Artikel Penelitian

Detection of Biofilm Formation from Enterococcus faecalis of Urine Catheters

Using Congo Red Agar and Tissue Culture Plate
Mohamad Yanuar Prasetyo Nugroho1 Wani Devita Gunardi2 Elisabeth D. Harahap2

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Strata 1 Program Studi Kedokteran Umum, Fakultas Kedokteran Universitas Kristen Krida Wacana

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Staf Pengajar Departemen Mikrobiologi, Fakultas Kedokteran Universitas Kristen Krida Wacana

Correspondence Address : Jalan Terusan Arjuna Utara No. 6 Kebon Jeruk, Jakarta Barat

Email : wani_gunardi@ukrida.ac.id
Abstract
The National Healthcare Safety Network reported that from 2006-2008, the genus
Enterococcal was the second most common Catheter Associated Urinary Tract Infections (CAUTI)
prior to Eschericia coli. The biofilm has an important role of the CAUTI pathogenesis. The role
of Enterococcus faecalis as the etiology of urinary tract infection has been related to its capability
to form the biofilm. There are several methods to detect biofilm production for example Tissue
Culture Plate (TCP), Tube method (TM), and Congo Red Agar method (CRA). The goal of this
study was to find the effective method to detect biofilm from E. faecalis. A total of thirteen E.
faecalis bacterial isolates from catheter culture were tested by the TCP method and CRA for
detection the biofilm formation. We obtained 61.5% and 69.2% of bacteria E. faecalis which were
able to produce biofilm by TCP and CRA. The sensitivity and specificity of the CRA compared
with TCP method were 75% and 40%. The CRA method was rapid and simple to do the detection
of biofilm formation but the specificity was not good enough, probably due to subjective paralax
reading of the results.

Key Words : Enterococcus faecalis, Urine Catheter, Biofilm, Congo Red Agar,
Tissue Culture Plate.

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Introduction
Enterococci is one of the normal intestinal flora of humans and animals, but these microbes
are important cause of nosocomial infection that infect various parts of the body and causing
bacteremia. Reported by 152 hospitals in United States, these bacteria has an important role in
nosocomial infections such as circulatory infections found as much as 11%, and as the most
pathogenic bacteria in surgical site infections found as much as 17%.1 For the last two decades,
the number of nosocomial infection by Enterococcus sp was rising. This can be caused by
excessive use of antibiotics, as well as the presence of biofilm factors that support the process of
the exchange of antibiotic-resistant determinant factors.2

One of the roles of biofilm is the ability of bacterial resistance in biofilm structures to increase
up to 1000 times more resistant to antibiotics than planktonic cells due to several mechanisms.3
Enterococci faecalis have the ability to express proteins on the surface that allow biofilm
colonization and formation on the host itself.4 According to Barbara, biofilms act as the main focus
of chateter-assosiated urinary tract infection (CAUTI).5 There were two studies on the prevalence
of biofilm producing bacteria which concluded that the majority of bacteria that produce biofilms
from a urine catheter were Enterococcus faecalis.6,7

The results of Enterococcus bacterial biofilm production also vary in many countries, such as
in the United Kingdom (UK), from 109 Enterococcus isolated from blood vessel catheters, E.
faecalis (100%) and E. faecium (50%) produce biofilms.8 Another study in Poland concluded that
59% of E. faecalis isolated from the urinary tract can form biofilms.9 In research from Pakistan,
75% of biofilm production was obtained from uropathogens from the urinary tract. The biofilms
were mostly detected from S. aureus (75%), E. faecalis (75%), and E. coli (40%).10 While in Japan,
all 352 E. faecalis samples from urinary tract infections can form biofilms classified as weak,
medium, or strong biofilm producer.11

There were several studies that mention methods for detecting biofilms such as Tissue Culture
Plate (TCP) was better at detecting biofilm-forming bacteria compared to Congo Red Agar
(CRA).6,12 Until now, there are still controversials about which method is most efficient for
detecting biofilms bacteria. There were two studies that mentioned CRA method have been carried
out, the result was this method is quite simple, easy, inexpensive, and the assessment is fast, but
the results can only be assessed based on qualitative visual analysis and subjective visual

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dependence.6,13 Therefore, this study wants to test whether the CRA method can be used for
biofilm detection test from Enterococcus faecalis bacteria from urine catheter isolates.

Method
The research design used was an observational research design, to determine the ability of
biofilm formation from thirteen Enterococcus faecalis bacteria from urinary catheter isolates
identified using Vitek2 (Biomeriux). Urine catheter samples were taken from August 2016 to
November 2016 in Tangerang private hospitals. Enterococcus isolates were made in culture stock
and stored at -80oC. This research was conducted in FK Ukrida laboratory, starting from August
2017 - October 2017.

1. Congo Red Agar Method 13,14

Agar medium in this method contains brain heart infusion broth (37 gr / L), sucrose (50 gr
/ L) and agar no.1 (10 g / L) and congo red dye (8 g / L) and congo red dye was dissolved
with aquadest on different erlenmeyer, both were carried out by autoclaving (121˚C for 15
minutes) separately. Congo red dye solution is then mixed into brain heart infusion sucrose
at 55˚C. Each CRA plate was then inoculated with each of the thirteen Enterococcus
faecalis and incubated for 24 hours at 37˚C. Strains that were able to form biofilms will
appear as black colonies, while strains that were unable to form biofilms will appear as red
colonies. Negative control in this study used sterile CRA, while positive control was used
by E. Coli ATCC e35218. This method is done with three repetitions.

2. Tissue Culture Plate Method. 15–18

Each test tube contained 10 ml of Luria-bertani Broth (LBB) (25 g / L) inoculated with
each of thirteen Enterococcus faecalis and incubated for 24 hours at 37˚C. The bacterial
suspensions then made with a standard 0.5 McFarland (1.5 x108 bacterial suspension / mL)
and each bacteria was diluted 1:11 using sterile LBB into each well of flat bottom tissue
culture plate. The suspensions in the plate were incubated for 48 hours at 37˚C. After the
incubation is complete, remove the suspensions by flipping the plate quickly and give 200 µl
of distilled water to each well, flip the plate back and waiting for the plate to dry. Give each
well 100 µl of 0.1% crystal violet solution for 15 minutes. The crystal violet solution then
discharged by flipping the microplate quickly, then each well is washed again using aquadest

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 200 µl each well. After washing, the microplate was dried to the same temperature as the
room. After the microplate was dry, give 150 μl of 96% ethanol, then the microplate must be
closed to reduce evaporation. The results will be read using ELISA plate reader at a
wavelength of 595nm. In this method, sterile LBB was used as a negative control. This method
was done with three repetitions, and the average value of Optical Densities (OD) of the three
will be taken as the result value.

Results
The results showed that 8 isolates (61.5%) from a total of 13 isolates detected by biofilm using
the TCP method as gold standard whereas with the CRA method detecting 9 (69.2%) strains were
biofilm producers from 13 E. faecalis isolates. CRA is a method used to look at the characteristics
of bacterial phenotypes in CRA media. The results obtained was in qualitative data in the form of
different colors in the colony. In Figure 1. shows the color of the colony produced in the CRA
method, there are difficulties in determining the interpretation of results based on colony color
similarities because subjective determination.

Figure 1. Color of Bacterial Colonies E. faecalis. (a, b, c); Color of Red Colonies, Non-Biofilm
Producing Colonies; (d), (e), (f); Color of Black Colony, Biofilm Producing Colonies

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 Table 1. Interpretation of Thirteen Isolates E. faecalis Based on OD from ELISA plate reader
results of Tissue Culture Plate
Formula Pembentuk Biofilm Perhitungan Jarak OD Jumlah Isolat
OD < ODc Non-Biofilm <0,462 5 (38,5%)

OD > ODc Lemah 5 (38,5%)

OD < 2ODc 0,462 - 0,924
OD>2ODc Sedang 3 (23%)
OD < 4ODc 0,924 - 1,848
OD > 4ODc Kuat >1,848 0 (0%)

In table 1. the formula for interpreting the results of the TCP method is explained according
to the criteria of Ibrišimović et al to divide a weak, medium and strong biofilm.18 This method
founds E. faecalis as biofilm producers were medium, weak and did not produce biofilm of 5
(38.5%), 3 (23%) and 5 (38.5%). In this study, there was no strong biofilm formation because no
OD value did not exceed four times of Optical Density control (ODc).

Table 2. Diagnostic Test Results from the CRA Method results with the TCP method
Metode TCP
TCP (+) TCP (-) Jumlah
Metode CRA (+) 6 (a) 3 (b) 9
CRA CRA (-) 2 (c) 2 (d) 4
Jumlah 8 5 13

Diagnostic test results to see the sensitivity and specificity values of the CRA method by
comparing the TCP method as gold standard were 75% and 40% (Table 2).
Table 3. Comparison of Biofilm Formation Detection Results from TCP and CRA (+) methods.
Klasifikasi biofilm Uji Deteksi Biofilm
TCP CRA (+)
Kuat 0 0
0% 0%
Sedang 3 3
23% 23%
Lemah 5 3
38,50% 23%
Non-Biofilm 5 3
38,50% 23%

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Discussion
From the results of this study, CRA method with a sensitivity value of 75% can be used to
detect biofilm formation from the Enterococcus faecalis and recommended as a screening method
for biofilm formation tests. These results are consistent with previous studies from Rajkumar who
tested 151 clinical isolates.19 The limitation of the CRA method is cannot determine its strength
degree (weak, medium, and strong) such as the TCP method. CRA method cannot detect biofilm
formation in all isolates whose ability to form biofilms includes a weak degree. Enterococcus
faecalis has the ability to ferment sugars such as glucose and lactose. The results of this
fermentation metabolite are needed to bind to the congo red to form a black colony. The
Enterococcus faecalis strain which has weak biofilm formation ability can be detected by the TCP
method because in this method, the growth medium is rich in nutrients which will help the
formation of biofilms through metabolites of sugar-fermenting. E. faecalis have an extracellular
matrix consisting of polysaccharides, proteins, DNA and RNA. CRA is a method that can detect
polysaccharide characteristics from the formation of E. faecalis biofilms. This shows a negative
result in CRA still have the possibility to form biofilms with a weak degree. This means that
bacteria that have the ability to form weak biofilms cannot necessarily be detected by the CRA
method. Another disadvantage is the error in eye parallax when interpreting the results on isolates
that should be negative. This shows that the CRA method is sensitive to detect medium and strong
biofilm producers, but in the weak biofilm form the sensitivity of CRA decreases.
Many factors to make biofilm structures adhere to a surface, one of which is the physical and
chemical environment and the location of biofilm attachments have a very big impact on biofilm
formation.20 Pantanella et al mentions, environmental conditions where biofilm growth is an
important factor that can affect the detachment or absence of bacterial cells from the attachment
site.21
On the other hand, the CRA method is an easy, fast, and inexpensive method to do. This
method is an agar media method that doesnt need tools for reading it, but the method of reading is
a weak point of human error which is a lack of the CRA method. In TCP, it takes more time than
the CRA method (CRA takes 24 hours, TCP takes 48 hours). TCP lacks itself in the way that work
requires special skills to work on this method.

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Conclusion
The results showed that 61.4% of E. faecalis from urinary catheter isolates formed a biofilm
with the TCP method as the gold standard, but for its implementation requires time, skills and
special tools.

CRA is a cheap, fast, and easy method to do. The difficulty of reading subjective results makes
CRA have a lack and resulting in less sensitive results. From the results of the diagnostic test, the
sensitivity and specificity of the CRA method is 75% and 40%, so the CRA method can be used
as a screening test for detection of biofilm formation in the Enterococcus faecalis.
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