The antimicrobial activity of plants in the vicinity of a geothermal area in Perak , Malaysia

We wish to report the study of the antimicrobial activity of plants collected in the vicinity of a geothermal area in Perak, Malaysia. This is the first report of these species in the vicinity of thextis geothermal area. The plants are locally known as Maman Pasir of the family Cleome icosandra, Cleomaceae, and species of the family Lamiaceae. Both Stachytarpheta are subshrubs and are believed to have specific biological activities as a result of living in such extreme areas. Methanol extracts of both plants revealed no antimicrobial activity against , Escherichia coli Staphylococcus and . aureus Pseudomonas aeruginosa


Introduction
Extreme environments provide a new scaffold for the study of natural products for drug discovery.These environments are a challenge to live in, therefore the plant species that are found there must have specific survival mechanisms.This increases the possibility of discovering unique biologically active compounds.The polar region is an example of an extreme environment that has been explored.In McMurdo Sound, Antarctica, benthic invertebrates are exposed to significant predation by sea stars and potentially infectious water-column microorganisms, which suggests that chemicallymediated defense strategies would be an advantage.For example, discorhabdin C (Figure 1), found in the sponge Lantrunculia apicalis, is alkaloid and possesses a unique structure with antitumor and antimicrobial activity, and plays a role in the sea star tube-foot retraction assay  6 , respectively.Locally, Hyptis suaveolens, located within the hot spring enclosure in the Tambun area of Perak is used to combat fever, and soothe headaches and skin rashes 7 .
Among the data presented in the 2nd edition of the National Antibiotic Guideline in conjunction with the Annual Scientific Meeting on Antimicrobial Resistance in 2015 in Putrajaya, Malaysia, was resistance of Escherichia coli to Ampicillin, which had risen to more than 50 percent.Streptococcus pneumoniae resistance towards Erythromycin had increased from 18.2 percent in 2013 to 28.1 percent in 2014, and Acinetobacter baumannii resistance against Meropenem had increased from 47.7 percent to 57.3 percent within seven years 8,9 .Additionally, P.aeruginosa is generally resistant to a large range of antibiotics and may demonstrate additional resistance after unsuccessful treatment 10 .Hence, research for the discovery of new antibiotics and drugs against these bacteria is very much needed.

Plant material
Plants were collected by hand on the 26 th of April 2016 in the vicinity of Ulu Slim, Perak, Malaysia (Figure 2).It should be noted that these plants do not have flowers or any significant morphology that allows their identification, so identifying them was a challenge.crude extract was obtained.A total of 56.8 mg crude extract of C.icosandra and 65.6 mg crude extract of Stachytarpheta sp. was obtained, after solvent removal using a rotary evaporator.

Antimicrobial assay
The antimicrobial assay consisted of the applied agar diffusion method, or Kirby-Bauer method 11,12 .The assay consists of inoculating an agar plate with the chosen microorganisms, followed by placement of paper discs which have been impregnated with a known concentration of antibiotic.After incubation, the inhibition zone is measured.
In our assay, three bacterial isolates of E.coli, S.aureus and P.aeruginosa were grown in nutrient broth at 37°C overnight, using a shaking incubator.The three bacterial isolates were subcultured from an existing culture, so we are unable to state the strain of bacterial isolates.Plates were swabbed with cotton wool impregnated with the microorganisms.Individual filter paper discs (Whatman, Cat No 1001 110; 6 mm diameter) were saturated with 10 µL solution containing approximately 1 mg/mL of crude extract and dried for 15 minutes in the laminar flow before they were placed on the top of cultured nutrient agar.The plates were then sealed with parafilm and inverted before being incubated at 37°C overnight.
After incubation, the inhibition halo was measured with a ruler (± 0.5mm); the measured distance was from the edge of the paper disc to the widest part of the inhibition halo.The average distance across the inhibition halos from the filter paper was taken as the level of inhibition of bacterial growth for each sample.

Results and discussion
Both plants were collected in the vicinity of the geothermal site at Ulu Slim, Perak, Malaysia.Water temperature was 90°C at the time of collection.Upon collection, plants were immediately identified by a botanist and then underwent methanol extraction.Both plants were identified to be C.icosandra (also called C.viscosa), locally known as Maman Pasir of the family Cleomaceae.In addition, Stachytarpheta species of the family Lamiaceae were also collected.Further species identification needs to be conducted for this particular plant; they are identified based on plant morphology and taxonomy.It should be noted that both plants are very small, subshrub-like and only consist of leaves that are absent of flowers.We believe that these plants may possess unique, biologically active compounds.We proceeded to evaluate their antimicrobial activity, but crude methanol extracts of both plants revealed no activity against the three tested bacteria E.coli, S.aureus and P.aeruginosa (Table 1, Figure 3)

Figure 2 .
Figure 2. Geothermal area in Ulu Slim, Perak, Malaysia, where the plants were collected.

Figure 3 .
Figure 3. Antimicrobial assay of crude methanol extracts of C.icosandra and Stachytarpheta sp.The figure displays petri dishes containing filter paper discs with saturated crude methanol extracts of C.icosandra �C�� and Stachytarpheta sp.�D��, in �i�� E.coli, �ii�� S. aureus and �iii�� P. aeruginosa.No inhibition halo was observed.
51][2][3].Malaysia, although the climate is tropical, is rich in various geography, including geothermal areas in the form of hot springs.The biodiversity of geothermal areas in Malaysia has yet to be fully studied, therefore, an opportunity to investigate the bioactivities of plants that grow near geothermal areas, has risen.Plants that survive here are unique because of their ability to adapt to extreme temperature.The most heat-tolerant plants are mosses and lichens, which can survive ground temperatures of 70°C.Prostrate kanuka (Kunzea ericoides var.microflorum) is a low, spreading variety of the kanuka shrub that only grows in geothermal areas.It can tolerate ground temperatures of up to 55°C 4 .Additionally, grasses such as Dichanthelium lanuginosum and Paspalum laeve Michx were also found in geothermally heated environments, namely in the Yellowstone National Park5and Hot Springs National Park, Arkansas