Induced resistance to S. aureus in marine environmental biofilms

John Lafleur

3/23/09

I. Background a) S. aureus epidemiology & MDR b) Common medical biofilms c) The problem with antibiotics d) Induced antibiosis in bacteria e) Putting it togetherII. Materials and methodsIII. ResultsIV. Australia

a) S. aureus epidemiology & MDR

Crum et al., 2003. The American Journal of Medicine 119:943-51

Crum et al., 2003. The American Journal of Medicine 119:943-51

Turnidge and Bell. 2000. Microbial Drug Resistance. 6(3):223-8

b) Common medical biofilms

Costerton et al.,1999. Science. 284:1318-22

Camargo et al., 2005. International Journal of Gynecology and Obstetrics . 90:148—9

von Eiff et al., Drugs 2005; 65 (2): 179-214

von Eiff et al., Drugs 2005; 65 (2): 179-214

• Central venous catheter related infections in the US:

• ¼ million per year

• ¼ die

• $25,000 each incident

c) The problem with antibiotics

Bacteria in biofilms up to 1000X more resistant to antibotics Costerton et al., 1985. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. 27(4):619-24

D'Costa, et al., 2006. Science. 311:374-7

• Bacteria have been around long enough to develop every possible kind of resistance to each other

d) Induced antibiosis in bacteria

Mearns-Spragg et al. 1998. Letters in Applied Microbiology 27:142–146

• If antibacterial activity can be induced in individual strains of bacteria, can it also be induced in whole biofilms?

Is there evidence that a complex, multi-species environmental biofilm might amplify any antibacterial

activity among individual members of its bacterial consortia?

Burmolle et al., 2006. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 76(6):3916– 23

e) Putting it together

• Multi-drug resistant biofilms on implantable medical devices are a growing problem with no obvious solution

a) antibiotics don’t work on biofilms

b) even if they did, there’s growing resistance

• Existing natural models show that nature has solutions to the problem of unwanted biofilm formation, and some of them involves preexisting (‘friendly) biofilms.

• If it is possible to induce resistance in an environmental biofilm to a problematic, biofilm-forming human pathogen, perhaps it would be possible to learn how this could also be done for an inanimate surface—such as the surface of an implantable medical device.

II. Materials and Methods

III. Results

S. aureus agar with biofilm treated with UV

S. aureus agar with biofilm no UV

S. aureus growth, % area per high-powered field

00.20.40.60.8

11.21.41.61.8

baseline S. aureus agarno UV

Plain agar noUV

S. aureus agarpositive UV

Plain agarpositive UV

Agar type and UV exposure

S.

aure

us

gro

wth

% a

rea

Percentage of area per high-powered field covered by S. aureus micro-colonies.

Comparison of percentages of area of S. aureus biofilm growth with associated P values

P value

Baseline (0.05%) vs. S. aureus agar with biofilm, no UV (0.07%) <0.001

S. aureus agar with biofilm no UV (0.07%) vs. Plain agar with biofilm, no UV (0.13%) <0.001

S. aureus agar with biofilm pos. UV (1.56%) vs. Plain agar with biofilm, pos. UV (1.30%)

0.14**

S. aureus agar with biofilm no UV (0.07%) vs. S. aureus agar with biofilm, pos. UV (1.56%)

<0.001

.

base line

Staph aureus biofilm no UV

Plain agar biofilm no UV

Staph aureus biofilm with UV

exposure

Plain biofilm with UV exposure

0

20

40

60

80

100

treatment type

% c

olo

nie

s w

ith

les

s th

an 4

ce

lls

Percentage of S. aureus microcolonies with less than 4 cells at baseline and after incubation by treatment type .

Percentage of S. aureus microcolonies with less than 4 cells at baseline and after incubation by treatment types (Sd =standard deviation).

Percent <4 cells per microcolony Sd

Baseline 78% +/-31%

S. aureus spent medium agar with biofilm

91% +/-24%

Plain agar with biofilm 68% +/-37%

S. aureus agar with biofilm, UV exposed

5% +/-14%

Plainagar with biofilm, UV exposed 53% +/-29%

S. aureus agar no biofilm nd nd

Plain agar no biofilm nd nd

Table 2. Comparison of percentages of microcolonies with fewer than 4 cells per micrcolony with associated p values

P value

Baseline (78%) vs. S. aureus agar with biofilm, no UV (91%) 0.02

Baseline (78%) vs. Plain agar with biofilm, no UV (68%) 0.10 **

S. aureus agar with biofilm no UV (91%) vs. Plain agar with biofilm, no UV (68%)

<0.001

S. aureus agar with biofilm no UV (91%) vs. S. aureus agar with biofilm, pos. UV (5%)

<0.001

IV Australia

Two day incubation--Dapi stain

Percent coverage S. aureus biofilm by treatment type after 48 hours incubation

0

2

4

6

8

10

12

Baseline S. aur. ag. pos. biofilm Plain ag. pos. biofilm S. aur. ag. pos. biofilm pos.UV

Plain ag. pos. biofilm pos.UV

Treatment type

Per

cen

t ar

ea c

ove

red

by

S.

aure

us

bio

film

Two day incubation--live/dead stain

Relative area covered by S. aureus biofilm by treatment type--live/dead stain

0

200

400

600

800

1000

1200

1400

1600

1800

2000

S. aur. ag. pos. biofilm Plain ag. pos. biofilm S. aur. ag. pos. biofilm pods UV Plain ag. pos. biofilm pos. UV

Treatment Type

Rel

ativ

e m

agn

itu

de

of

area

co

vere

d

Two day incubation—live/dead stain

relative area covered by S. aureus biofilm by treatment type--live/dead stain

0

50

100

150

200

250

300

350

Baseline S. aur. ag. pos. biofilm Plain ag. pos. biofilm S. aur. ag. pos. biofilm podsUV

Plain ag. pos. biofilm pos.UV

treatment type

rela

tive

are

a co

vere

d b

y S

. au

reu

s b

iofi

lm

P values for comparison S. aureus area coverage

S. aureus agar versus plain agar:

• #1: P=0.001

• #2: P=0.01

• #3: P=0.02

Next steps

• DGGE to get an idea of differences in biofilm consortia between S. aureus and plain agar

• Isolate members of consortia and attempt to recreate induced S. aureus inhibition in lab-based biofilm culture

• Many thanks to Professors S. Kjelleberg and S. Rice, and to all the kind people on the sixth floor

• Prof. M. Shiaris

• Dr. M. Yasuda

• Prof. G. Burgess

Questions?