamber at 37 °C for 30 min. After incubation, wash the coverslips in 4 changes of PBS.
    3. After the final wash, return the coverslip to the labeled slide. Stain with 4′,6-diamidino-2-phenylindole (DAPI; moisture chamber; 37 °C for 30 min). After incubation wash the coverslips in 4 changes of PBS, then affix to the labeled slide with hard set mounting medium.
    4. Allow the mounting medium to cure for 24 hr at RT in the dark. Examine the coverslips under oil objective on either a bright field microscope or fluorescent microscope with FITC and DAPI cutoff filters. Examine pictures of at least 50 fields (100 cells per organism minimum) for co-localization (Figure 4).

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Representative Results

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The level of robustness of data obtainable from system described in this report is shown in Figure 2 a-f 38. Through the use of this system the modulation of staphylococcal and fungal interaction with virally infected cells and their effect on each other's adherence can be delineated. These types of studies require microscopic examination of the interaction as shown in Figures 3 and 4 38 in order to determine whether the polymicrobial interaction is occurring on the same cells. In this study differential cell interaction is observed as a result of HSV-modulation of staphylococcal and fungal adherence that is viral species specific.

S. aureus and C. albicans (GT and YF) adhered to the same HSV-uninfected HeLa control cells. This co-localization on cells indicates a lack of physical inference with each other's HeLa cell adherence and that the measured levels of differential adherence measured were likely HSV-mediated (Figure 3A, A1) 38. However, upon HSV-1 or HSV-2 infected HeLa cells no co-localization of staphylococci and C. albicans was observed. (Figures 3B, B1, B2, B3, C1, C2). Using fluorescent microscopy (FITC-conjugated anti-HSV-gD monoclonal antibody) further confirmed that S. aureus did not appear to co-localize with C. albicans nor HSV-1 or HSV-2 (Figures 4A, A1, A2, A3, A4, B1, B2, B3). This co-operation between HSV and Candida extended to both yeast and germ tube forms (Figures 2A-F) of C. albicans. This specificity of association between the triad of microbes reflects the specificity of colonization seen on a much broader scale in the host oronasopharynx mucosa.

Figure 1
Figure 1. X-gal Staining Pictures of Dosage Dependent HSV-1 Infection in HeLa Cells. HeLa cells infected with HSV-1 at various MOI and X-Gal stained. (A) HeLa cells in well of 96 well plate with X-Gal stained mock-infected HeLa cell control; 20x initial magnification; (B) HeLa cells in well of 96 well plate with X-Gal stained HeLa cell infected with HSV-1 at an multiplicity of infection (MOI) of 10; 20x initial magnification; (C) HeLa cells in well of 96 well plate with X-Gal stained HeLa cell infected with HSV-1 at an multiplicity of infection (MOI) of 50; 20x initial magnification; scale bar applies to all. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Effect of HSV-1 (panels A, C, E) and HSV-2 (panels B, D, F) at Multiplicities of Infection (MOI) of 50 and 10 on Adherence of S. aureus and/or C. albicans to HeLa Cells. (A) S. aureus (Sa) binding to HSV-1 infected cells in the presence of C. albicans germ tubes (GT) or yeast forms (YF); (B) S. aureus (Sa) binding to HSV-2 infected cells in the presence of C. albicans germ tubes (GT) or yeast forms (YF); (C) C. albicans germ tubes (GT) binding to HSV-1 infected cells in the presence of S. aureus (Sa); (D) C. albicans germ tubes (GT) binding to HSV-2 infected cells in the presence of S. aureus (Sa); (E) C. albicans yeast forms (YF) binding to HSV-1 infected cells in the presence of S. aureus (Sa); (F) C. albicans yeast forms (YF) binding to HSV-2 infected cells in the presence of S. aureus (Sa). All data points are Mean +/- SEM, n= 16 normalized to virus-free control. * = significantly different (p< 0.05) from uninfected HeLa cell control. # = significantly different (p< 0.05) from paired point indicated by bracket; scale bar applies to all.38 Please click here to view a larger version of this figure.

Figure 3
Figure 3. Lack of S. aureus and C. albicans Interactions on HSV-1 and HSV-2 Infected HeLa Cells. HSV-1 and HSV-2 infected (MOI 50) HeLa cell monolayers with S. aureus and C. albicans (5:1 target to cell). For bright field microscopy, cell monolayers were stained with Gram's crystal violet, then examined by light microscopy. Cells that were positive for Candida or S. aureus (100 individual cells per microbe signal/per coverslip) were secondarily scanned for the presence of additional microbe co-localization signals (1,000x initial magnification). (A & A1) S. aureus (Sa) and C. albicans yeast forms (YF) or germ tube forms (GT) co-localize on uninfected HeLa cells; (A2, insert). Percent of HeLa cells with co-localized or individual microbes; (B - B3). Lack of S. aureus and C. albicans co-localization in the presence of HSV-1; (C - C2) Lack of S. aureus and C. albicans co-localization in the presence of HSV-2. Mean ± SEM; scale bar applies to all.38 Please click here to view a larger version of this figure.

Figure 4
Figure 4. Lack of Co-localization of S. aureus with C. albicans on HSV-1 or HSV-2 Infected HeLa Cells. HSV-infected HeLa cell monolayers challenge with S. aureus and C. albicans (5:1 target to cell) stained (FITC-conjugated anti HSV gD antibody, and DAPI). Pictures are representative of findings from screening of cells that were signal positive for HSV then scanned for Candida and S. aureus (100 individual cells per microbe signal per coverslip) that were then secondarily scanned for the presence of additional microbe co-localization signals (1,000x initial magnification; Nikon). (A - A4) C. albicans (A2, insert; DAPI staining) co-localize with HSV-1; (B - B3) C. albicans co-localized with HSV-2 (B1, insert, C. albicans DAPI staining). Mean ± SEM; scale bar applies to all.38 Please click here to view a larger version of this figure.

1 2 3 4 6 7 8 9 10 11 12

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. Please click here to download this file.

Plate 1 1 2 3 4 5 6
Control HeLa cells only HeLa + GT HeLa + YF HeLa + Sa HeLa + GT+Sa HeLa + YF + Sa
Plate 2
HSV HeLa cells + HSV HSV + HeLa + GT HSV + HeLa + YF HSV + HeLa + Sa HSV + HeLa + GT+Sa HSV + HeLa + YF + Sa

Table 2.
General Template for Visual Analysis of Polymicrobial Interactions with HSV-infected and Uninfected HeLa Cells. GT= Candida albicans germ tube phenotype; YF= C. albicans yeast form phenotype; SA= methicillin sensitive Staphylococcus aureus; HSV=herpes simplex virus. Please click here to download this file.

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Currently no information is available on complex interactions between permanent to semi-permanent members of the host microbiome that cross multiple taxonomic domains, i.e., prokaryotic, eukaryotic and viral. Therefore we developed a novel in vitro model system to study biofilm initiation by S. aureus and C. albicans on HSV-1 or HSV-2 infected HeLa 229 (HeLa) cells 38. The HeLa cell model system presents a unique advantage. This is due to their lack of surface fibronectin expression, which serves as a receptor for both S. aureus and C. albicans39-41. Since the apical 42surface of mucosal epithelia normally lacks fibronectin, this system more closely mimics that observed in natural infection and colonization43-47. Thus, we are able to more directly examine the role specific viral entry receptor turnover plays in subsequent adherence by other members of the microbiome.

Using entry proficient non-spreading HSV-1 and HSV-2, these findings show that cell entry of HSV-1 or HSV-2 renders cells refractory to super-infection with S. aureus, while enhancing C. albicans adherence in a virus concentration dependent manner (Figure 4). Interestingly, the effects of HSV-1 on GT forms vs. YF, was the reverse of that measured for HSV-2, a finding which may have a significant impact on promotion of vaginal candidiasis in clinical presentations48-51. From a pathogenesis perspective, it is generally accepted that the GT phenotype of C. albicans is the pathogenic form, with the YF the commensal state51-54. Adherence of C. albicans GT that were co-incubated with S. aureus showed an altered pattern of binding to HSV-infected HeLa cells, as compared to that measured for YF - S. aureus binding. HSV-1 enhanced adherence of GT to HeLa cells, but to a significantly lesser extent (p< 0.05) than that measured for YF adherence, as discussed above, i.e., 270 % control for YF adherence and 190% control for GT adherence. In addition, while S. aureus had no effect on HSV-1 enhancement of GT binding, the coccus negated the enhanced adherence mediated by the presence of HSV-2. The reverse pattern was observed for S. aureus effect on YF interaction with virus infected cells. This predilection for different fungal forms by HSV-1 (YF) vs. HSV-2 (GT) may play a role directing maintenance of the commensal state in vivo. With regards to the effect of the GT form on S. aureus binding, the GT almost completely abolished the HSV-1 inhibition of staphylococcal adherence to HeLa cells. In contrast, although the ability of GT to associate with HSV-2 infected cells was significantly increased as compared to HSV-1, the presence of S. aureus blocked the HSV-2 mediated increase in adherence.

The question of whether any changes in microbe adherence are the result of altered specific putative receptors present, or due to mechanical-steric hindrance can be answered in this model. Through the combined use of quantitation of microbe-cell interactions (CFU count) and microscopic examination of the interactions, we were able to determine that HSV-1 and HSV-2 appear to block S. aureus-cell interaction, since S. aureus adhered solely to uninfected HeLa cells. Furthermore, microscopic examination of cells show that Candida and HSV-1 and HSV-2 co-localized. Used together here the findings from this study parallel the in vivo observations site specificity for colonization indicating the utility of this model for the study of polymicrobial interactions.

Effective use of the protocol described herein is dependent on a variety of factors. First, the use of spread deficient virus. This enables a clean examination of the effect viral entry and cell signaling has on subsequent microbe interactions, without the complication of changes that can occur due to envelop-acquisition prior to leaving the cell. In addition, use of defective virus allows for a safer environment for the handling of Biosafety Class Two Pathogens. Secondly, this protocol allows for the use of both viral and microbe adherence variants. Use of variants that only bind to specific receptors permits delineation of shared receptors between microbes, or, alternatively the detection of novel receptors. Through the use of monoclonal antibodies to the receptors, there is the potential for visualization of adhesin localization on the microbe surface and provides a tool to study altered adhesin expression. This protocol is not suitable for the study of microbes that cannot be selectively isolated on differential medium. The methodology is also dependent on the availability of monoclonal antibody to virus-specific proteins. Although analysis in this study was enhanced by the size differential between yeast and bacteria, use of differentially fluorescently tagged, e.g. Texas red, microbe specific monoclonal antibody would be an effective work-around should the microbes in question be similar in morphology and size.

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The authors have nothing to disclose.


This project was supported by Midwestern University, IL Office of Research and Sponsored Programs (ORSP) and Midwestern University College of Dental Medicine-Illinois (CDMI).


Name Company Catalog Number Comments
BBL Sabouraud Dextrose BD 211584
Fungisel Agar Dot Scientific 7205A
Mannitol Salt Agar Troy Biologicals 7143B
Sheep blood agar Troy Biologicals 221239
Hela cells
1x DMEM (Dubelcco's Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate Corning 10-017-CM
Gentamicin 50 mg/ml Sigma 1397 50 µg/ml final concentration in the complete DMEM
Trypsin EDTA (0.05% Trypsin, 0.53 M EDTA)Solution 1x Corning 25-052-CI
Fetal Bovine Serum Atlanta Biologicals S11150 10% final concentration in the complete DMEM
Other medium and reagents
ONPG Thermo Scientific 34055
Ultra-Pure X gal Invitrogen 15520-018
1x HBSS (Hanks' Balanced Salt Solution) Corning 20-021-CV
1x PBS Dot Scientific 30042-500
RIPA Lysis Life Technologies 89901
Methanol Fisher Scientific A433P-4
HSV 1&2, specific for gD ViroStat 196
Gram Crystal Violet Troy Biologicals 212527
Petri dish 100 x 15 Dot Scientific 229693 
Petri dish 150 x 15 Kord Valmark 2902
96-Well plates Evergreen Scientific 222-8030-01F
24-well plates Evergreen Scientific 222-8044-01F
Culture tubes 100 x 13 Thomas Scientific 9187L61
Cover slip circles, 12 mm Deckglaser CB00120RA1



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Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi
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Plotkin, B. J., Sigar, I. M., Tiwari, V., Halkyard, S. Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi. J. Vis. Exp. (113), e54162, doi:10.3791/54162 (2016).More

Plotkin, B. J., Sigar, I. M., Tiwari, V., Halkyard, S. Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi. J. Vis. Exp. (113), e54162, doi:10.3791/54162 (2016).

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