Studying quorum sensing inhibition on BMG LABTECH microplate readers

августа 14, 2020

The lab of Associate Professor Dr Crystale Lim Siew Ying focusses on antibiotic resistance profiling. A key area of their research is quorum sensing inhibition as an alternative way to address this problem.

Dr Angela Curtis | BMG LABTECH
Dr Angela Curtis
PhD, Head of Marketing, APAC
BMG LABTECH Australia

Table of contents

Quorum sensing inhibition: An alternative approach to suppress bacterial virulence

Antibiotic resistance has been recognized as a global healthcare crisis resulting in 700,000 deaths annually. It has been estimated that antibiotic resistant infections will cause 10 million annual deaths by 20501. While the race is on to identify and develop new antibiotics, scientists have turned to alternative approaches to address disease, such as the inhibition of bacterial virulence factors. This research has opened the door to the development of anti-pathogenic drugs.

 

One approach to suppress bacterial virulence is via quorum sensing inhibition.  Quorum sensing is a mechanism that bacteria use to monitor and respond to changes in environmental conditions, in a population density dependent manner2, 3.  In this process, bacteria ‘communicate’ by secreting small, diffusible signaling molecules called autoinducers. This system has been shown to regulate a variety of processes in bacteria, including virulence factor production, surface protein expression and biofilm development4.  

 

Quorum sensing was first described in Vibrio fischeri, a Gram-negative marine bacterium. At low cell density, V. fischeri is non-bioluminescent, but becomes bioluminescent as cell density increases via quorum sensing signal receptors, which activate a gene cluster responsible for light (Fig. 1)5

Identifying candidates for quorum sensing inhibition 

Crystale Lim Siew Ying is an Associate Professor and the Deputy Dean at the Faculty of Applied Sciences of UCSI University, Malaysia. Her laboratory works on community antibiotic resistance profiling as part of the Malaysian Antimicrobial Stewardship Programme6.  As an extension of this area of research, she also works on quorum sensing inhibition. Vibrio fischeri (unpublished work) and Chromobacterium violaceum are used as bacterial models to identify potential inhibitory compounds from plant sources2.  

 

The identification of a potential candidate that induces quorum sensing inhibition without interfering with bacterial growth could be a promising target for anti-pathogenic drugs, as it places less selective pressure on pathogens and avoids the development of resistant bacteria7. One such inhibitor is the compound isoprenyl caffeate originally found in manuka propolis6. Research continues in further understanding this inhibitor in pathogenic bacteria to elucidate the molecular mechanisms of its action.

 

Crystale described how BMG LABTECH’s FLUOstar® Omega microplate reader has supported her research. “The FLUOstar Omega has been useful in antimicrobial and quorum sensing inhibition assays on model bacteria of various colors, including bioluminescence. Antimicrobial studies using Minimum Inhibitory Concentration (MIC) are relatively straightforward, usually using turbidity as a measure of bacterial growth at OD600. Things are a little different for quorum sensing inhibition assays using bacteria models such as C. violaceum that is based on the production of a violet-colored pigment, violacein, as an indicator of active quorum sensing, or V. fischeri that is based on bioluminescence. For quorum sensing inhibition, both the growth and color indication of the bacteria model should be assayed simultaneously during treatment with a potential inhibitor to show that the inhibitor does not interfere with bacterial growth (examples in Fig. 2 and 3). 

Crystale’s innovative work on quorum sensing inhibition earnt her the John David Williams Memorial Award from the International Society of Chemotherapy in 20138.  Her achievements are many, with a solid focus to improve health outcomes and the environment through her research, networks, and broader community projects. This dedication earnt her the inaugural L’Oreal-UNESCO for Women in Science (FWIS) National Fellowship (Malaysia) in 2006.

Crystale´s experience with the FLUOstar Omega 

The FLUOstar Omega is regarded as an “essential laboratory work horse”. Crystale’s research has included cytotoxic and apoptotic assays for studies into cellular health and mechanisms of action9, 10, where the FLUOstar Omega is the laboratory’s “one-stop shop”. 

 

“Most common absorbance-, luminescence- and fluorescence-based assays are able to be run without having to hunt high and low for spectrophotometers, luminescence readers and fluorometers”, Crystale explained. Crystale has also worked with fluorescence-based promoter analysis assays in E. coli (unpublished data) and works extensively in RNA and DNA isolations during the course of her research in real-time PCR gene expression studies. In these studies, Crystale finds the 16-spot LVis plate of great use when processing many samples for concentration and purity determination.  

Crystale’s first encounter with a microplate reader was during her PhD candidature where she extensively used plate readers for her research. Since then, Crystale has continued to appreciate the capabilities and reliability of BMG LABTECH’s microplate readers. Indeed, Crystale’s reader is now 6 years old and still performing exceptionally well. BMG LABTECH’s local distributor Progene Link Sdn Bhd is “… essentially like your friendly neighborhood service provider – always happy to help, suggests workshops and webinars, gently reminds you to maintain the reader properly, and is there for you in your time of need”, explains Crystale. 

 

The FLUOstar Omega is also used by UCSI University for teaching purposes, as BMG LABTECH’s user-friendly MARS data analysis software is a boon for even undergraduate students to easily learn the capabilities of the microplate reader. The FLUOstar Omega’s multi-mode capabilities and broad range of analysis tools allow a wide variety of assays to be run on a single reader and the instrument’s reliability and longevity ensure that the core needs of the teaching facility are met. In addition, the multi-user software can be installed on as many computers as the user requires without the need to purchase licenses. 

 

Microplate readers for quorum sensing inhibition studies

BMG LABTECH microplate readers are ideal for researching quorum sensing inhibition in bacteria, as the multi-mode readers allow fully-automatic and simultaneous reading to monitor microbial growth and bioluminescence in parallel2, 5, 11. The robust shaking mechanism and accurate temperature control, various well scanning options, and the ability to independently regulate the atmospheric environment make BMG LABTECH readers ideal for bacterial research. 

 

The application note Monitoring bacterial cell-to-cell communication "quorum sensing" using a BMG LABTECH microplate reader shows a multiplex approach to elucidate the effect of changes in environmental condition on the growth and bio-luminescence in V. fischeri. Further peer-reviewed papers on quorum sensing inhibition assays with BMG LABTECH microplate readers can be found in BMG LABTECH’s scientific publication database.

 

Download BMG LABTECH’s Microbiology Brochure for further information.

Get in touch with Crystale

If you would like to contact Associate Professor Dr Crystale Lim Siew Ying and her team to learn more about their research, please email crystalelim@ucsiuniversity.edu.my or connect with Crystale on LinkedIn at www.linkedin.com/in/crystale-siew-ying-lim-6866218a

Further Information

 

References

1. World Health Organization (29th April 2019). New report calls for urgent action to avert antimicrobial resistance crisis. World Health Organization. Date viewed 9/7/20. <https://www.who.int/news-room/detail/29-04-2019-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis>
2. Gemiarto, A.T., Ninyio, N.N., Lee, S.W. et al. (2015). Isoprenyl caffeate, a major compound in manuka propolis, is a quorum-sensing inhibitor in Chromobacterium violaceum . Antonie van Leeuwenhoek 108, 491–504. doi.org/10.1007/s10482-015-0503-6
3. Paluch, E., Rewak-Soroczyńska, J., Jędrusik, I. et al. Prevention of biofilm formation by quorum quenching. Appl Microbiol Biotechnol 104, 1871–1881 (2020). doi.org/10.1007/s00253-020-10349-w
4. Waters, CM and Bassler BL (2005). Quorum sensing: Cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol 21:319-346. doi.org/10.1146/annurev.cellbio.21.012704.131001
5. Eboigbodin, K.E and Robinson, G.K (2009). Monitoring bacterial cell-to-cell communication quorum sensing using a BMG LABTECH microplate reader. BMG LABTECH. Date viewed 9/7/20. www.bmglabtech.com/monitoring-bacterial-cell-to-cell-communication-quorum-sensing-using-a-bmg-labtech-microplate-reader
6. CSC Chong, ZM Bo, WK Tan, S Parmasivam, ST Pang, BLH Sim, CKC Lee, RLH Lim, CSY Lim. (2020). Antibiotic resistance of ESKAPE bacteria in acute pharyngitis patients from private clinics in central Malaysia. Asia Pacific Journal of Molecular Biology & Biotechnology 28(3):52-58  https://doi.org/10.35118/apjmbb.2020.028.3.06 
7. Scutera S, Zucca M, Savoia D. (2014). Novel approaches for the design and discovery of quorum-sensing inhibitors. Expert Opin Drug Discov.; 9:353–66.
8. Gemiarto, Adrian & Chan, Eric & Lim, Crystale. (2013). Propolis as a potential quorum sensing inhibitor in Chromobacterium violaceum.. International Journal of Antimicrobial Agents. 42. S108. 10.1016/S0924-8579(13)70450-3). DOI: 10.1016/S0924-8579(13)70450-3)
9. MN Ismail, YH Tan, RLH Lim, SR Yusof, CSY Lim. (2020). Late-breaking Abstracts for 6th Congress of European Academy of Neurology: Tiger Milk Mushroom has Neuroprotective and Neuroregenerative Properties in Human Neuroblastoma Cell Line.  European Journal of Neurology, 27 (Suppl. 1), pg1303 LB131 doi.org/10.1111/ene.14363
10. Ng TJ, Teo MYM, Liew DS, Effiong PE, Hwang JS, Lim CSY, In LLA. (2019). Cytotoxic and apoptosis-inducing effects of wildtype and mutated Hydra actinoporin-like toxin 1 (HALT-1) on various cancer cell lines. PeerJ 7:e6639 doi.org/10.7717/peerj.6639
11. Topa, S. H., Palombo, E. A., Kingshott, P., & Blackall, L. L. (2020). Activity of Cinnamaldehyde on Quorum Sensing and Biofilm Susceptibility to Antibiotics in Pseudomonas aeruginosa. Microorganisms, 8(3), 455. doi.org/10.3390/microorganisms8030455