healthmedicinet

Sample

Eighty isolates of S. epidermidis that had been referred to the medical laboratory of Kashani Hospital, Imam Ali Hospital
and Hajar Hospital in Shahrekord, Iran, including 42 from urine (52.5%) from cases
of urinary tract infection, 23 from blood (28.75%) from patients of septicemia, and
15 from dialysis bags (18.75%) from kidney failure patients undergoing peritoneal
dialysis. Biofilm production studied by phenotypic characterization, and microtiter
plate assay. Virulence genre for biofilm formation were investigated by PCR.

Phenotypic characterization

The method employed was that described by Freeman et al. (1989]). The Congo red agar medium comprised BHI (37 g/L), sucrose (50 g/L), No. 1 agar
(10 g/L) and Congo Red stain (0.8 g/L). Plates of the medium were inoculated and incubated
in aerobic environment for 24 h at 37°C. Under such condition, biofilm producers form
black crusty colonies on CRA, whereas non-producers form red colonies.

Microtiter Plate Assay for detection of biofilm

Biofilm production was detected using microtiter plate assay, following the procedure
described by O’ Toole (O’ Toole 2011]). The isolates of S. epidermidis were inoculated in 10 mL of tryptic soy broth with 0.25% glucose and incubated overnight
with shaking at 37°C. Next, the cultures were diluted 1:100, and 200 µL of the diluted
cultures, per well, were inoculated into 96-well polystyrene microtiter plates. After
48 h incubation at 37°C under aerobic conditions, the plates were washed three times
with 300 µL distilled water. Subsequently, the plates were stained with 200 µL of
1% crystal violet, per well, for 10 min. Excess crystal violet was removed by gently
washing the plate twice with distilled water. Finally, a volume of 250 µL of 95% ethanol
solution, per well, was added to the plate and the optical density was measured at
570 nm. The absorbance of destaining solution was measured at 570 nm in an Elisa reader
(Stat fax-2100). A well with sterile TSB or LB served as controls, whereby their ODs
were subtracted from that of the experimental strains. The mean OD 570 nm value was
determined using four replicates, and was considered to be adherence positive at OD
570 nm greater than or equal to 0.300 high biofilm formation, between 0.200 and 0.299,
and adherence negative at OD 570 nm less than 0.100.

Investigation of virulence genes

The virulence genes in S. aureus were investigated by PCR. The primer sequence used, the annealing temperature and
the PCR program employed are given in Table 1. Purification of DNA was achieved using a Genomic DNA purification kit (Fermentas,
GmbH, St. Leon-Rot, Germany) according to the manufacturer’s instruction. The total
DNA was measured at 260 nm optical density according to the method described by Sambrook
and Russell (2001]). The PCR reactions were performed using Accupower PCR PreMix kit (BioNEER), following
essentially the procedure described by Arciola et al. (2005]). The PCR mix contained 20 ?L of PCR PreMix. Accupower PCR PreMix component of 1U
Taq DNA Polymerase, 250 ?m Each dNTP (dATP, dCTP, dGTP, dTTp), 10 mM Tris–HCl (pH = 9),
30 mM KCl and 1.5 mM MgCl2. In each reaction add 5–50 ng Templet DNA and 5–10 pmol
primer. The PCR reaction for detection of 16srRNA, icaA, ica B, ica R and Ica C, D genes were performed using 10 pmol of each primer and 50 ng DNA of reaction mix.
The PCR was performed using a DNA thermal cycler (Master Cycler Gradiant, Eppendrof,
Germany). The amplicons were stained with ethidium bromide and electrophoresed in
1.5% agarose gel at 80 V for 30 min. PCR products were visualized and photographed
using UVIdoc gel documentation systems (Uvitec, UK). The PCR products were compared
against a 100 bp DNA marker (Fermentas, Germany). Staphylococcus epidermidis PTCC 1435 was used as a positive control.

Table 1. Primers used genes in Staphylococcus epidermidis

Statistical analysis

The data on production of biofilms by the strains of S. epidermidis was analyzed by the statistical software SPSS
^®
version 19.0 (SPSS Inc., USA). P values were calculated using the Chi square test.
P  0.05 was considered to be statistically significant.