INEB - Instituto de Engenharia Biomédica
UNIVERSIDADE DO PORTO
Barros1,2,
J.
L.
Grenho1,2,
C.M.
Manuel2,4,5,
Effect of the initial inoculum concentration in Staphylococcus epidermidis RP62A biofilm formation
C.
Ferreira2,4, F.J. Monteiro1,2, L.F. Melo2,4, O.C. Nunes2,4
RESULTS AND DISCUSSION
and M.P. Ferraz1,3 1INEB
- Instituto de Engenharia Biomédica, Universidade do Porto, Porto –
Portugal. 2FEUP
- Faculdade de Engenharia – Universidade do Porto, Departamento
Surface preparation and characterization
de Engenharia Metalúrgica e Materiais, Portugal. 3CEBIMED
- Centro de Estudos em Biomedicina, Universidade Fernando
Pessoa, Portugal. 4LEPAE
- Laboratório de Engenharia dos processos, ambiente e energia,
Departamento de Engenharia Química, Faculdade de Engenharia – Universidade do Porto, Portugal. 5ULP
- Universidade Lusófona do Porto, Portugal.
[email protected]
INTRODUCTION Nanohydroxyapatite (nanoHA), due to its chemical properties, has appeared as an exceptionally promising bioceramic to be used as bone regeneration material. Staphylococcus epidermidis is a major nosocomial pathogen associated to infections of implanted medical devices1,2. This organism presents unique problems of diagnosis and infection treatment involving biofilm formation on implanted biomaterials, such as dental and orthopedic devices3. Biofilm development processes are influenced by multiple factors, such as the initial inoculum concentration and material surface features. Rimondini et al.1 referred that critical suspension concentrations ranging from 4 103 to 4 107 CFU/ml are necessary to develop infections in rabbit models after orthopedic implants. The present study aim is to evaluate the effect of initial inoculum concentration on biofilm formation under conditions simulating human body environment (fed-batch system, light absence, 37ºC).
B
A
Figure 1 – Preparation, characterization and chemical composition of nanoHA1000 disks. (A) preparation of nanoHA disks sintered at 1000ºC. (B) SEM-EDS images of nanoHA1000 disks. Image area was 5 5 µm with 15000X magnification. Table 1 – Contact angle measurements, zeta potential, porosimetry parameters, surface area and roughness of the HA has exceptional biocompatibility and bioactivity properties with
tested nanoHA1000 disks.
Contact Angle (°)
18.1 6.3
Zeta potential (mV)
-18.1 2.5
Mean Pore diameter (m)
9.8 10-9
Total surface area (m2/g)
9.8
Theoretical total porosity (%)
6.8
Ra 2 2 µm2 (nm)
respect to bone cells and tissues, perhaps due to its similarity with the mineral component of body hard tissues. The structural characteristics of this synthetic biomaterial may influence their response in a physiological environment, such as the biological apatite deposition and bone bonding4.
36.4 8.9
MATERIALS & METHODS S. epidermidis RP62A biofilm formation
7
e
Log10 (Cells density/mm2)
cd
c
c
e de
5
a
24 hours
b
ab
48 hours
3 5
A
×10-1
5
100
×102
5
h
×105
g
f
80 e d
60 c
24 hours
b
40
48 hours
Applied Biomaterials &
20
B
5 × 10-1
5 × 102
initial inoculum concentration 5 10-1 cells/ml
CFUs/mm2 B105_72h B105_72h B105_72h B105_48h B105_48h B105_48h 2
Cells/mm
B102_72h
B10-1_72 B10-1_72 B10-1_72 B105_24h B105_24h B105_24h
Biofilm formation data on nanoHA1000 for B10-1_48 B10-1_48
B10-1_24 B10-1_24 B10-1_24
initial inoculum concentration 5 102 cells/ml Biofilm formation data on nanoHA1000 for initial inoculum concentration 5 105 cells/ml
B10-1_48
B102_72h B102_72h
-1.5
1.0 Principal component 1 (PC1 = 98.3%)
cultivable cells on nanoHA1000 disks over time after inoculation with
5 × 105
three different initial inoculum concentrations). The two Principal Components (PC1 and PC2) explained 100% of the total variance
Medical Microbiology,
Figure 2 – Attached cells per unit surface area for biofilms growth on nanoHA1000 disks in a system fed-batch. (A)
ACKNOWLEDGMENTS
Surface density (Cells density/mm2). (B) Percentage of
This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia in the framework of the project NaNOBiofilm (PTDC/SAU-BMA/111233/2009).
CFUs/mm2.
Biofilms 5 International Conference, 10 to 12 December 2012, Paris – France.
Biofilm formation data on nanoHA1000 for
Figure 3 – PCA biplot diagram with the biofilm data (cells density and
a a
Initial inoculum concentration (Cells/ml)
COMPETE
B102_48h B102_48h B102_48h
72 hours
Applied Microbiology,
Colloid and Interface
component 2 (PC2 = 1.7%) Principal -1.0
72 hours
4
0
L. et al., 2005, Journal of Biomechanics, (3)1:1-10. 2Cerca, N. et al., 2004, Letters in 39(5):420–424. 3O' Gara, J.P. et al., 2001, Journal 50:582-587. 4Ribeiro, N. et al, 2010, Journal of Science, 351:398-406.
B102_24h B102_24h B102_24h
REFERENCES 1Rimondini,
1.0
6
Percentage (% CFUs/mm2)
NanoHA disks, previously sintered at 1000ºC (nanoHA1000) were characterized by measuring zeta potential, contact angle, surface topography, total surface area and porosimetry. A culture of S. epidermidis RP62A at the exponential growth phase in Tryptic Soy Broth (TSB) was used to prepare three different densities: 5×105 cells/ml, 5×102 cells/ml and 5×10-1 cells/ml. NanoHA1000 disks were placed inside each suspensions and incubated for 24, 48 and 72 hours at 37ºC and 150 rpm. Every 24 hours, the medium was carefully replaced by fresh TSB. At each sampling time, biofilms on nanoHA disks surface were quantified by total cells using a blue fluorescent nucleic acid stain (DAPI) and cultivable cells numbers (colony forming units, CFUs). All tests were run in triplicate. The results of these methods were compared by applying the Tukey HSD multiple comparison test using SPSS® Statistics (vs. 19.0, Chicago). In all cases p < 0.05 was chosen to denote the significance level. The biofilm formation data on nanoHA1000 from the different initial concentrations were also compared using Principal component analysis (PCA) test performing in CANOCO® software for windows statistics (vs. 4.55, Netherlands).
Different lowercase letters indicate significant
among samples. For initial inoculum concentration 5 102 cells/ml the variance of biofilms formed on nanoHA1000 disks over time was lower than when the initial inoculum concentration was 5×10-1 cells/ml.
differences (p < 0.05) according to Tukey HSD.
CONCLUSIONS • Independently from the initial inoculum, an increase on biofilm cell density over time was observed, indicating that even at the lowest concentration, S. epidermidis was able to form biofilm under the tested conditions. • For the lowest concentration, a lag conditioning period up to 48 h before biofilm growth on nanoHA surfaces was observed, probably due to the mass transfer of bulk macromolecules to the surface and the formation of an adsorbed layer. • For the other concentrations tested (5 102 and 5 105 cells/ml) the biofilm formed sooner , probably due to the number of bacteria in contact with the material surface. Given the high number of biofilm cells after 24 h of incubation, the steady-state
MOD S20/03
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was reached.
