HERB’s POTENCY

AGAINST
BACTERIAL BIOFILM

Sri Winarsih
Department of Pharmacy
Faculty of Medicine Universitas Brawijaya

March 4, 2023 Malang International Conference on Herbs and Toxicants (MIC – HT) 1
BIOFILM
 ▪ Biofilms are now believed to be the primary
mode of existence for bacteria in aqueous
environments
▪ 1,000 – 10,000x greater populations than
planktonic
▪ Defined as a consortium of microorganisms
encased in a complex 3-D gelatinous matrix
of extracellular material secreted by the
BIOFILM inhabiting organisms.
▪ The establishment, maintenance and
existence of biofilm communities are highly
complex, socially organized processes
▪ Can be formed in low nutritional conditions, a
neutral ph of 7-7.5 and an adverse
temperature, namely a temperature range of
20 ° -40 ° C

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 • The development of biofilms has important impacts:
• bacterial properties
Biofilm • virulence
impact • survival
• diversity

• Enhanced scavenging of nutrient from bulk liquid

and surface
• Physiological alterations
– enhance growth rates
Biofilm – enhanced resistance
advantages – enhanced virulence
– greater local diversity
• physical protection and stabilization
• high densities – provide framework for coordinated
and socialized behavior

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Multiple studies have shown that during the time a biofilm is being
created, the pathogens inside it can communicate with each other
thanks to → a phenomenon called quorum sensing.

Although the mechanisms behind quorum sensing are not fully

understood, the phenomenon allows a single-celled bacterium to
perceive how many other bacteria are in close proximity.

If a bacterium can sense that it is surrounded by a dense

population of other pathogens, it is more inclined to join them and
contribute to the formation of a biofilm.

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In a study published in Nature, Singh et al. (2000) developed a sensitive test
which shows P. aeruginosa from cystic fibrosis lungs produces quorum-
sensing molecules that are the signals for biofilm formation.

- It turns out that P. aeruginosa secretes two signaling molecules, one

that is long, and another that is short.

- Using the new test, the team was able to show that planktonic forms of P.
aeruginosa produce more long signaling molecules. Alternately, when they
tested the P. aeruginosa strains isolated from the lungs of patients with
cystic fibrosis (which were in biofilm form), all of the strains produced the
signaling molecules, but in the opposite ratio – more short than long.

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Bacterial pioneers facilitate the arrival of other pathogens by providing more
diverse adhesion sites.

They also begin to build the matrix that holds the biofilm together. If there are
species that are unable to attach to a surface on their own, they are often able to
anchor themselves to the matrix or directly to earlier colonists.

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 Biofilm life cycle of
P. aeruginosa in
the minimum glucose media

(Rasamiravaka et al., 2014)

(Stage I) planktonic cell attachment , (Stage II) irreversible attachment,

(Stage III) microcolony formation, (Stage IV) biofilm maturation, (Stage V) biofilm disperse

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 A bacterial biofilm is
“A structured community of bacterial cells enclosed in a self-
produced polymeric matrix and adhere to an inert or living surface”

that means that bacteria can join together on essentially any surface and start to
form a protective matrix around their group. The matrix is made from polymers –
substances composed of molecules with repeating structural units that are
connected by chemical bonds → exopolysaccharides

“When bacteria are under stress - which is the story of their lives - they team up and form this
collective called a biofilm. They are like cities with channels for nutrients to go in and waste to go out.”
(Cho et al.,2007)

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 Common sites of biofilm infection on human body
One biofilm reach the bloodstream they can spread to any moist surface of the human body
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 HERBS AGAINST BIOFILM

03/03/2023 11
 - Staphylococcus
aureus
Ocimum sanctum Alpinia purpurata Allamanda cathartica - Escherichia coli
- Pseudomonas
aeruginosa
- Klebsiella
pneumonia

Camelia sinensis Mimosa pudica Malus sylvestri peels

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 METHODS

Extraction: by maceration process using ethanol 96%

Tube dilution → serial of the herb’s extract concentration

Crystal violet → to stain the biofilm formation
Mean Gray Value (MGV) using Adobe Photoshop CS 6
→ to measure the thickness of biofilm
→ the thicker of biofilm is indicated by the smaller
of MGV
(Hassan et al., 2011)

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 • bacteria + extract → biofilm
• Minimum Biofilm Inhibitory Concentration
Biofilm (MBIC) determined by finding the MGV of the
inhibition treatment group not less than 10% more the
MGV value of the negative control tube
(Macià et al., 2014)

• bacteria → biofilm + extract

Biofilm
degradation • potency of degradation determined by
herb’s concentration and time

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 RESULTS

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 6.25% 12.5% 25% 50%
1.55% 3.125% 200
180
160
140
120
100
80
60
40
20
0
0% 1,56% 3125% 6,25% 12,5% 25% 50% 50% +
Medium
0% (bacteria without extract) extract without bacteria Mean Grey Value

Red galangal extract vs P. aeruginosa (biofilm inhibition) → MBIC is 25%

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 6.25% 12.5% 25% 50%
0%

Extract without bacteria

Green tea leaves extract vs P. aeruginosa (biofilm degradation) → based on concentration

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 120

100 90,89
76,53 80,33
80
57,55
MGV

60 48,53
40

0% 2 hours
20
0% 4 hours
0
0% 6,25% 12,50% 25% 50%
Green tea extract concentration

140

120 108,01
100
81,16
80
MGV

60
44,05 2 jam
12.5% 2 hours 12.5% 4 hours
35,394
40 4 jam
20

0
0% 12,50%
Green tea concentration

Green tea leaves extract vs P. aeruginosa (biofilm degradation) → based on time

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 Ocimum Alpinia Allamanda Camelia Mimosa Malus
sanctum purpurata cathartica sinensis pudica sylvestri
(red galanga) (green tea peels
rhizome) leaves)
Biofilm MBIC Biofilm Biofilm Biofilm Biofilm
degrad degrad degrad degrad degrad

S.aureus 25% 4 12.5% 12.5% 12.5% 25%

hours 4 hours 4 hours 4 hours
MRSA 15%,
2 hours
E.coli 50%

P.aeruginosa 6.25% 25% 12.5% 12.5%

2 hours 4 hours 4 hours
K.pneumonia 25%

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 The content contained in red galangal rhizome that can potentially inhibit
biofilm formation is flavonoids and tannins which inhibit the signal in the
quorum sensing mechanism by disrupting the interaction between cyl-
homoserine lactone (signal molecules used by Gram-negative bacteria)
with their receptors (TraR and RhIR receptors) (Paczkowski et al., 2017)

Flavonoids inhibit biofilms by binding to DNA, proteins, and sulfhydryl

groups and interfere with the activity of AKT, ATPase, and superoxide
dismutase which will decrease the citric acid cycle and metabolism microbe
(Silvi et al., 2016)

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 Summary
Many herbs are potential to
develop as bacterial
antibiofilm on clinical setting
such as a catheter lubricating
gel, and as a complementary
treatment with antibiotics on
persistent infection

20XX presentation title 21

 Our team

Yohana Hartya Abu Hasan An Nisaa Auberta Callista Rizvidella Ardhy Ihza Estyka Ratu

Graziela Putri Jeanne Gabrielle Oywa Cantika Rachma Ayu Umi Qulsum
Sri Winarsih

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thank you for attention
sri winarsih
wien23.fk@ub.ac.id