ABSTRACT:

Biofilms are sessile microbial communities attached to surfaces that can be biotic or
abiotic and cause many medical problems. A bacterial biofilm is formed by the
gram-negative bacteria Pseudomonas aeruginosa in the patients with cystic
fibrosis.Aim of the study was to use matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-TOF MS) profiling as a new methodology
to monitor P. aeruginosa biofilm development. Biofilms were grown within
polypropylene tubes containing a glass slide, and were harvested after 3, 5, 7, 9, or 12
days of inoculation. Planktonic cells were obtained separately by centrifugation as
control. Two independent MALDI-TOF experiments were performed, one by
collecting biofilms from both the glass slide and the polypropylene tube internal
surface, and the other by acquiring biofilms from these surfaces separately. Scanning
electron microscopy (SEM) and atomic force microscopy (AFM) were used to
evaluate the morphological progression of the biofilm. Results showed that MALDI
profiling can distinguish between different biofilm stages. We can conclude that
MALDI profiling can serve as a promising tool for the clinical diagnostic and
prognostic workup of biofilms formation and control.

INTRODUCTION:

Bacterial biofilms are sessile colonies grown on the biotic and abiotic surfaces. These
biofilms protect the microbial cells against antibiotics action and other physical or
chemical challenges, making them difficult to eliminate. Ultimately, host exacerbated
responses may cause chronic inflammation and extensive tissue damage, without
being able to remove the bacteria.More recently, matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been successfully
used to investigate the molecular profile of bacteria but has not yet reported to
discriminate the different stages of bacterial biofilms formation.A Gram-negative
bacteria Pseudomonas aeruginosa forms biofilm in lungs of patients with cystic
fibrosis.This cause acute infections in hospitalized people, immunocompromised
patients and neutropenic patients due to chemotherapy treatments. The aim of the
present study was to evaluate possible changes in the molecular profile of biofilms
from P. aeruginosa in varying stages of maturity in two distinct surfaces (glass and
polypropylene) using MALDI-TOF MS examination of the morphology of such
 biofilm stages by scanning electron microscopy (SEM) and atomic force microscopy
(AFM).

Materials and Methods:

Evaluation of biofilm development stages by MALDI-TOF MS

Two experiments were performed first one was to check the efficiency of MALDI technique
and Second experiment was performed to evaluate any phenotypical differences among films
grown on different substrates.

Growing planktonic cells and biofilms:

 20 ml of Mueller Hinton (MH) culture medium was added in five 50ml polypropylene
plastic tubes.
 A glass slide was placed vertically inside these tubes.
 A pre-inoculum of P. aeruginosa strain ATCC 27853 was incubated in MH culture
medium at 37 C during 24 h.
 10 ml of this inoculum was added to the polypropylene tubes and were placed in a shaking
incubator at 37 C at 170 rpm.

Analysis of biofilms:
 Collected biofilms from each tube 3, 5, 7, 9, and 12 days after the inoculation.
 Washed tubes and slides two times with distilled water
 Bacteria were collected from the tube internal surface with a sterile toothpick.
 Centrifuging 300 ml of the medium of the 5-day tube at 3000 g for 3 min to collect
planktonic cells.
 Dissolved the pellet in 25 ml of distilled water and used 1ml of this suspension for
analysis.
MALDI-TOF MS:
 In a polished 96-well MALDI target plate, poured the sample in 24 wells for each sample.
 Covered every sample with 1ml of a-cyano-4-hydroxycinnamic acid saturated matrix solution
(10 mg/ ml, dried at room temperature before MALDI-TOF MS analysis.
 After crystallization, the samples were analyzed onto a commercial MALDI-TOF mass
spectrometer and spectra were acquired automatically using a standard procedure.

Use of MALDI-TOF MS to evaluate bacterial biofilm growth on glass and on polypropylene

surfaces
 18 polypropylene plastic tubes were prepared and analyzed by MALDI-TOF mass
spectrometer and spectra were acquired.

 36 spectra were acquired in every group, and one was selected as the most representative
based on its common features and displayed for comparison among the experimental groups
using FlexAnalysis 3.0 software.

 With the MALDI Biotyper 3.0 software, 11 MSPs were created.


Morphological characterization of the biofilms and planktonic cells

 Prepared 15ml polypropylene plastic tubes as discussed above.

 Took 2ml of planktonic cells and dried on coverslip.
 P. aeruginosa biofilms on coverslips were fixed using 1 ml of modified Karnovsky fixative
for 3 h at room temperature.

 Mounted the sample on a metal sample support using double-sided tape.

 Using an atomic force microscope sample was analyzed.

Scanning electron microscopy (SEM)

 P. aeruginosa biofilms and planktonic cell samples were grown and fixed as previously
described for AFM.
 Washed coverslips with sodium cacodylate buffer. And post-fixed in 1% osmium tetroxide
in the absence of light for 1 h.
 Washed the sample with distilled water and dehydrated in acetone baths the critical point
drying was done using liquid CO2.

 Mounted the samples in stubs and sputter coated with a thin gold layer. Then analyzed
samples using a Jeol® JSM 840A scanning electron microscope.