Submitted : 22-06-2024 Revised : 25-08-2024 Accepted : 10-03-2024 Majalah Obat Tradisional (Trad.
Med.
January-April 2025 Vol.
, p 110-120 ISSN-p : 1410-5918 ISSN-e : 2406-9086 Antibacterial and Antibiofilm of Cinnamomum burmanii Bark Oil (CbBO) against Klebsiella pneumoniae ATCC 700603: In Vitro Study Inayati Inayati1,2*.
Hartono Hartono1,3.
Dono Indarto1,4.
Betty Suryawati1,5 Doctoral Program of Medical Sciences.
Faculty of Medicine.
Universitas Sebelas Maret Surakarta.
Indonesia 2 Department of Microbiology.
Faculty of Medicine and Health Sciences.
Universitas Muhammadiyah Yogyakarta 3 Department of Physiology.
Faculty of Medicine.
Universitas Sebelas Maret Surakarta.
Indonesia 4 Department of Department of Physiology and Biomedical Laboratory.
Faculty of Medicine.
Universitas Sebelas Maret Surakarta.
Indonesia 5 Department of Microbiology.
Faculty of Medicine.
Universitas Sebelas Maret Surakarta.
Indonesia
ABSTRACT
Many cases of multidrug-resistant (MDR) bacterial infections are caused by Klebsiella pneumoniae (K.
, an infectious disease bacterium.
Hospital inpatients can be exposed to this occurrence.
MDR has 96% of infections at Klaten Hospital into highly infectious biofilms, while 54.
49% of infections have produced biofilms.
Approximately 80% of bacterial illnesses resistant to antibiotics are caused by biofilm-forming bacteria.
To reduce biofilm formation, antibacterial compounds, one of which comes from natural products, are necessary.
Renowned for its essential oil.
Cinnamomum burmanii Bark Oil (CbBO) has been utilized extensively in herbal medicine to combat pathogenic bacteria such as Pseudomonas aeruginosa.
Candida albicans.
Enterobacter spp.
, and Staphylococcus aureus.
This study aims to evaluate CbBOAos antibacterial capabilities using the microdilution method and its antibiofilm properties against K.
pneumoniae ATCC 700603 using the MTT test.
A CbBO minimum bactericidal concentration (MBC) of 0.
25 mg/mL and a minimum inhibitory concentration (MIC) of 0.
125 mg/mL were employed for antibacterial activity.
The antibiofilm potential was determined by measuring the minimum biofilm eradication concentration (MBEC) at 0.
5 mg/mL and the minimum biofilm inhibition concentration (MBIC) 25 mg/mL.
In conclusion.
CbBO demonstrated antimicrobial and antibiofilm qualities.
However, a greater concentration of CbBO was required for antibiofilm formation than for antibacterial purposes.
Keywords: Antibacterial.
Antibiofilm.
Cinnamomum burmanii.
Essential Oil.
Klebsiella pneumoniae INTRODUCTION Klebsiella pneumoniae (K.
is a gram-negative rod bacterium infecting the bloodstream and tissues.
A recent study discovered that multidrug-resistant K.
has caused community-acquired infections (Murphy & Clegg, 2.
pneumoniae belongs to the Enterococcus faecium.
Staphylococcus aureus.
Klebsiella pneumonia.
Acinetobacter baumannii.
Pseudomonas aeruginosa.
Enterobacter spp.
, and Escherichia coli (ESKAPEE) bacteria category.
Those bacteria have been reported to be multidrug-resistant (MDR), extensively drugresistant (XDR), and even pan-drug-resistant (PDR), identified by the World Health Organization (WHO) as a priority pathogen requiring strict management (Yu et al.
, 2.
The 2019 global epidemic led to a prevalence of 50% of carbapenem-resistant pneumonia (CRKP) infections and 4.
1% to 79.
of ciprofloxacin-resistant XDR K.
infections (GLAS, 2.
In Singapore, the mortality rate from K.
pneumoniae bacteremia reached 20% to 26% (Chew et al.
, 2.
*Corresponding author : Inayati Inayati Email : inayati@umy.
Meanwhile, in Indonesia, a study conducted at RSUP Dr.
Soeradji Tirtonegoro Klaten disclosed 49% of patients had MDR K.
infection, and 85.
63% of those isolates produced biofilms with a high intensity of 26.
95% (Nirwati et , 2.
The emergence of biofilm-forming bacteria has triggered the development of antibiotic resistance and chronic diseases.
Compared to nonbiofilm-forming bacteria, biofilm-forming bacteria antimicrobials (Shadkam et al.
, 2.
Antibiotic sensitivity of biofilm bacteria differs from that of planktonic bacteria.
for example, the minimum inhibitory concentration (MIC) value of ampicillin for Escherichia coli ATCC 25922 is 2 AAg/mL in the planktonic phase, but it escalates to 512 AAg/mL in the biofilm phase (Fux et al.
, 2.
Planktonic bacteria are the primary target of antibiotic development efforts aimed at combating multidrug-resistant pathogenic bacteria and host immunity (Bjarnsholt et al.
, 2.
There is still a need for effective biofilm inhibition, eradication techniques, and new antibacterial sources.
Antibiofilm strategies encompass the prevention of biofilm development and the physical, chemical.
DOI: 10.
22146/mot.
97396 | Majalah Obat Tradisional, 30.
, 2025 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.
0 International License.
Antibacterial and Antibiofilm of Cinnamomum burmanii Bark Oil (CbBO) and biological destruction of biofilms.
Examples of such mechanisms include the use of chelating phytochemicals, and quorum sensing inhibitors (QSI) (Borges et al.
, 2.
Essential oil (EO) possesses substantial antibacterial properties that enable it to prevent and manage microbial illnesses, making it a significant natural product ( Tariq et al.
, 2019.
Yu et al.
, 2.
Cinnamon (Cinnamomum burmani.
is a medical herb frequently utilized due to its abundance of beneficial compounds and essential oils, including cinnamic acid, eugenol, cinnamaldehyde, and cinnamate ( Tung et al.
, 2010.
Vangalapati et al.
Rao & Gan, 2.
This plant is indigenous to Indonesia.
Its bark extract exhibits antibacterial activity against various pathogenic pathogens, such as Pseudomonas aeruginosa.
Candida albicans, and Enterobacter spp.
(Silvia Novita, 2.
Cinnamaldehyde, the primary ingredient in cinnamon oil, inhibits the same target on the plasma membrane as chlorhexidine does (Nuryastuti et al.
, 2.
The essential oil derived from C.
verum leaves possesses a high eugenol content and strong antibacterial and antibiofilm properties against K.
Staphylococcus aureus, and Pseudomonas aeruginosa, with an MIC 5 mg/mL and an MBC of 1.
0 mg/mL.
Minimum biofilm inhibition concentrations (MBIC.
mg/mL and minimum biofilm eradication concentrations (MBEC) of 4.
0 mg/mL were SEM revealed reduced biofilm density, cell shrinkage, and cell wall damage (Wijesinghe et al.
, 2.
Cinnamon oil could effectively inhibit and kill germs and has an antibiofilm effect against Methicillin-resistant Staphylococcus (MRSA), with MIC and MBC values of 0.
25 mg/mL and MBIC and MBEC values of 1.
0 mg/mL (Cui et al.
, 2.
The literature currently lacks information on natural compounds with antibacterial properties that prevent the growth of pneumoniae biofilm, especially cinnamon oil derived from Cinnamomum burmanii.
Accordingly, this study aims to assess the antibacterial and antibiofilm capabilities of CbBO against the standard isolate K.
pneumoniae ATCC 700603.
polystyrene plate tip, spectrophotometer (Thermo Scientific TM), sterile test tube (Iwaki TM), sterile 96well-flat-bottom microplate (Iwaki TM), and microtiter plate reader (Bio-rad TM).
MATERIALS AND METHODS
Preparation of Klebsiella pneumoniae Biofilm Model pneumoniae isolates grown statically for three hours in TSB at 37 AC were adjusted to a bacterial concentration of 1x1010 CFU/mL.
Three iterations of the biofilm test were performed using 24-well polystyrene plates containing 400 AAL of high glucose TSB in each well.
Each well received 100 AAL of bacteria, which were then statically incubated for 24 hours at 37 AC.
The biofilm was Instrument This study utilized several instruments, encompassing mortar or pestle, round-necked flask, distillation flask, drip funnel, separating funnel, rotary evaporator (IKA TM), biosafety cabinet class II (Thermo Scientific TM), petri dish (Iwaki TM), ose needle, electric bunsen, microscope (Olympus TM), slide, incubator (Memmert TM), 96well microplate (Iwaki TM), micropipette, 24-well Majalah Obat Tradisional, 30.
, 2025 Material This study employed several materials, including Cinnamomum burmanii (Nees & T.
Nee.
Blume-type cinnamon bark oil and a standard isolate from the Lauraceae.
pneumoniae subsp.
Pneumoniae ATCCTM 700603 Thermo ScientificTM Culti-LoopsTM 700603 K.
pneumoniae ATCCTM R4603074.
MacConkey medium (Oxoid TM).
API-E identification kit (Biome Rieux TM).
TSB with 1% glucose (TSBG) (Oxoid TM), phosphate buffered saline solution (PBS, pH 7.
(Merck TM).
HuckerAos crystal violet of 2% .
1% w/.
(Merck TM), sterile distilled water, acid glacial acetate (Merck TM), menadione crystallin (Sigma MCLS TM).
MTT (Bio basic TM), dimethyl sulfoxide (DMSO) (Sigma TM), 2% formaldehyde (Sigma TM).
BHI broth (Oxoid TM).
Mueller Hinton agar (Oxoid TM), and sterile distilled water (DW).
Method Preparation of Cinnamomum burmanii Bark Oil using Steam Distillation Cinnamon bark (Cinnamomum burmani.
was ground using a mortar and pestle and then transferred into a 100 mL round-necked flask.
Distillation began after adding 40 mL of water.
After gathering about 20 mL of the hazy distillate, the distillation flask was filled with an additional 20 mL of water using the drip funnel.
After collecting 20 mL of distillate, the procedure was Subsequently, the distillate was placed onto a separating funnel and extracted with 2 y 20 mL of diethyl ether.
The fine layer was dried over anhydrous magnesium sulfate, and the mixture was filtered.
The ether was removed, and the cinnamon essential oil was collected using a rotary One kg of Cinnamomum burmanii bark yielded 3 mL of CbBO.
Hence, a CbBO concentration 3% .
signifies that every 100 g of Cinnamomum burmanii bark contains 0.
3 mL of CbBO or 3 AAL/g (Prajapati et al.
, 2.
Inayati Inayati preserved with 1 mL of 2% formaldehyde for 20 minutes, rinsed three times with PBS, and then stained for 20 minutes with 0.
5% crystal violet.
To achieve absorbed staining, 1 mL of 95% ethanol was applied to the plates after they were properly cleaned with distilled water.
A spectrophotometer read the staining at 570 nm, and the biofilm intensity was calculated using optical density (OD) and optical density control (OD.
OD O ODc indicates a negative biofilm intensity.
OD < 2
signifies a weak biofilm.
OD < 4 implies a medium biofilm, and OD < 4 represents a strong biofilm (Vuotto et al.
, 2014.
Alcyntar-Curiel et al.
, 2.
Determination of Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration (MIC) All test isolates were utilized to create standard cell suspensions in sterile BHI broth using 24-hour bacterial isolates.
After that, several colonies were inoculated on 10 mL of TSB with 1% glucose (TSBG) and kept at 37 AC for the entire day.
To achieve the desired concentration, each sample was diluted 1:100 in new TSB to 0.
5 McFarland .
5 x 108 CFU/mL).
To generate serial dilutions of CbBO .
AAL/wel.
, 32 mg/mL of the bark oil was put into a 96-well sterile flat-bottom microtiter plate and subsequently diluted with sterile BHI broth.
Then, a standard cell suspension that had been separately produced .
AAL/wel.
was added to the microtiter plate as an inoculant .
egative control .
rowth contro.
: 100 AAL BHI broth standard cell suspensio.
MIC was determined by inoculating 5 AAL of solution from each well on a Mueller Hinton agar plate and incubating aerobically at 37 AC for another 24 Determination of Minimum Biofilm Inhibitory Concentration (MBIC.
TSB/BHI liquid medium of 180 L and cinnamon oil of 20 L were added into the well of Meanwhile, the wells of column 2 and so on were filled with 100 L of TSB/BHI liquid As much as 100 L of fluid from column 1 was put in column 2 to create serial delusions.
Several wells were utilized for control: negative control .
rowth control, 100% growt.
, positive control .
L of liquid medium containing a certain concentration of CbBO), solvent control, and media control.
Subsequently, 10 L of 108 CFU/mL bacterial suspension was added.
The microtiter plate was incubated for 24 to 48 hours at 37 oC, and biofilm staining was performed with MTT.
The absorbance was read with an ELISA reader at a wavelength of 560 nm .
to 700 n.
The experiment was carried out in six repetitions at two different times (Wijesinghe et al.
, 2.
Determination of Minimum Biofilm Eradication Concentration (MBEC) The concentration (MBEC) was determined using the method from Ramage et al.
The MBEC determination principle involves initially growing biofilm cells and subsequently subjecting them to treatment with CbBO.
The steps involved growing biofilm isolate K.
pneumoniae ATCC 700603 in a MacConkey selective medium for 48 hours at 37 oC.
The supernatant and planktonic cells were carefully discharged and washed three times with 200 L of PBS.
Serial samples of CbBO were created using another 96-well plate.
As much as 100 L of liquid medium containing the concentration of CbBO was added to the plate on which the biofilms were grown.
Then, it was incubated at 37 oC for 24 The medium solution was then discharged and washed three times with 200 L of PBS.
A total of 100 AAL of MTT solution was added to each 96well-containing biofilm cell.
The plate was closed and incubated at 37 oC for two hours.
The plate cover was removed, and the solution was thrown away after the incubation.
A total of 100 L of isopropanol acid at 5% was poured and incubated for one hour at room temperature.
The absorbance was determined using an ELISA reader set to 560 nm in wavelength (Ramage & Wickes, 2.
RESULTS
CbBO contains several chemicals, with cinnamaldehyde being the most prevalent .
78%), followed by alpha copaene .
42%).
The chemical content of CbBO obtained through steam distillation using gas chromatography-mass spectrometry (GC-MS) was analyzed qualitatively, as displayed in Figure 1.
The CbBO MIC value had the least amount of CbBO concentration able to prevent the growth of the standard isolate K.
pneumoniae ATCC 700603.
Based on the OD value, as depicted in Figure 2, the MIC value of CbBO against the standard isolate pneumoniae ATCC 700603 was 0.
125 mg/mL.
The minimal bactericidal concentration (MBC) was determined based on the lowest CbBO concentration not demonstrating any growth of K.
pneumoniae colonies on the MacConkey selective Figure 3 exhibits the MBC CbBO values for the standard isolate K.
pneumoniae ATCC 700603, the negative control, and the positive control.
Antibiofilm potency of CbBO was measured using the MBIC and MBEC values.
Treatment with 25 mg/mL CbBO inhibited the biofilm of the Majalah Obat Tradisional, 30.
, 2025 Antibacterial and Antibiofilm of Cinnamomum burmanii Bark Oil (CbBO) Figure 1.
Percentage of qualitative GC-MS test results of CbBO containing 86.
05% 3-phenyl-(CAS) Figure 2.
The MIC of CbBO at different concentrations against the standard isolate K.
pneumoniae ATCC 700603 (K.
pn ATCC) comparisons to negative and positive controls (Kn and K.
The CbBOAos percentage inhibition MIC against the isolate K.
pneumoniae ATCC 700603.
The OD value was measured with six repetitions at two different times.
standard isolate K.
pneumoniae ATCC 700603.
Figure 4 illustrates the MBIC value of CbBO against the reference isolate K.
pneumoniae ATCC 700603 25 mg/mL.
The results of measuring MBEC CbBO values at concentrations of 0.
5 mg/mL, 0.
25 mg/mL, 125 mg/mL, and 0.
06 mg/mL against the standard isolate K.
pneumoniae ATCC 700603 biofilm were compared with the negative control and positive control.
The MBEC CbBO value against the standard isolate K.
pneumoniae ATCC 700603 was measured based on the OD value, as displayed in Figure 5.
The MBEC value for CbBO treatment at a concentration of 0.
5 mg/mL against the standard isolate K.
pneumoniae ATCC 700603 biofilm Majalah Obat Tradisional, 30.
, 2025 depicted a lower OD value than the negative It indicates that CbBO at a concentration of 5 mg/mL can eradicate the biofilm of the standard isolate K.
pneumoniae ATCC 700603.
DISCUSSION