8 513 Billy Cris (Short Com) ... SHORT COMMUNICATION Screening of Quorum Quenching Activity of Bacteria Isolated from Ant Lion BILLY CHRISTIANTO YOGIARA*AND Faculty of Biotechnology, Universitas Katolik Indonesia Atma Jaya, Jalan Jenderal Sudirman 51, Jakarta 12930, Indonesia g B . Key words: g K . Kata kunci: acterial intercellular communication or quorum sensing controls the pathogenesis of many medically important organisms. Therefore, it is important to isolate bacteria that can disintegrate the communication, in a process called quorum quenching. Bacteria from ant lions ( sp.) were grown on Luria agar, and approximately 1.85 x 109 CFU mL was obtained. Seven morphologically different colonies were screened for quorum quenching activity using wild type as an indicator. One isolate (Myr7) was found to possess quorum quenching activity, was later identified as by employing 16S rRNA ant lion, quorum sensing, quorum quenchin omunikasi intersel bakteri atau telah diketahui mengendalikan sifat patogen dari beberapa organisme yang penting secara medis. Oleh karena itu, isolasi bakteri yang dapat memutus rangkaian komunikasi intersel-yang disebut dengan -menjadi sangat penting. Bakteri dari undur-undur ( sp.) yang ditumbuhkan pada media agar-agar Luria, menghasilkan sejumlah 1.85 x 109 CFU mL . Sebanyak tujuh koloni yang berbeda secara morfologi ditapis aktivitas -nya dengan sebagai bakteri indikator. Penapisan tersebut menghasilkan satu isolat (Myr7) yang memiliki aktivitas yang diidentifikasi sebagai menggunakan gen penyandi 16S rRNA undur-undur, , Myrmeleon Chromobacterium violaceum Aeromonas quorum sensing quorum quenching Myrmeleon quorum quenching Chromobacterium violaceum quorum quenching, Aeromonas quorum sensing quorum quenchin -1 -1 Antimicrobial agents, including antibiotics and related medicinal drugs, have long been used for treatments to reduce the threat posed by infectious diseases. Unfortunately, many pathogenic bacteria have become resistant to several antibiotics, for i n s t a n c e , p e n i c i l l i n - r e s i s t a n t (Bacquero 1995; Goldstein and Garau 1997), vancomycin-resistant enterococci (Dixson . 1985), methicillin-resistant (Sutherland and Rolinson 1964; Kareiviene 2006; Martins and Cunha 2007), multi-resistant salmonellae (Newell . 2010), and multi-resistant (Espinal . 2001). The increasing resistance of pathogenic bacteria to antibiotics may lead to public health risk. To overcome this problem, innovative strategies were needed to discover novel antibiotic targets or antivirulent drugs as alternatives to classical antibiotics (Baron 2010). It has been known that the nature of bacterial pathogenesis is controlled by quorum sensing mechanisms (Kievit . 2000; Williams 2000). To be able to communicate with other cells, the bacteria produce, detect, and respond to a small signal molecule called autoinducer. The autoinducer is responsible to S t re p t o c o c c u s pneumoniae et al Staphylococcus aureus et al. et al Mycobacterium tuberculosis et al et al et al. induce particular gene expression, including virulent genes. According to Finch . (1998), quorum sensing mechanism can be disrupted and has become a potential target for antiinfection therapy. Antiquorum sensing activity is often called quorum quenching. This activity can be useful to prevent colonization of pathogenic bacteria that use quorum sensing to regulate virulent genes. In recent years, quorum quenching enzyme and inhibitor from various sources have been studied, both from prokaryotic and eukaryotic organisms. There are two types of prokaryotic quorum quenching enzymes such as AHL- lactonase and AHL-acylase. AHL-degrading enzymes from eukaryotic organisms can be found on pig kidney (acylase I) and on airway epithelian humans (lactonase) (Dong and Zhang 2005). In this study we used ant lion ( sp.) whose bacterial community and potential activity are underexplored. Dunn and Stabb (2005) performed culture-independent 16S rRNA gene sequence analysis on the bacteria associated with the tissues of an ant lion, . All 222 sequences obtained by Dunn and Stabb (2005) were identified as These sequences could be subdivided into two main groups, the - with 75 clones similar to spp. and the - with 144 clones similar to the family et al Myrmeleon Myrmeleon mobilis Proteobacteria. Proteobacteria Wolbachia Proteobacteria α γ*Corresponding author, Phone: +62-21-5731740, Fax: +62-21-5719060, E-mail:yogiara@atmajaya.ac.id ISSN 1978-3477, eISSN 2087-8575 Vol 5, No 1, March 2011, p 46-49 I N D O N E S I A Available online at: http://www.permi.or.id/journal/index.php/mionline DOI: 10.5454/mi.5.1.8 E n t e ro b a c t e r i a c e a e Enterobacteriaceae Wolbachia et al. Bacillus cereus B. sphaericus, Morganella morganii, Serratia marcescens, Klebsiella Chromobacterium violaceum et al Chromobacterium violaceum et al et al et al C. violaceum . T h e y f o u n d t h a t t h e -like 16S rRNA gene sequences were most commonly isolated from gut tissue, and -like sequences were predominant in the head and body tissues. Nishiwaki (2007) has reported insecticidal activity of bacterial isolates of ant lion. Isolated , and species killed 80% or more cutworms when injected at a dose of 5 x 10 cells per insect. This study explored the possibility of finding antiquorum sensing molecule from ant lion-associated bacteria. The aim of this study is to obtain bacterial isolates that possess quorum quencing activity. About 20 ant lions were rinsed and surface- sterilized three times using sterile 0.85% of NaCl and vortexing. The specimen was put into a 50 mL conical tube and ground. About 1 mL of 0.85% of NaCl was a d d e d a n d s e r i a l d i l u t i o n s w e r e a p p l i e d . Approximately 100 µL sample was spread onto modified Luria agar (0.25% (w/v) trypton, 0.125% (w/v) yeast extract, 0.25% (w/v) NaCl, and 1.5% (w/v) bacteriological agar), and then incubated at 30 C for 2 - 3 d. Plate screening assay was used to evaluate quorum quenching activity with as an indicator (Adonizio . 2006). In brief, Luria agar plates spread with followed by spotting of tested bacteria. Plates were incubated for 2 d at 30 °C, and quorum sensing inhibition was detected by a ring of colorless, but viable, cells around the bacterial isolate colony (Adonizio . 2006). Molecular identification using 16S rRNA gene sequencing was carried out at Eijkman Molecular Biology Institute, Jakarta-Indonesia. DNA sequences were aligned to 16S-rRNA gene database provided by Ribosomal Database Project (RDP) web site (http://rdp.cme.msu.edu/index.jsp) (Cole . 2007, 2009). Phylogenetic tree was constructed using Treebuilder software provided by RDP and viewed by MEGA4 software (Tamura . 2007). A total of approximately 1.85 x 10 CFU mL bacteria were observed and seven isolates were successfully isolated from ant lions. One out of seven isolate were detected to possess quorum quenching activity (Fig 1). Both isolate produced extracellular compound that may degrade the signal molecule required for quorum sensing activity. The degradation was indicated by colorless surround- tested isolates. These colorless bacteria were confirmed to produce purple pigment when streaked back onto another plate (data not shown). Partial 16S-rRNA gene sequences of isolate Myr7 wes submitted to Genbank database 5 9 -1 o (www.ncbi.nlm.nih.gov) under accession number HQ453362. Molecular identification showed that isolate Myr7 had similarity to the genus . The isolate Myr7 had 98% similarity to strain M10 DQ200865. Phylogenetic tree analysis (Fig 2) showed phylogenetic position of isolate Myr7 among members of order. The tree revealed that the isolate Myr7 was clustered in the genus cluster and shared the same branch with subsp. ( D S M 3 0 1 8 7 ) a n d s t r a i n M 1 0 (DQ200865). These results are different from the study conducted by Dunn and Stabb (2005), who successfully identified the family from the South American ant lion as stated above. Quorum quenching activity was found in several bacteria, both Gram positive and Gram negative. It was reported that (Leadbetter and Greenberg 2000) and (Uroz . 2005) had the ability to use AHL molecules as nitrogen and carbon sources, respectively, as well as energy sources. Six bacteria isolated from the leaf surface of , i.e. , , , and , were proved to actively degrade acylhomoserine lactone as a signal molecule in quorum sensing mechanism (Morohoshi 2009). The quorum quenching activity were also shown by other bacteria including sp. strain 240B1 , , sp. IBN110., , strain PAI-A, PAO1, strain XJ12B (Dong and Zhang 2005). Not only prokaryotic organisms possess anti quorum Aeromonas Aeromonas Aeromonadales Aeromonas Aeromonas hydrophilla hydrophilla A e ro m o n a s Enterobacteriaceae Variovorax paradoxus Rhodococcus erythropolis et al Solanum tuberosum Agrobacterium larrymoorei R. erythropolis, B. silvestris Microbacterium testaceum B. cereus, Eschericia coli Bacillus , B. thuringiensis, B. cereus, B. mycoides, B. anthracis A. tumefaciens Arthrobacter K. pneumoniae Pseudomonas P. aeruginosa Ralstonia T Fig 1 Screening of quorum quenching activity of isolates Myr7 and MyrB. DH5 was used as a negative control.Escherichia coli Volume 5, 2011 Microbiol Indones 47 sensing activity, eukaryotic organisms have also been known to have a quorum quenching activity. Extracts of pea plants ( ) and crown vetch ( ) (Teplitski 2000), , , , , and (Adonizio 2006) have been tested of their abilities to inhibit the expression of quorum sensing activity byAHL inactivation. spp. has not been reported to possess quorum quenching ability. Therefore, this result was the first of such findings. Despite the ability to inhibit quorum sensing, member of the genus has been known to its quorum sensing machinery such as AhyRI and AsaRI that are homologous to LuxRI. produces C4-HSL and N-hexanoylhomo serine lactone (C6-HSL) (Swift . 1997). In conclusion, we have successfully isolated two bacterial isolates that possess antiquorum sensing activity against quorum sensing-regulated pigment production of . According to partial 16S- rRNA gene analysis, the bacterial isolate had similarity Pisum sativum Coronilla variations et al. C. erectus, Chamaecyce hypericifolia Callistemon viminalis Bucida burceras Tetrazygia bicolor Quercus virgiana et al. 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