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Antibacterial Activity of Java Turmeric (Curcuma xanthorrhiz

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sylvester william silan

on 18 May 2014

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Transcript of Antibacterial Activity of Java Turmeric (Curcuma xanthorrhiz

Curcuma xanthorrhiza
Klebsiella pneumoniae
Sylvester Anak William Silan
GS 35808
Master of Science (Food Science)

Supervisory Committee

Professor Madya Dr. Yaya Rukayadi (FSTM, UPM)
Professor Dr. Son Radu (FSTM, UPM)
Klebsiella pneumoniae is ubiquitous in environment such as vegetation, soil and surface water and furthermore the isolates from the environment were found to be as virulent as the clinical strains (Bagley, 1985; Struve and Krogfelt, 2004; Balestrino, 2008)

Outbreaks of gastrointestinal disease from the fresh produce were also found to be responsible from the bacterial contamination especially associated with Enterobacteriaceae family (Falomir et al., 2010)

Lawley et al., (2012) reported that fresh sprout products in Canada had been found to be contaminated with K. pneumoniae
According to Puspanadan et al., 2012 that some vegetables such as carrot, tomato, cucumber, lettuce and parsley has been contaminated by this bacteria.

Our interest mainly is on the
Curcuma xanthorrhiza
Roxburgh also known as temulawak in Malaysia.

The bioactive compound in
C. xanthorrhiza
, xanthorrhizol exhibited anticariogenic activity against
mutants (Rukayadi and Hwang, 2006), antifungal against
C. albicans
C. glabrata
C. guilliermondii
C. krusei
C. parapsilosis
C. tropicalis
(Rukayadi et al., 2006), anti-Malassezia activity against
M. furfur
M. pachydermatis
(Rukayadi and Hwang, 2007a) and antimycotic against opportunistic filamentous fungi (Rukayadi and Hwang, 2007b).

The antibacterial of xanthorrhizol against resistant
Klebsiella pneumoniae
has not been done.

Food poisoning in food products

The use of natural disinfectant which is safe and edible

May cause harm to human in long term

Chemical disinfectant and antibiotics
Klebsiella pneumoniae
1. To determine the susceptibility of temulawak extract againts resistant
K. pneumoniae
isolated from vegetables.

2. To assess the biofilm formation ability of resistant
K. pneumoniae
isolated from vegetables.

3. To analyse antibiofilm activity of temulawak extract and xanthorrhizol against resistant
K. pneumoniae
biofilms in vitro.

Highest concentration of extract
Lowest concentration of extract
Positive control
Negative control
Sample Collection
The rhizome of temulawak or Curcuma xanthorrhiza Roxb. were collected from Laboratory of Natural Product, Institute of Bioscience (IBS), Universiti Putra Malaysia, Serdang.

Plant Extraction
(Rukayadi et al., 2008)
100 g of dried powdered C. xanthorrhiza was soaked in 400 ml of 100% (v/v) methanol for 48 hours at room temperature.

The extract was filter and further treated with rotary vacuum evaporator to yield concentrated methanol extract at 50˚C 150 rpm.

0.1 g of extract was dissolved in 1 ml dimethylsufoxide (DMSO) to obtain stock solution. 10% DMSO does not kill the tested bacteria in this study.

Inoculum Preparation
24 isolates of Klebsiella pneumoniae was obtained from Bacteriology Food Safety Laboratory, Universiti Putra Malaysia, Serdang. The isolates were isolated from raw vegetables (Puspanadan et al., 2012).

Klebsiella pneumoniae ATCC 13773 from the American Type Culture Collection (Rockville, MD, USA) was used as a references control throughout the study

Disc Diffusion Assay
Methanolic extracts of C. xanthorrhiza were screened for antibacterial activity using standard paper disc diffusion assay according to CLSI method.

Bacteria strain was spread on Mueller Hinton agar plate using sterile cotton swab and 10 µl of extract was pipetted on the bacteria on agar plate for 24h at 37° C
Observed for any clear zone and perfomed two times in duplicate

Minimal Inhibitory Concentration (MIC)
The lowest concentration of seeds extracts that shows complete inhibition of visible growth.

Minimal Bactericidal Concentration (MBC)
The lowest concentration of antimicrobial agent at which no growth occur in the MHA agar plate

Kill-Time Curve
The concentration of C. xanthorrhiza used were 0× MIC, 1× MIC, 2× MIC and 4× MIC for resistant K. pneumoniae

Cultures with final volume 1ml was incubated at 37° C with agitation. A 1 µl aliquot was transfered to centrifuge tube containing 1 ml phosphate buffer (PBS) at predetermined time (0 min, 30 min, 1, 2, and 4 hours).

The aliquot (1 µl) was mix with the phosphate buffer and transfered into microcentrifuge tube containing 990 µl of phosphate buffer for serial dilution.

An appropriate volume (10 µl) was spread onto MH agar and incubated at 37° C for 24 hours to determine the numbers of cfu/ml.

Kill – Time Curve

Time-kill curves were plotted for resistant
K. pneumoniae
following exposure to
C. xanthorrhiza
extracts at 4 different concentration (0× MIC, ½× MIC, 1× MIC, 2× MIC and 4× MIC) with predetermine time of 0, 0.25, 30, 1, 2 and 4 h

Disc Diffusion Assay

The range for the inhibition zone for the resistant
K. pneumoniae
isolates was 9.0 to 10.0 mm

K. pneumoniae
ATCC 13773 have the inhibition zone of 12.0 mm

C. xanthorrhiza
extract was effective against standard
K. pneumoniae
ATCC 13773 and resistant
K. pneumoniae
isolates from several vegetables

Positive control
Chlorohexidine (CHX)
10% DMSO
C. xanthorrhiza
Table 1: Diameter of inhibition zone for
C. xanthorrhiza
extract on 24 isolates of resistant
K. pneumoniae
K. pneumoniae
ATCC 13773

Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)

The determination of MIC and MBC using the microdilution method according to CLSI 2012

The MIC and MBC value for K. pneumoniae ATCC 13773 was 156.25 mg/ml and 5000 mg/ml respectively

For all K. pneumoniae isolates, the MIC and MBC were 1.25 mg/ml and 2.5 mg/ml respectively

The isolates were cultured on 96-well microtitre plate for MIC determination

The colony then growth on MHA plates for MBC determination

Table 2: Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for
C. xanthorrhiza
against 24 isolates of resistant
K. pneumoniae
Time-kill curve shows that the reduction of resistant
K. pneumoniae
was fast acting;
> 3 log10 within less than 15 min at 4× MIC (5.0 mg/ml) for all isolates.
> 3 log10 after 4h at 2× MIC (2.5 mg/ml) for isolates from tomato
Figure 1: Methanolic extracts activity of C. xanthorrhizol as antimicrobial agents against 24 isolates of K. pneumoniae. Figure 2 (a)(b)(c)(d) was the activity of C. xanthorrhiza against K. pneumoniae isolated from cucumber, tomato, carrot and lettuce respectively.

Results and Discussion
Methanolic extract of
C. xanthorrhiza
have the antibacterial activity against resistant
K. pneumoniae

The MIC and MBC values of the
C. xanthorrhiza
extract was 1.25 mg/ml and 2.5 mg/ml

C. xanthorrhiza
extract have killed the resistant
K. pneumoniae
in 0.5h at 4× MIC (5.0 mg/ml) for all isolates and killed after 4h at 2× MIC (2.5 mg/ml) for isolates from tomato

Further studies should be aiming on the understanding the antibacterial mechanisms of the compound found in plant extract against the Gram-negative bacteria

Conclusion and Recommendation
1. Bagley, S. T. (1985). Habitat association of Klebsiella species. Infection control, 52-58.
2. Balestrino, D., Ghigo, J. M., Charbonnel, N., Haagensen, J. A., & Forestier, C. (2008). The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides. Environmental microbiology, 10(3), 685-701.
3. Falomir, M. P., Gozalbo, D., & Rico, H. (2010). Coliform bacteria in fresh vegetables: from cultivated lands to consumers. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, 2, 1175-1181.
4. Lawley, R., Curtis, L., & Davis, J. (2012). The food safety hazard guidebook. Royal Society of Chemistry. Pp124
5. Rukayadi, Y., & Hwang, J. K. (2006). In vitro activity of xanthorrhizol against Streptococcus mutans biofilms. Letters in applied microbiology, 42(4), 400-404.
6. Rukayadi, Y., & Hwang, J. K. (2007a). In vitro anti‐Malassezia activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb. Letters in applied microbiology, 44(2), 126-130.
7. Rukayadi, Y., & Hwang, J. K. (2007b). In vitro antimycotic activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb. against opportunistic filamentous fungi. Phytotherapy Research, 21(5), 434-438.
8. Rukayadi, Y., Yong, D., & Hwang, J. K. (2006). In vitro anticandidal activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb. Journal of Antimicrobial Chemotherapy, 57(6), 1231-1234.
9. Struve, C., & Krogfelt, K. A. (2004). Pathogenic potential of environmental Klebsiella pneumoniae isolates. Environmental microbiology, 6(6), 584-590
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