Transcript Slide 1

Characterization and inhibition of medical silicone biofouling by
Candida spp. in mixed-species biofilms
Brandon Childers1, Clare McCoy2, Karl Malcolm2 & Alexandra C. Brand1
1. Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen,
Foresterhill, Aberdeen, AB25 2ZD, UK
2. School of Pharmacy, Queen's University Belfast, Belfast, BT9 7BL,
Northern Ireland, UK.
Introduction
Soft silicones are used for the manufacture of indwelling medical devices such as PEG tubes, gastronasal tubes and voice prostheses, which require frequent
replacement due to the build-up of mixed-species biofilms, which obstruct device function (1,2). From a clinical prosthetic device, we isolated 3 bacteria (Proteus mirabilis, Klebsiella oxytoca,
and Micrococcus luteus) and 2 Candida species (dubliniensis, and parapsilosis) in order to investigate interactions between co-colonising species and optimise assays to evaluate antimicrobial
medical silicones developed by our collaborators. In planktonic culture, individual bacteria demonstrated antagonistic effects against C. dubliniensis and C. parapsilosis, in a manner similar to
that observed between P. aeruginosa and C. albicans (3). The antagonistic effects were abrogated in the context of a mixed-species biofilm. A robust biofilm composed of all five organisms,
recapitulating the original explant biofilm, was established over the course of six weeks. To test our hypothesis that silicone modified with mobile head groups would reduce microbial
adhesion, a hydrophilic, linear polymer (AMPEG450) was grafted to Nusil silicone and tested for microbial colonisation in vitro. Initial adhesion of Candida spp and bacteria was reduced by
90% and 70 %, respectively, on the modified silicone. Biofilm formation after 48 h by C. albicans was also significantly decreased compared to unmodified Nusil. This model will be used to
further characterize the mechanisms involved in medical polymer biofouling and assess a range of silicone-modification strategies to inhibit biofilm formation and thereby increase the useful
life of medical devices.
1. Bacterial killing of Candida spp. in planktonic culture
.
.
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4. Five clinical co-isolates form a robust biofilm in vitro
In yeast growth conditions,
C. parapsilosis but not
C. dubliniensis was sensitive to
P. mirabilis.
A
Figure 1. Candida dubliniensis or C. parapsilosis yeast cells (106 cells/mL) were inoculated into 10 mL RPMI-1640 or YPD and incubated for 3 h at
37°C or 30°C, to produce hyphae or yeast cells, respectively. Cultures were incubated for 7 days at 37°C or 30°C after addition of 2.5mL of a midlog
culture of bacterial cells (P. aeruginosa, P. mirabilis, K. oxytoca, or M. luteus). Hypoxic conditions were created by withholding oxygen from cultures
grown under hypha-inducing conditions (RPMI, 37°C). The viable fungal cell population was determined daily by plating on YPD solid medium
containing antibacterial agents.
Figure 4. A five-species biofilm was formed in artificial saliva
medium using an initial inoculum of 106 cells/ml/yeast and 107
cells/ml/bacteria in a culture flask containing a 1.5 cm2 silicone
coupon. Cultures were incubated for 6 weeks at 37°C. The
medium was replaced once each week and ‘re-seeded’ with fungal
and bacterial species. A. Biofilms were mechanically disrupted
and the cell suspension used to determine CFUs on bacterial
selective media and/or YPD containing antibiotics . B. Mixedspecies biofilm formation was visually assessed by scanning
electron microscopy.
2. Variable ability of bacteria and Candida spp to form
single-species biofilms
C.
B.
A.
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A
B
C. parapsilosis
C
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‘Re-seeding’ of biofilms was required for the
establishment of a stably-attached structure,
suggesting that in vivo biofilms may result
from more than one infective exposure event.
Biofilms formed by the five co-isolates were denselystructured. Although higher, the numbers of cells
present at 6 weeks was in proportion to the number
of cells present at 48 h.
Adhesion by Candida spp
B
Adhesion by bacteria
C
Biofilm formed by C. albicans
No overall correlation
between initial adhesion
and biofilm formed at 48 h.
The clinical isolates formed
poor biofilms compared to
the two laboratory strains,
C. albicans SC5314 and
P. aeruginosa PA01.
3. In biofilms, fungi co-exist with antagonistic bacteria
C. dublinienses
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5. Silicone surface modification inhibits adhesion and
biofilm formation
A
Figure 2. A. Candida spp. adhesion to silicone was assessed by incubation of a 1.5 cm2 silicone coupon in RPMI medium containing 106 cells/mL at
37°C for 40 min. Adhered cells were enumerated by light microscopy from 10 randomly selected visual frames.
B. Bacterial adhesion to silicone was assessed by incubation of a 1.5 cm2 silicone coupon in RPMI medium containing 107 cells/mL at 37°C for 4 h.
Cells were removed from the silicone and CFUs determined per coupon.
C. Biofilms formed by individual species generated in tryptic soy broth using 106 cells/ml yeast and 107 cells/ml bacteria in a 96 well microtiter
plate. Plates were incubated at 37°C for 48 h and biomass quantified by crystal violet staining.
Composition of biofilm
on silicone
cl
B
Hypha formation correlates
with susceptibility of
C. dubliniensis to killing by 3
bacteria.
Under hypoxic conditions,
P. aeruginosa did not kill
C. dubliniensis but P. mirabilis killed
both Candida spp.
Cross-section of a 6-week-old C. albicans
biofilm formed on silicone elastomer.
Blue fluorescent strain courtesy of Karen Strijbus.
Figure 5. The medical silicone, Nusil, was modified by the addition of mobile, long-chain headgroups using a novel grafting method. A. Candida spp. adhesion to Nusil and AMPEG450 was
assessed by incubation of 1.5 cm2 silicone coupons in RPMI media containing 106 cells/mL at
37°C for 40 min. Adhered cells were enumerated by light microscopy, using 10 randomly
selected visual frames. B. Bacterial adhesion to the silicones was similarly assessed in RPMI
medium containing 107 cells/mL with incubation at 37°C for 4 hours. Adhesion was quantified by
determining CFUs on selective media to determine number of cells per silicone coupon. C. C.
albicans biofilms were formed on the silicones in RPMI medium, as above. Biofilms were
incubated at 37°C for 48 hours and biomass assessed by crystal violet staining.
.
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6. Summary & conclusions
C. dubliniensis is less abundant
in the present of50bacteria
than
µm
C. parapsilosis.
C. dubliniensis remains viable in
biofilms in the presence
of the 3 bacteria that killed it in
planktonic conditions.
Figure 3. A and B. Combinations of ex-plant species were used to form biofilms in tryptic soy broth using an initial inoculum of 106 cells/ml
yeast and 107 cells/ml bacteria in a 96-well plate. Cultures were incubated for 48 h at 37°C. Biofilms were mechanically disrupted and fungal
CFU determined by growth on YPD containing antibiotics. C. A 48 hour biofilm was formed using the 5 ex-plant species in artificial saliva in a
culture flask containing a 1.5 cm2 silicone coupon. Biofilms were mechanically disrupted and CFU determined by plating on bacterial selective
media and/or YPD containing antibiotics.
References
1. Ramage, G., Martinez, J.P., and Lopez-Ribot, J.L. (2006). Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS yeast
research 6, 979-986.
2. Ticac, B., Ticac, R., Rukavina, T., Kesovija, P.G., Pedisic, D., Maljevac, B., and Starcevic, R. (2010). Microbial colonization of tracheoesophageal voice
prostheses (Provox2) following total laryngectomy. European archives of oto-rhino-laryngology 267, 1579-1586.
3. Brand, A., Barnes, J.D., Mackenzie, K.S., Odds, F.C., and Gow, N.A. (2008). Cell wall glycans and soluble factors determine the interactions between
the hyphae of Candida albicans and Pseudomonas aeruginosa. FEMS Microbiology Letters 287, 48-55
•
During growth as planktonic cells, bacterial explant isolates exhibit antagonism in a
growth-condition-dependent manner against Candida spp isolated from the same device.
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Bacteria-Candida antagonistic effects are abrogated in biofilms.
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Single-species biofilms form poorly compared to multi-species biofilms (Fig. 6).
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Production of a robust and stable in vitro mixed-species biofilm requires ‘reseeding’.
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The stability of biofilms containing C. albicans may involve hyphal anchoring within the
silicone substratum.
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Silicone surface modification using mobile end-groups shows potential as a method of
reducing microbial adhesion and the rate of biofilm formation.
Acknowledgements
Adhesion was reduced by 90 % for
Candida spp and by 70 % for the 3
bacterial species co-isolated from
the voice prosthetic.
C. albicans biofilm film formation
by was reduced by 75 % on
AMPEG450 silicone compared to
untreated Nusil.
fjfj
Figure 6: A 6-week-old single-species
C. albicans biofilm formed on
silicone elastomer.