Detection of quorum sensing molecules is often performed by using quorum sensing reporter bacteria strains, of which most are sensitive to either autoinducer-1 (AHL's) or autoinducer-2 (Steindler and Venturi, 2007; Rai et al., 2015). As mentioned previously, these bacterial reporters can be based on non-genetically modified or genetically-modified bacteria.

Genetic modifications consist of altering the bacterial genome, in order to obtain a detectable signal in the presence of the quorum sensing molecule of interest. This genetic modification consists of coupling a suitable promoter to a desired reporter gene, and hence the desired detection signal. The detection signal is produced by a reporter protein, e.g., bioluminescence, chemiluminescence, fluorescence, colorimetric response, etc. Production of the reporter protein depends on activation of the promoter, which initiates transcription of the reporter gene.

Inherent to genetic modifications of the bacteria, is the need of selecting those bacteria that are successfully genetically modified [e.g., those bacterial cells that have acquired the plasmid(s)]. Selecting these correctly, genetically modified bacteria can be performed by for example antibiotic resistance genes (O'Connor et al., 2016).

Table 1 provides an overview of plasmid-based AHL reporter bacteria.

The Vibrio harveyi BB170 bioluminescence assay is an example of a reporter bacteria constructed by modifications of the bacterial genome to prevent production of and/or response to certain quorum sensing molecules. It is a frequently used reporter strain for quantification of autoinducer-2 molecules. Genetic modifications of the BB170 strain ensure it does not produce endogenous autoinducer-1 (AHL's) or autoinducer-2 and is insensitive for autoinducer-1. Hence, bioluminescence only occurs when autoinducer-2 is added, where the light intensity correlates with the amount of autoinducer-2 that is added (O'Connor et al., 2016).

A variety of approaches are applied to proof the presence of, and in rare cases, eventually identify quorum sensing peptides (see Table 2). Currently, ~300 quorum sensing peptides and structural analogs have been published (Wynendaele et al., 2013). The majority of these peptides have been tested by the researchers on a system considered a reporter bacteria based biosensor system. While peptide structure elucidation was frequently performed by liquid chromatography-mass spectroscopy and/or Edman degradation. Analogs of the cognate quorum sensing peptides are also frequently evaluated using a specific biosensor.

For example, two reporter strains in S. mutans GS5, capable of detecting the competence stimulating peptide (21-CSP) UA159sp4 have been described. The CSP-mediated quorum sensing sytem of S. mutans involves the genes comCDE, comAB, and comX. In brief, the CSP precursor, encoded by comC, is cleaved and exported to form CSP by an ABC-transporter encoded by comAB. Additionally, the comDE gene encodes a two-component signal transduction system that recognizes CSP and initiates a response. ComX encodes a sigma factor that regulates transcription of several genes needed for uptake of foreign DNA and the ComE protein is activated by autophosphorylation upon binding of CSP with the ComD histidine kinase receptor. Phosphorylated ComE activates target genes such as comCDE, comAB, and comX, thereby generating a positive feedback quorum sensing system (Syvitski et al., 2007). The reporter strain is composed of a comC mutant S. mutans GS5, resulting in a mutant strain which is unable to produce CSP itself, but still able to respond to exogenous CSP, named SMdC. Mutation of the comC gene is obtained by inserting a spectinomycin (Specr) resistance gene in the comC gene of S. mutans GS5. This mutant was used to develop two lacZ transcriptional reporter strains in which the lacZ gene is fused to the comDE promoter or the nlmAB promoter. To generate the first reporter strain, a vector pYH2 was formed by fusion of the comDE promoter with a promoterless lacZ gene in the Streptococcus—Escherichia coli shuttle vector pSL; subsequently, pYH2 was transformed into SMdC, resulting in the SMdC-pYH2 reporter strain. The second reporter strain is constructed by fusion of the nlmAB promoter with a promoter-lacking lacZ gene and transformation of this pOMZ47 plasmid into SMdC, resulting in the SMdC-PnlmAB reporter strain (Steindler and Venturi, 2007). Following addition of exogenous CSP (UA159sp), due to transcription of the lacZ gene, both SMdC-pYH2 and SMd-C-PnlmAB express β-galactosidase (Syvitski et al., 2007; Tian et al., 2009). Cleavage of O-nitrophenyl-β-D-galactoside by β-galactosidase renders O-nitrophenol, with the yellow color proportional to the enzyme activity and hence the quorum sensing peptide concentration (Syvitski et al., 2007).

The ability of CSP (0.2–100 nM) to induce competence in S. intermedius NCTC 11324 was tested: overnight cells of S. intermedius NCTC 11324 were diluted 1:200 and subsequently, quorum sensing peptide and a plasmid expressing erythromycin resistance genes were added. After incubation for 24 h, transformants were selected by growth on THB agar plates containing erythromycin. The amount of transformants/mL increased when increasing concentrations of CSP were added (Wynendaele et al., 2015a). Comparable transformation assays were performed by Håvarstein et al. (1997) with two other synthetic CSPs from Streptococcus milleri and from Streptococcus gordonii (Håvarstein et al., 1997) and by Pozzi et al. (1996).

The reporter strain for the Gram-negative E. coli quorum sensing peptide “extracellular death factor” (EDF) was described by Kolodkin-Gal et al. (2007). They studied the influence of the quorum sensing peptide on mazEF-mediated cell death in E. coli. The mazEF module encodes the stable toxin MazF, which exerts its toxic effects by cleavage of single-stranded mRNA at ACA sequences, and the labile antitoxin MazE, which counteracts the effect of MazF. The mazEF module is induced by stress. Stressful conditions that prevent transcription/translation of mazEF, such as inhibition of transcription and/or translation by antibiotics (e.g., rifampicin, chloramphenicol, spectinomycin) or DNA damage, reduces the level of the labile MazE, hereby reducing the counteracting effect on MazF. However, mazEF-mediated cell death only occurs in dense cultures since it requires the quorum sensing peptide EDF. Addition of rifampicin provokes mazEF-mediated cell death in dense cultures, but not in diluted cultures. The mazEF-mediated cell death in these diluted cultures could be restored by addition of supernatant of a dense culture. The bioassay for detection and quantification of EDF activity is based on this principle. Different dilutions of the supernatant of a dense culture of the E. coli strain MC4100relA+, containing EDF, were added to a diluted culture of MC4100relA+, after which stressful conditions (e.g., addition of chloramphenicol) were induced. The percentage of surviving colony forming units (CFUs) was determined after overnight incubation (Kolodkin-Gal et al., 2007).

Quorum sensing peptides can also be detected quantitatively (limit of detection 10 aM) by demonstrating a product of which formation is regulated by a quorum sensing peptide. Nilsen et al. (1998) studied the production of the bacteriocins enterocin A and enterocin B in Enterococcus faecium CTC492, regulated by the quorum sensing peptide EntF. Without EntF, a non-bacteriocin-producing culture (Bac–) of Enterococcus faecium CTC492 was obtained. Upon addition of induction factor EntF, production of enterocin A and enterocin B is induced and after 20–24 h incubation, bacteriocin activity is assayed by addition of sterilized supernatant to a culture of an indicator organism, e.g., Lactobacillus sakei NCDO 2714 (sensitive to both enterocin A and enterocin B), Pediococcus pentosaceus FBB 63 (sensitive to enterocin A) or Lactobacillus sakei FVM 148 (sensitive to enterocin B). Growth inhibition of these strains indicates presence of bacteriocins in the supernatant and thus, presence of induction factor EntF in the sample added to the non-bacteriocin-producing culture (Nilsen et al., 1998).

A different approach uses the phenomenon of plasmid transfer to demonstrate the presence of quorum sensing peptides. Genes located on plasmids and encoding for hemolysins, bacteriocins, and antibiotic resistance genes are transferable. Several Enterococcus faecalis strains are able to transfer plasmids, facilitated by sex pheromones (i.e., quorum sensing peptides) (Dunny et al., 1979). Certain quorum sensing peptides, excreted by recipient strains, induce donor strains, containing conjugative plasmids, to produce a proteinaceous substance on their surface. Thereby, mating aggregates are formed, facilitating plasmid transfer. These quorum sensing peptides are also called clumping-inducing agents (CIA) since they cause self-aggregation of donor cells when culture filtrate of recipients is added (Dunny et al., 1979). Mori et al. (1984, 1986a, 1988), Suzuki et al. (1984) and Nakayama et al. (1995a), isolated and determined the sequence of several of these CIAs. Once the recipient strain acquires the conjugative plasmid, the excretion of the specific CIA, corresponding with the acquired plasmid, stops and the strain becomes sensitive to exogenous quorum sensing peptides. However, production of other CIAs, specific for donor strains containing other conjugative plasmids, still continues (Ike et al., 1983). Quantification of CIA can be performed by means of a microtiter system. Serial dilutions of recipient strain filtrates (or sample) are added to responder cells (donor cells with the appropriate conjugative plasmid) and incubated for 90–120 min, after which clumping is observed in dilutions (samples) containing sufficient amount of CIA (quorum sensing peptide) (Dunny et al., 1979). The same principle can be applied to other quorum sensing peptides facilitating plasmid transfer in E. faecalis e.g., cAD1 (Mori et al., 1984), cCF10 (Mori et al., 1988), cPD1 (Suzuki et al., 1984), and cOB1 (Nakayama et al., 1995a). Quorum sensing peptides cOB1 and cAD1 are an exception to the assumption of one pheromone (cX) specifically activating plasmid transfer of its corresponding plasmid (pX) since both pOB1 and pY11 respond to cOB1 and both pBEM10 and pAD1 respond to cAD1 (Murray et al., 1988; Nakayama et al., 1995a).

The production stop of quorum sensing peptide, once the recipient strain acquires the corresponding plasmid, is due to the production of a quorum sensing inhibitor encoded on the plasmid, which inhibits the activity of its corresponding quorum sensing peptide (Mori et al., 1987). Inhibitory activity of iPD1 (Mori et al., 1986b, 1987) and iAM373 (Nakayama et al., 1995b) can be quantitatively measured by modifying the microtiter system described above. Using iAD1 as example, serially diluted sample solutions containing iAD1 are added to a mixture of responder cells OG1S(pAD1::Tn917) and cAD1. After incubation for 2–3 h, inhibition of cAD1-induced clumping of responder cells is observed (Mori et al., 1986b).

A variety of liquid-chromatographic methods have been reported, especially for the N-acyl homoserine lactones (see Table 3) (Morin et al., 2003; Li et al., 2006; Fekete et al., 2007; Kumari et al., 2008; Ortori et al., 2011; Wang et al., 2011; Cataldi et al., 2013), including methods allowing the simultaneous analysis of various N-acyl homoserine lactones via a non-targeted LC-MS/MS method (Patel et al., 2016). Generally, these methods are capable of generating limits of detection (LOD) in the broad nM-range (median LOD of 2.3 nM for 19 N-acyl homoserine lactone standards; range: 0.6 nM–2838.9 nM), with some compounds owing a limit of detection around 0.6 nM (for C10-HSL) (Patel et al., 2016).

Besides the closely related cyclodipeptides demonstrated qualitatively, the presence of quorum sensing peptides has not yet been demonstrated in vivo (Marchesan et al., 2015), except for one report (Darkoh et al., 2015) where the presence of a thiolacton peptide, without structural elucidation, is suggested in feces of patients suffering from C. difficile infection. Nevertheless, the presence of these peptides in cell culture broths has already been proven via reporter strains (see Table 2). Additionally, various authors have reported quorum sensing peptide levels in bacterial cell culture broths via chromatographic techniques, hence applying an orthogonal detection method compared to the biological reporter bacteria.

Additional to reporter bacteria based biosensors, several quorum sensing peptides have been qualitatively or quantitatively determined in bacterial cell culture broths (see Table 4). For example, the currently reported E. faecalis quorum sensing peptides have been quantified in cell culture broths. Based on the reports, the concentration of these quorum sensing peptides is in the broad picomolar range, though caution should be paid to the often elaborate sample preparation methods that have been applied, mostly intended for a qualitative approach.

Besides the presence of E. faecalis quorum sensing peptides in cell culture broths, other authors have developed chromatographic methods to demonstrate quorum sensing peptide presence of other species in their respective cell cultures (Junio et al., 2013; Todd et al., 2016). For example, the presence of Staphylococcus autoinducing peptide I was demonstrated using a ultra-high performance liquid chromatographic system coupled to a mass spectrophotometer in methicillin-resistant Staphylococcus aureus cell cultures with a limit of detection of 3.5 nM and a LOQ of 0.10 μM (Todd et al., 2016). Junio et al. (2013) demonstrated quantitatively (LOD 0.25 μM; LOQ 2.6 μM) the time- and strain dependent synthesis of AIP-1 in cell culture medium in the μM-magnitude, with concentrations as high as 13 ± 2 μM at 16 h incubation of the S. aureus LAC strain (Junio et al., 2013).

Additionally, the extracellular death factor quorum sensing peptide from E. coli has also been demonstrated in cell culture broths via a qualitative chromatographic method (Kolodkin-Gal et al., 2007).

Quorum sensing is not limited to bacteria, but eukaryotic yeasts have also shown to possess quorum sensing mechanisms. The C. albicans quorum sensing molecules farnesol and tyrosol are via a LC-MS/MS method quantifiable with limits of detection of respectively 15.2 and 3.0 nM (Gregus et al., 2010).

Via gas chromatography coupled to a mass spectrometer (GC-MS), an enantiomer-selective method was achieved for the analysis of C8-HSL (LOD: 0.1 mg/L ≈ 0.4 μM for L-C8-HSL), C10-HSL (LOD: 0.1 mg/L ≈ 0.4 μM for L-C10-HSL), and C12-HSL (LOD: 0.3 mg/L ≈ 1.1 μM for L-C12-HSL). By applying this method to Burkholderia cepacia LA3 cell culture supernatant, the authors demonstrated the in vitro production of D-C10-HSL (Malik et al., 2009). N-3-oxoacyl homoserine lactones were determined by derivatizing them into their pentafluorobenzyloxime derivatives with subsequent gas chromatography-mass spectrometry analysis. ODHL (biofilm: 3 ± 2 μM; effluent: 1 ± 0.1 nM), OdHL (biofilm: 632 ± 381 μM; effluent: 14 ± 3 nM), OtDHL (biofilm: 40 ± 15 μM; effluent: 1.5 ± 0.7 nM), and OOHL (effluent: 0.1 ± 0.1 nM) were by applying this method found in biofilms and/or effluent (Charlton et al., 2000). Cataldi et al. (2007) also reported a GC-MS method capable of detecting AHL's in the μM-range with an average LOD of 4.4 μM for the 6 investigated AHLs (Cataldi et al., 2007). The autoinducer-2, following silylation, has also proven to be analyzable by gas chromatography-mass spectrometry (LOD 5.3 nM and limit of quantification (LOQ) 16 nM) (Thiel et al., 2009).

Besides liquid and gas chromatography, (high performance-) thin-layer chromatography has also been used in the analysis of certain quorum sensing molecules (Shaw et al., 1997; Bala et al., 2013). Shaw et al. (1997) achieved LODs ranging from 0.5 fmol to 300 pmol depending on the specific AHL (Shaw et al., 1997), whereas Bala et al. (2013) reported a lower limit of detection of 0.006 nmol/spot and a lower limit of quantification of 0.01 nmol/spot for PQS (Bala et al., 2013). A hybrid technique, combining bacterial reporters with thin-layer chromatography has been described (Andersen et al., 2001; Charlesworth et al., 2015) (LODs ranging from 0.012 to 1,710 ng depending on the specific AHL) (Charlesworth et al., 2015).

Besides liquid and gas chromatography, some authors reported capillary electrophoresis as a suitable technique to quantify quorum sensing molecules. With partial filling micellar electrokinetic chromatography–electrospray ionization-ion trap mass spectrometry, two acyl homoserine lactone derivatives (i.e., N-octanoyl-L-homoserine lactone and N-decanoyl-L-homoserine lactone) molecules originating from Burkholderia cepacia could be qualitatively detected in bacterial cell culture broths down to a concentration of approximately 1 μM (Frommberger et al., 2003). The same authors were capable of determining quantitatively (LOQ 0.05 μg/ml) C8-HSL (0.26 μg/ml ≈ 1.1 μM), C10-HSL (1.93 μg/ml ≈ 7.6 μM), and C12-HSL (0.16 μg/ml ≈ 0.6 μM) in Burkholderia cepacia cell culture broths by means of capillary zone electrophoresis-mass spectrometry following hydrolysis of the homoserine lactones into their corresponding organic acids (Frommberger et al., 2005).

Several P. aeruginosa-quinolones are also detectable using capillary electrophoresis with LODs of 65 nM (PQS), 94 nM (HHQ), 61 nM (“Quinolone 1”) and 79 nM (“Quinolone 2”) (Zhou et al., 2012). Noteworthy is the eukaryotic quorum sensing molecule from C. albicans farnesol, which is also detectable via capillary electrophoresis. By applying this capillary electrophoresis method, the authors demonstrated the concentration of farnesol to be approximately 2 nM in a cell culture supernatant (Kubesová et al., 2010).