Theodora 

et al. BMC Res Notes (2019) 12:732

https://doi.org/10.1186/s13104-019-4775-1 BMC Research Notes

RESEARCH NOTE Open Access

Screening and quantification of anti‑quorum

sensing and antibiofilm activities
of phyllosphere bacteria against biofilm forming
bacteria
Nadine Amabel Theodora, Vania Dominika and Diana Elizabeth Waturangi*

Abstract 
Objective:  The objectives of this research were to screen anti-quorum sensing activity of phyllosphere bacteria and
quantify their antibiofilm activity against biofilm forming bacteria (Bacillus cereus, Staphylococcus aureus, Enterococcus
faecalis, Salmonella typhimurium, Vibrio cholerae, Pseudomonas aeruginosa).
Results:  We found 11 phyllosphere bacteria isolates with potential anti-quorum sensing activity. Most of the crude
extracts from phyllosphere bacteria isolates had anti-quorum sensing activity against Chromobacterium violaceum at
certain concentration (20 and 10 mg/mL), but not crude extract from isolate JB 7F. Crude extract showed the larg-
est turbid zone (1,27 cm) using isolate JB 14B with concentration of 10 mg/mL and the narrowest turbid zone isolate
(1 cm) using JB 18B with concentration of 10 mg/mL. Crude extracts showed various antibiofilm activities against all
tested pathogenic bacteria, it showed the highest biofilm inhibition (90%) and destruction activities (76%) against S.
aureus.
Keywords:  Antibiofilm activity, Chromobacterium violaceum, Phyllosphere, Quorum sensing, Violacein

Introduction Phyllosphere bacteria, which lives the most on the leaves

Nowadays, we know that about 65% of all bacterial infec- surface area, reported to have potential in quorum quench-
tions were associated with bacterial biofilms [1]. Biofilm ing activity with produce molecules such as AHL lactonase
is an organized aggregate of microorganisms like bac- enzyme [4–6]. High populations of phyllosphere bacteria
teria within an extracellular polymeric matrix that they show activities such as antimicrobial and antibiofilm that
produce [1, 2]. Several pathogenic bacteria form a bio- produced to survive on the leaves surface area [7]. Many
film using a mechanism called quorum sensing. Quo- research have been conducted to analyze anti-quorum
rum sensing is a communication form among bacteria sensing activity from phyllosphere bacteria. The objectives
by various types of extracellular signal molecules called of this research were to screen anti-quorum sensing activ-
autoinducer (AI). Bacteria in biofilm were more resistant ity of phyllosphere bacteria using Chromobacterium vio-
to antibiotic because biofilm matrix can help with inter- laceum as indicator bacteria and quantify their antibiofilm
fering the penetration of antibiotic. Therefore we need activity against biofilm forming bacteria (Bacillus cereus,
to explore compound that have capability to inhibit or Staphylococcus aureus, Enterococcus faecalis, Salmonella
destroy biofilm as well as anti-quorum sensing to control typhimurium, Vibrio cholerae, Pseudomonas aeruginosa).
attack of biofilm-forming pathogenic bacteria [3].
Main text
Methods
*Correspondence: [email protected] Bacterial cultivation
Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, The phyllosphere bacteria were from Atma Jaya Cath-
Jenderal Sudirman 51 Street, South Jakarta, 12930 DKI Jakarta, Indonesia olic University of Indonesia culture collections in

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Theodora et al. BMC Res Notes (2019) 12:732 Page 2 of 5

cryopreservation. These bacteria were from previous were incubated at 37  °C for 24  h. This assay was per-
research and recovered from Psidium guajava, Averrhoa formed in triplicate [12].
carambola, and Anredera cordifolia leaves [8, 9]. Bacte-
ria were grown in Luria–Bertani Agar (LA) and were Detection of anti‑quorum sensing activity
incubated at 28 °C for 48 h. After that, single colony was The crude extracts were tested for anti-quorum sens-
picked and grown in King’s B medium and incubated at ing activity against C. violaceum by agar well diffusion
28 °C for 48 h. method. C. violaceum was streaked on LA with a ster-
Pathogenic bacteria used were B. cereus ATCC 14579, ile cotton swab. Then the extracts (50  μL) with various
S. aureus ATCC 29213, E. faecalis ATCC 33186, P. aer- concentration (5, 10, and 20  mg/mL) were applied to
uginosa ATCC 27853, S. typhimurium, V. cholerae. All the well. DMSO was used as a control. The plates were
pathogenic bacteria were from cryopreservation and incubated at 28 °C for 24 h. Anti-quorum sensing activity
were streaked onto LA then incubated 37 °C overnight. was observed through a turbid halo zone against a back-
ground of violacein pigment. This assay was performed in
Primary screening of anti‑quorum sensing activity triplicate [10].
The monitor strain C. violaceum was grown separately
in 50  mL of LB broth medium and incubated at 28  °C, Quantification of antibiofilm activity
120  rpm for 48  h. Phyllosphere bacteria were streaked The pathogenic bacteria were inoculated into BHIB and
onto LA in a straight line then incubated at 28 °C for 24 h. incubated overnight. After that, for biofilm inhibition
After that, 100 μL of monitor strain (­ OD600 = 0.132) were test, 100  µL of crude extracts and 100  µL of bacterial
put into 2 mL semisolid agar (0.75% agar) for overlay on cultures ­(OD600 = 0.132) were transferred into 96-well
top of the phyllosphere isolates which had been streaked microtiter plates (polystyrene) then incubated at 37  °C
before. These plates were incubated at 28  °C overnight. for 24 h. Meanwhile for biofilm destruction test, 100 µL
A positive result indicated by inhibiting violacein pig- of bacterial culture were transferred into 96-well micro-
mentation (opaque zone) of the C. violaceum around the titer plates then incubated. After that, 100  µL of crude
streak of the phyllosphere isolates [10]. extracts will be added and incubated at 37  °C for 24  h.
Then planktonic cells and media were discarded. Adher-
Production of crude extract ent cells were rinsed gently twice with distilled water and
Isolates that had given positive result from the primary allowed to air dry. The biofilms were stained by 200 μL of
screening of anti-quorum sensing activity were extracted 0.4% (w/v) crystal violet solution for 30  min. After that,
by using liquid–liquid extraction. The bacterial culture the dye were discarded and the wells were rinsed twice
were inoculated into 100  mL of Luria–Bertani Broth with distilled water. The wells were air dried and then
(LB) then incubated in orbital shaker incubator at 28 °C 200 µL of ethanol were used to solubilize the crystal vio-
for 48  h 120  rpm. After that, centrifuged at 13,888×g let. The optical density were determined at 595 nm using
for 15  min and cell-free supernatant was harvested and a microplate reader. BHIB was used as blank and bacte-
mixed with an equal volume of ethyl acetate. The solvent rial cultures without extracts were used as control. This
layer was harvested and evaporated in a rotary evapo- test was performed triplicate [13].
rator. After that, extract evaporated in an oven vacuum Percentage biofilm inhibition
overnight to obtain the crude extract. To this, 1% of
Dimethyl Sulfoxide (DMSO) will be added to obtain a (Control OD595 − Treated OD595)
= × 100%
final concentration of 5, 10, and 20  mg/mL stock (w/v) (Control OD595)
and kept at − 20 °C [11].
Microscopic observations
Antibacterial activity assay This step was done using Scanning Electron Microscope
The crude extracts that had been obtained were tested (SEM) at Dexa Laboratories of Biomolecular Sciences.
against pathogenic bacteria such as B. cereus, E. faeca- First, B. cereus and S. typhimurium were grown in BHIB
lis, and S. aureus, P. aeruginosa, S. typhimurium, and V. and incubated overnight. Then, bacteria were spotted to
cholerae using agar well diffusion method. Pathogenic a steril cover glass and incubated overnight to form bio-
bacteria were streaked continuously on Brain Heart Infu- film. After that, crude extracts were spotted into the bio-
sion Agar (BHIA). Then, the extracts were applied 50 μL film and incubated at 37  °C overnight. At the last step,
of 5, 10, and 20 mg/mL solution to the well. Streptomy- the results were analyzed using SEM at DLBS [14].
cin (Merck; 10  mg/mL) were used as positive control,
whereas DMSO was used as negative control. The plates
Theodora et al. BMC Res Notes (2019) 12:732 Page 3 of 5

Results Detection of anti‑quorum sensing activity

Primary screening of anti‑quorum sensing activity We found out that each of phyllosphere isolate has their
There were 11 out of 60 phyllosphere isolates from previ- own optimal concentrations but most of them showed
ous study showed an anti-quorum sensing activity. Those activity at concentration of 20  mg/mL and all of them
positive isolates were extracted and continued to the next have no activity at concentration of 5  mg/mL Crude
step. extract showed the largest turbid zone (1.27  cm) using
isolate JB 14B with concentration of 10  mg/mL and the
Antibacterial activity assay narrowest turbid zone isolate (1  cm) using JB 18B with
From this assay, we know that 1 out of 11 positive phyllo- concentration of 10 mg/mL (Table 1).
sphere isolates crude extract, EJB 7B, showed antibacte-
rial activity against all Gram positive pathogenic bacteria Quantification of biofilm activity
which used in this research. Meanwhile, control positive According to the result of quantification of biofilm
(Streptomycin) showed various turbid zone depending (inhibition) activity assay, the results showed that crude
on the pathogen bacteria used. Average clear zone V. extracts had various inhibition activity against all patho-
cholerae is 2 cm, P. aeruginosa is 3 cm, S. typhimurium is genic bacteria used in this study, with the most positive
2.3 cm. Average clear zone B. cereus is 4 cm, S. aureus is results against S. aureus and the least against P. aerugi-
3.5 cm, E. faecalis is 3 cm. nosa. (Table  2). Crude extracts that showed the highest
biofilm inhibition activity against S. aureus (90%) is from
Table 1 Result of  detection of  anti-quorum sensing isolate JB 19B.
activity Meanwhile, different results were obtained from quan-
Phyllosphere Origin of isolates Concentrations (cm) tification of biofilm (destruction) activity assay. Accord-
isolates ing to biofilm destruction activity data (Table  2), the
5 mg/mL 10 mg/mL 20 mg/mL
results showed that crude extracts had various destruc-
JB 3B Psidium guajava 0 0 0.83 tion activity against all pathogenic bacteria used in this
JB 11B Psidium guajava 0 0 1.13 study, with the most positive results against S. aureus and
JB 14B Psidium guajava 0 2 1.1 E. faecalis and the least against P. aeruginosa (Table  2).
JB 15B Psidium guajava 0 0 1.4 Crude extracts showed the highest biofilm destruction
JB 16B Psidium guajava 0 0 1.27 activity against S. aureus (76%) using isolate JB 18B.
JB 18B Psidium guajava 0 1 1.2
JB 19B Psidium guajava 0 0 1.07 Microscopic observations
JB 20B Psidium guajava 0 1.2 1.7 Regarding the results of biofilm destruction we can
AF3 Anredera cordifolia 0 0 1.1 determined morphological changing, which destruction
JB 7F Psidium guajava 0 0 0

Table 2  Results of biofilm activity quantification against pathogenic bacteria

Pathogens Activity Isolates activity (%)
JB 3B JB 11B JB 14B JB 15B JB 16B JB 18B JB 19B JB 20B AF3 JB 7F EJB 7B

S. aureus Inhibition 87 61 67 80 72 65 90 58 35 86 X
Destruction 73 74 0 72 62 76 59 65 23 2 X
E. faecalis Inhibition 19 0 42 0 0 27 0 0 0 0 X
Destruction 56 4 54 71 35 64 45 23 37 0 X
B. cereus Inhibition 67 30 0 42 58 0 0 34 0 0 X
Destruction 45 55 0 26 10 23 9 9 0 0 X
V. cholerae Inhibition 87 0 14 0 56 18 0 80 0 0 63
Destruction 71 72 0 58 59 48 73 0 0 0 0
S. typhimurium Inhibition 29 8 37 21 33 0 0 27 8 0 5
Destruction 0 4 0 11 11 0 23 9 15 2 12
P. aeruginosa Inhibition 68 0 0 0 0 0 0 0 0 0 0
Destruction 20 4 0 0 0 0 23 0 0 0 0
X: no test was performed
Theodora et al. BMC Res Notes (2019) 12:732 Page 4 of 5

Fig. 1  SEM images of S. typhimurium biofilm destruction by extract of isolate JB 19B with (a) pathogen control and (b) control + extract (×250) and
Bacillus cereus biofilm destruction by extract of isolate JB 18B with (a) pathogen control and (b) control + extract (×500)

activity of extract from isolate JB 18B and JB 19 B against bacteria [16]. We also can conclude that quorum quench-
mature biofilm of B. cereus and S. typhimurium. ing activity is affected by bacteria producer and extract
concentration that we used [10].
Discussion Biofilm is a cell function whose gene expression is reg-
Based on primary screening of anti-quorum sensing ulated by quorum sensing [17, 18]. Therefore, quorum
activity results, we found 11 out of 60 phyllosphere iso- quenching mechanisms might be a good alternative to
lates were potential to be used as anti-quorum sensing overcome biofilm problems [19]. Based on quantifica-
agent. It might be happened because phyllosphere bac- tion of biofilm activity both in inhibition and destruction
teria need survival strategy in the stressful environment steps (Table 2), these extracts showed various results. The
due to the fluctuations in physical conditions and limited biofilm inhibition activity might happen because quorum
and highly heterogenous availability of nutrients [15]. sensing process of pathogenic bacteria was disturbed by
Based on antibacterial activity assay result, we found interfering autoinducer synthesis, cell to cell exchange,
that only isolate EJB 7B extract had antibacterial activ- autoinducer’s reception and transduction, and degrading
ity against Gram positive-biofilm forming bacteria. The autoinducer [7, 20].
result showed that most of them had no bactericidal The biofilm destruction activity might be the result of
activity towards biofilm-forming pathogenic bacteria enzyme that could hydrolyze the compound of biofilm or
which is would not lead to antibiotic resistance [10]. small molecule that induce biofilm destruction [21]. EPS
Based on the results, at 5  mg/mL concentration all of composition of pathogenic bacteria biofilms were diverse
the phyllosphere extracts have no activity. It might be due depending on the bacteria [22]. Various EPS compounds
to because the concentration were relatively small. JB 7F can be degraded by specific enzymes like proteases,
extract showed no activity at any concentrations because deoxyribonucleases, glycoside hydrolase [23].
it needed higher concentration for quorum quenching From SEM analysis, we can determine morphological
activity. In this study, inhibition of violacein pigments changing which showed destruction activity (Fig.  1). It
could happened because AHL from C. violaceum were indicated there is reduction of extracellular matrix and
degraded by metabolites that produce by phyllosphere this result approved quantification of antibiofilm assay
Theodora et al. BMC Res Notes (2019) 12:732 Page 5 of 5

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