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Annotated Bibliography:

Maisuria, V. and Nerurkar, A. (2015) Interference of Quorum Sensing by Delftia sp. VM4 Depends on the Activity of a Novel N-Acylhomoserine Lactone-Acylase. PLOS ONE, 10(9), p.e0138034.

This study aimed to identify the chemical produced by Delftia tsuruhatensis VM4 responsible for the degradation of the quorum-sensing molecules N-acylhomoserine lactones (AHLs) in Pectobacterium carotovorum. An AHL bioassay was performed with the biosensor strain Chromobacterium violaceum CV026 and bioluminescence was used to detect luxRI and lasRI expression via AHL presence. Anionic-exchange column chromatography was used to purify the protein isolates responsible for AHL degradation and multiple amino acid sequence alignment was used to determine the identity of the protein. The VM4 AHL degrading enzyme (82 kDa) was shown to degrade AHL via quorum-sensing attenuation as well as prevent the virulence of Pectobacterium carotovorum BRI without affecting the growth of the bacteria. This paper called for future work to analyze the application of the discovered AHL acylase against other pathogens. This work contributes to the discussion of Delftia tsuruhatensis’s ability to inhibit quorum-sensing and attenuate virulence while providing a potential therapeutic target, the AHL degrading enzyme (82 kDa).

Malešević, M., Di Lorenzo, F., Filipić, B., Stanisavljević, N., Novović, K., Senerovic, L., Polović, N., Molinaro, A., Kojić, M. and Jovčić, B. (2019) Pseudomonas aeruginosa quorum sensing inhibition by clinical isolate Delftia tsuruhatensis 11304: involvement of N-octadecanoylhomoserine lactones. Scientific Reports, 9(1).

To address growing concerns surrounding Pseudomonas aeruginosa’s high patient mortality rate, this study analyzed the mechanisms underlying Delfia tsuruhatensis 11304’s inhibitory abilities against problematic clinical isolates of P. aeruginosa. The 11304 strain was cultured with Chromobacterium violaceum CV026 to measure the strain’s ability to inhibit violacein, a known quorum-sensing molecule. Additionally, a microdilution method was used to inoculate the P. aeruginosa MMA83 with the 11304 strain in which: biofilm production was quantified by measuring absorbance, the production of virulence factors was measured with corresponding assays, and the quorum-sensing genes expressions were quantified using reverse transcription PCR. The 11304 ethyl acetate extract was shown to debilitate the MMA83’s quorum-sensing, to restrict its ability to form a biofilm, and to suppress its ability to produce virulence factors such as elastase, rhamnolipid and pyocyanin by transcriptional inhibition of the las, rhl and pqs genes, without affecting bacterial cell growth or disrupting existing biofilm. While this study isolated a novel long-chain dihydroxy-N-octadecanoylhomoserine lactone that was speculated to be responsible for the observed quorum-sensing inhibition, the researcher acknowledge that further studies need to be dedicated to the identification of the compounds from the 11304 strain responsible for quorum-sensing inhibition. This report provides details regarding D. tsuruhatensis’s dihydroxy-N-octadecanoylhomoserine lactone’s potential for inhibiting biofilm production and quorum-sensing while decreasing the virulence of clinically dangerous drug resistant bacteria.

Pesci, E., Milbank, J., Pearson, J., McKnight, S., Kende, A., Greenberg, E. and Iglewski, B. (1999) Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences, 96(20), pp.11229-11234.

Since Pseudomonas aeruginosa can use quorum-sensing to control virulence factors, this study aimed to identify a novel regulatory signal used by the bacterium in cell-to-cell signaling. Thin Layer Chromatography and High Performance Liquid Chromatography were performed to fractionate the P. aeruginosa ethyl acetate extract and 1H and 13C NMR, infrared spectroscopy, melting point analysis, and mass spectroscopy were used to identify chemical that exhibited quorum-sensing activity in the bioassay. This study discovered a cell-to-cell signaling molecule in P. aeruginosa that’s expression is regulated by the las quorum sensing system and requires RhlR in order to control the production of LasB elastase, a major virulence factor. Furthermore, the study called for additional research into the mechanism by which this new quorum-sensing molecule regulates lasB in order to better understand the relationship of signaling molecules and virulence. This report enforces the importance of cell-to-cell signaling molecules in bacterium’s virulence, or the severity of their harmfulness.

Priya, K., Yin, W. and Chan, K. (2013) Anti-Quorum Sensing Activity of the Traditional Chinese Herb, Phyllanthus amarus. Sensors, 13(11), pp.14558-14569.

This study sought to establish the existence of anti-quorum sensing properties in a traditional chinese medicinal herb, Phyllanthus amarus. To identify the presence of anti-quorum sensing activity, a methanol extract of the P. amarus was prepared and cultured overnight with the purple pigmented indicator bacteria Chromobacterium violaceum. Bioluminescence was used to quantify the inhibition of Pseudomonas aeruginosa PAO1 and virulence factor assays and swarming plates were prepared as well. The P. amarus methanol extract exhibited anti-quorum sensing activity against C. violaceum and PAO1 without inhibiting bacterial growth. P. amarus also significantly reduced expression of a gene that plays a role in biofilm production, lecA; swarming of PAO1, which is controlled by quorum-sensing ability; and inhibition of the production of the toxin Pyocyanin via the lasR-lasI, rhlR-rhlI, and mvfR-haq quorum system complex. This study called for future work analyzing the anti-quorum sensing activity of P. amarus on the RhlR/C4-HSL quorum sensing system and called for the characterization of the active molecule responsible for anti-quorum sensing in P. amarus. This study demonstrates a real world application of an anti-quorum sensing bacteria prior to the discovery of the mechanistic principles governing the herbs effectiveness; many medical advances have similar origins as the detection and understanding of the biochemistry behind already proven useful substances can drive the development of more efficient therapeutics.

Singh, V.K., Mishra, A. and Jha, B. (2017) Anti-quorum Sensing and Anti-biofilm Activity of Delftia tsuruhatensis Extract by Attenuating the Quorum Sensing-Controlled Virulence Factor Production in Pseudomonas aeruginosa. Frontiers in Cellular and Infection Microbiology, 7.

As an avenue to research new methods for fighting multi-drug resistant bacteria, this study explored the anti-quorum and anti-biofilm potential of the Delftia tsuruhatensis SJ01 strain, investigated key regulatory genes in quorum sensing, and hypothesized a possible inhibition mechanism. After isolating a bacteria for its anti-quorum sensing properties, identifying the Delftia tsuruhatensis SJ01 strain using Basic Local Alignment Search Tool (BLAST), and preparing a bacterial extract in methanol, the extract’s inhibition of a quorum sensing activity indicator, violacein, when introduced to Chromobacterium violaceum was quantified; the extract was introduced in various quantities to cultures of P. aeruginosa PAO1 and P. aeruginosa PAH to measure the effect on biofilm formation; fluorescence microscopy, scanning electron microscopy, and atomic force microscopy were used to visualize the effect of the extract on individual cells; a bacterial motility assay was performed to determine the effect of the extract on the swimming ability of the P. aeruginosa strains; and the amount of pyocyanin and rhamnolipid and elastase and protease activities were measured to determine the effect of the extract on the virulence factors of the P. aeruginosa strains. Furthermore, fractionation, mass spectrometry, and electrospray ionization were used to identify the active compound in anti-quorum sensing and anti-biofilm activity, 1,2-benzenedicarboxylic acid, while RNA isolates were compared between test groups to identify the genes involved (LasI, LasR, RhlI, and RhlR) in quorum sensing using a microarray. With no harm to the individual bacterial cells, the Delftia strain was shown to reduce the production of virulence factors in the PAO1 and PAH strains, inhibit the quorum sensing indicator violacein by 98% with 0.1 mg/ml extract, and reduce motility in the PAO1 and PAH strains, likely contributing to the observed significant decrease of biofilm production in these strains. This article shows the potential of this specific bacterium and 1,2-benzenedicarboxylic acid to serve as a model for designing therapeutics to attack the quorum sensing and biofilm ability of multi-drug resistant bacteria. The article even called for future research dedicated to investigating the potential applications of the new insights it provided.

Stickler, D.J., Morris, N.S., McLean, R.J.C. and Fuqua, C. (1998) Biofilms on Indwelling Urethral Catheters Produce Quorum-Sensing Signal Molecules In Situ and In Vitro. Applied and Environmental Microbiology, 64(9), pp.3486–3490.

This study’s purpose is to determine if bacterial biofilms produce Acylated homoserine lactones (AHLs), a quorum-sensing signal, in a clinical setting. In order to test for the presence of AHLs in clinical bacterial strains, the researchers isolated the bacteria from catheters and placed them on a plate with an AHL reporter stain, Agrobacterium tumefaciens. The strains were also introduced to synthetic catheters wherein biofilm formation was observed using electron microscopy. Pseudomonas aeruginosa biofilm existed on both clinical and synthetic catheters and was shown to produce AHLs in both cases. This study called for a need for future research to establish the precise role of AHLs in biofilms. This report provides background for the relationship between biofilm and the quorum sensing chemicals referenced in other reports and contributes relevance to the topic by showing the existence of these biofilms in a clinical setting.

Summarization:

Bacterial resistance is outpacing the development of new antibiotics, engendering the design of novel therapeutic approaches to fighting these multi-drug resistant bacteria. The ability of bacteria to form biofilms contributes to their antibiotic resistance. Furthermore, these biofilms are able to participate in a cell-to-cell signaling mechanism, quorum sensing (QS), that can increase the pathogen’s virulence. Certain bacterial species have demonstrated an ability to inhibit quorum sensing during bacterial competition. Since QS plays a role in biofilm formation, this ability to inhibit QS can serve as a model for designing new therapeutics to fight multidrug resistant bacteria.

Pseudomonas aeruginosa is a highly infectious bacterial pathogen that grows in biofilms and possesses high antibiotic resistance in clinical settings; therefore, it has commonly been used as a model for understanding the role of QS and biofilm production in antibiotic resistance and virulence. D. tsuruhatensis has been shown to be able to significantly inhibit QS, suppress biofilm formation, and attenuate virulence in P. carotovorum and Chromobacterium violaceum without damaging individual bacterial cells. While many studies have conferred the ability of D. tsuruhatensis to attenuate virulence through QS and biofilm inhibition, an agreement has yet to be reached as to the exact mechanism the bacteria uses to achieve this effect.

 Article 

Abstract:

Delftia tsuruhatensis’s anti-quorum sensing capabilities against the highly drug resistant bacteria P. aeruginosa holds possible implications for designing novel therapeutic drugs. Quorum sensing mechanisms are responsible for biofilm production, virulence, and antibiotic resistance; therefore, quorum sensing inhibition is potentially a critical pathway for attenuating the harmfulness of drug resistant bacteria. While D. tsuruhatensis’s inhibition is well established by current literature, little agreement exists regarding the precise mechanism employed by the bacteria to achieve these effects. Consequently, future research should be aimed at defining these mechanisms in order to provide comprehensive therapeutic models.

Key words: biofilm, quorum sensing, virulence, antibiotic resistance, inhibition, attenuation

Introduction:

Bacterial resistance is outpacing the development of new antibiotics, engendering the design of novel therapeutic approaches to fighting these multi-drug resistant bacteria. The ability of bacteria to form biofilms contributes to their antibiotic resistance. Furthermore, these biofilms are able to participate in a cell-to-cell signaling mechanism, quorum sensing (QS), that can increase the pathogen’s virulence. Certain bacterial species have demonstrated an ability to inhibit quorum sensing during bacterial competition. Since QS plays a role in biofilm formation, this ability to inhibit QS can serve as a model for designing new therapeutics to fight multi-drug resistant bacteria.

Pseudomonas aeruginosa is a highly infectious bacterial pathogen that grows in biofilms and possesses high antibiotic resistance in clinical settings; therefore, it has commonly been used as a model for understanding the role of QS and biofilm production in antibiotic resistance and virulence. D. tsuruhatensis has been shown to be able to significantly inhibit QS, suppress biofilm formation, and attenuate virulence in P. carotovorum and Chromobacterium violaceum without damaging individual bacterial cells. While many studies have conferred the ability of D. tsuruhatensis to attenuate virulence through QS and biofilm inhibition, an agreement has yet to be reached as to the exact mechanism the bacteria uses to achieve this effect. This review looks at nine original research articles in order to detail the current comprehensive knowledge of D. tsuruhatensis’s ability to inhibit QS and biofilm production to ultimately attenuate virulence in bacterial pathogens. While these properties lend to D. tsuruhatensis’s potential to serve as a model for designing new drugs, this review illuminates the need for future studies in order to confirm this prospect and discover the exact mechanisms behind this ability.

D. tsuruhatensis Inhibits Quorum Sensing

Bacterial species like Pseudomonas aeruginosa are able to participate in a cell-to-cell signaling mechanism called quorum sensing by sending out small diffusible molecules. Pesci et al. discovered that there are at least three different signaling molecules that P. aeruginosa uses to achieve quorum sensing. D. tsuruhatensis 11304 ethyl acetate extract was also shown to possess quorum sensing inhibition properties against P. aeruginosa and the widely used quorum sensinsing indicator strain Chromobacterium violaceum CV026. In this case, a novel long-chain dihydroxy-N-octadecanoylhomoserine lactone was isolated and speculated to be the agent responsible for interfering with the N-acyl-homoserine lactones (AHL)s of the quorum sensing bacteria. In another study, it was demonstrated that the Delftia tsuruhatensis VM4 strain is able to quench Pectobacterium carotovorum’s quorum sensing ability. The enzyme thought to be responsible for degrading N-acyl-homoserine lactones (AHL), a quorum sensing signaling molecule, was isolated from VM4 using anionic-exchange column chromatography and was identified to be 82 kDa with a multiple amino acid sequence alignment tool. In yet another study, fractionation and mass spectrometry identified 1,2-benzenedicarboxylic acid to be responsible for D. tsuruhatensis SJ01’s ability to interfere with the cell-to-cell signaling mechanism of CV026 by 98% with just 0.1 mg/ml of extract. Although quorum sensing inhibition by multiple D. tsuruhatensis strains has been demonstrated decisively, due to dissension regarding the causative agent, future studies should be directed at identifying the mechanistic principles governing this ability.

D. tsuruhatensis Suppresses Biofilm Production

It has been established that QS systems play a role in biofilm formation in P. aeruginosa. This relationship perhaps explains in part how D. tsuruhatensis 11304 ethyl acetate extract was able to restrict P. aeruginosa MMA83’s ability to form biofilm; however, it is noteworthy that the 11304 strain was unable to disturb existing biofilm. In an alternative, but perhaps related, fashion, D. tsuruhatensis SJ01 was able to decrease biofilm formation in P. aeruginosa PAO1 and P. aeruginosa PAH by decreasing the swarming mobility of the P. aeruginosa strains. It is clear that QS plays a vital role in biofilm production, so it is no surprise that D. tsuruhatensis’s ability to inhibit QS can in turn suppress biofilm production; however, further research needs to be conducted to determine an understanding of the precise mechanisms that guide this relationship.

Biofilm Production is Linked to Increased Antibiotic Resistance

P. aeruginosa in biofilm were shown to have an increased antibiotic resistance. ndvB-dependent β-(1→3)-linked cyclic glucans discovered in P. aeruginosa were linked to biofilm antibiotic resistance. A novel substance, furanone C-30, was shown to significantly reduce biofilm production in P. aeruginosa by targeting genes associated with QS. Moreover, this compound made P. aeruginosa 2-3 times more susceptible to antibiotics following biofilm reduction.

D. tsuruhatensis Attenuates Virulence

It was established that a cell-to-cell signaling molecule in P. aeruginosa controls the production of a major virulence factor, LasB elastase. D. tsuruhatensis anti-quorum sensing properties attenuated virulence of P. carotovorum and P. aeruginosa.

Conclusion:

D. tsuruhatensis has been shown to be able to inhibit QS, suppress biofilm formation, and attenuate virulence in multi-drug resistant pathogenic bacteria. Moreover, D. tsuruhatensis achieves these effects without damaging individual bacterial cells, an important characteristic in avoiding the selection that leads to antibiotic resistance. Using the natural ability of D. tsuruhatensis to quench QS in highly drug resistant bacterial pathogens holds a great promise to medicine, therefore future research should be directed at unlocking the key mechanisms behind this ability as no current agreement exists.

References: