Research title: Inhibition of Biofilm Formation of Isolated Sphingomonasspp.

by Marine
Actinomycete spp.
Chapter 1
Background
Sphingomonas spp. are aerobic Gram-negative, oxidase-positive, non-fermentative rod
and capable of biofilm production. Sphingomonas was divided into four genera:
Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis.
Sphingomonas spp. are widely distributed in the environment. They have been isolated
from sea water, sea ice, riverwater, waste water, mineral water, water equipment used in
hospital or even hospital equipments such as cateters. Also they are isolated in various
drinking water distribution systems.
Statement of the problem
The problem of the research was that the previous researcher did not clearly state on
how the different Marine Actinomycytes inhibits the biofilm production of Sphingomonas
spp. Also the identification of the different Marine Actinomycetes used was not clearly
identified due to lack of time.
Objectives
This research aims:
1. To isolate Sphingomonas spp. from dental water pipes, household faucets, drinking
fountains, medical devices and stagnant water
2. To perform molecular methods to identify the Sphingomonasspp. and Actinomycete
spp. isolated up to the species level
4. To test the ability of the Marine Actinomycetespp. to inhibit biofilm formation of
Sphingomonasspp. using crude extract and secondary metabolites
5. To perform antimicrobial susceptibility test on the isolated Sphingomonasspp.
Scope and limitations
1. This research focuses on isolating Sphingomonasspp. from the potential sources
listed above, culturing the organism using different medium that renders it nutrients for
its maximum growth, and performing molecular methods to identify the
Sphingomonasspp isolated.
2. Biofilms are composed of a variety of microorganisms. With that being said, this
research will also consider those organisms but only Sphingomonasspp. will be
subcultured and purified for future use.
3. There are no existing literatures on the antimicrobial sensitivty of Sphingomonasspp.,
with that, this research will perform antimicrobial susceptibility test using antibiotic disks
of known concentration, measure the corresponding ZOI and arrive at an interpretation
unique to the species isolated.

Prepared by: Domingo N., Gabac M., Garcia F.

Research title: Inhibition of Biofilm Formation of Isolated Sphingomonasspp. by Marine
Actinomycete spp.
4. All throughout the research, only the crude extract and the secondary metabolites of
the Marine Actinomycete spp isolated will be used against biofilm formation of
Sphingomonasspp. Being able to determine the mechanism of reaction that these
secondary metabolites do since the study where this research is pattered was not able
to do so.

Methodology
Isolation of Sphingomonasspp
The samples were collected from dental water pipelines, household faucets,
drinking fountains, medical devices such as oxygen masks and catheters, and stagnant
water of bedside water bottles and water tubs. These samples were cultured in a
nutrient agar and incubated for 24 hours. Colonies of suspected Sphingomonasspp
were sub-cultured and purified. Further identification of the species was done using API
20NE and DNA kit for molecular identification.

MicrotiterPlate Biofilm assay

A. Biofilm growth
A 24-hour old culture of Sphingomonasspp were diluted into fresh medium
for biofilm assays. The M63 minimal medium supplemented with magnesium
sulfate and arginine as the sole source of carbon and energy source was
used as a biofilm-promoting medium that stimulates less planktonic growth
and a more robust biofilm. A 100 L of the dilution was put on each well of the
96-well microtiter plate, this was done on triplicates. The microtiter plates
were incubated for 24 hours at room temperature.

B. Biofilm staining
After incubation, the planktonic cells and other solid media components
were removed by turning the plate over and shaking out the liquid content.
The plates were gently submerged in a small tub of water, and the water was
then removed. This process was repeated twice to remove unattached cells
and media components that would otherwise be stained and significantly
lower the background staining. A 125 L of a 0.1% solution of crystal violet in
water was added to each well of the microtiter plate. The plates were
incubated at room temperature for 10-15 minutes. The microtiter plates were
then rinse by submerging it again on a tub of water, removing the water, and
allowing it to dry by turning it upside down.

C. Biofilm Quantification
A 125 L of 30% acetic acid in water was added to each well of the
microtiterplale to solubilize the crystal violet. The plates were incubated at

Prepared by: Domingo N., Gabac M., Garcia F.

Research title: Inhibition of Biofilm Formation of Isolated Sphingomonasspp. by Marine
Actinomycete spp.
room temperature for 10-15 minutes. The 125 L solubilized crystal violet was
transferred to a new flat bottomed microtiter dish. The absorbance at 550
nmwas measured using a spectrophotometer with 30% acetic acid in water as
the blank solution.

Extraction of crude extracts

Submerged state fermentation method
A 1000 mL of yeast and malt extract broth was prepared in which 100 m was
dispensed into 500 mL Erlenmeyer flask, sterilized and cooled. At room temperature,
the broth was inoculated with 2 mL suspension of isolates and kept for 14 days in a
rotary shaker at 200 r/min. Then the culture broth was harvested by centrifugation at
4,000 r/min for 15 minutes. The supernatant was collected and added equal volumes of
ethyl acetate at a ratio of 1:1 v/v, then shaken vigorously for 1 hour and repeated twice.
The solvent phase was separated from aqueous phase by using a separating funnel
and subjected to rotary vacuum evaporator at a water bath temperature of 60 C at 100
r/min to remove solvent and to get crude extracts. (Gebreyohannes et al, 2013).

Antibiotic Disc Diffusion Assay

Antimicrobial activities of the crude extracts were tested against isolated
Sphingomonassppusing the Kirby-Bauer disc diffusion method. The inoculum was
prepared by mixing 1 mL of the 24 hour old culture with 9 mL sterile distilled water and
the turbidity was compared with that of the standard 0.5 McFarland solution. To obtain
an even growth, the entire Mueller Hinton Agar (MHA) was swabbed uniformly by the
cotton swab dipped into the properly adjusted inoculum. The inoculated plates were
allowed to dry under the laminar flow before applying the extract. Five filter paper disc
(6 mm diameter) was impregnated with crude extracts of different isolated marine
Actinomycetes, and placed on the surface of the inoculated MHA plates. Another plate
was swabbed with the same inoculum and tested its susceptibility against various
antibiotics: _______________ Plates were incubated for 24 hours and the zones of
inhibition were measured.

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Actinomycete spp.
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Prepared by: Domingo N., Gabac M., Garcia F.

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Actinomycete spp.
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Prepared by: Domingo N., Gabac M., Garcia F.

Research title: Inhibition of Biofilm Formation of Isolated Sphingomonasspp. by Marine
Actinomycete spp.

Prepared by: Domingo N., Gabac M., Garcia F.