This project has focused on the relationship between the green seaweed Ulva, commonly found in the intertidal zone of the UK coastline and its cognate bacterial community. It has previously been reported that motile Ulva zoospores are attracted to N-Acylhomoserine lactones (AHLs), signalling molecules utilised by Gram-negative bacteria in a density dependent form of cellular communication termed quorum sensing (QS) and produced by several biofilm dwelling species of marine bacteria. The species represented in the bacterial community associated with Ulva spp. were identified by generating a 16S rDNA phylogenetic clone library from bacterial DNA isolated from the surface of the seaweed. These data revealed that the majority of the population belonged to the Proteobacteria or Bacteroidetes phyla. In order to investigate whether QS signalling affected the rate of zoospore germination in addition to zoospore attraction, Ulva zoospores were settled and allowed to grow on synthetic AHLs, biofilms derived from AHL-producing model organisms and strains relevant to the Ulva epiphytic population which were shown to produce AHLs. Results from these experiments revealed that AHLs affected zoospore germination and the early growth of the Ulva germling as zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. We therefore hypothesise that reduced germling growth in the presence of AHLs allows Ulva to obtain a healthy epiphytic bacterial community that is vital for the seaweed’s later development. Further understanding of Ulva growth biology could have potential applications in preventing marine biofouling by this genus of seaweed. This study progressed to characterise AHL production in a number of strains of Shewanella and Bacteroidetes bacteria, which, for differing reasons were deemed relevant to Ulva biology. Although data presented by this thesis showed AHL production in these bacterial groups, AHL synthase and response regulator sequences could not be identified in the published genome sequences from either Shewanella or the Bacteroidetes. This study also identified an AHL inactivating acylase enzyme in an environmental Shewanella isolate. This acylase, AacS, was shown to degrade a variety of synthetic AHLs and the AHLs produced by Yersinia pseudotuberculosis. This study has therefore increased the range of marine bacteria known to be producing AHLs, however the lack of AHL synthase and response regulator genes in the genomes of these bacteria leads to the conclusion that many marine bacteria possess novel, yet to be characterised AHL-mediated QS systems. Finally, this study screened a number of extracts from marine microalgae for compounds that act as agonists or antagonists to AHL-mediated QS. Although no AHL mimics were identified data presented by this thesis showed extracts to affect the luminescence produced in lux-based AHL bio-reporters in the presence of exogenously added signal, affect a number of QS regulated phenotypes in marine pathogens and effect QS regulated genes in the human pathogen Pseudomonas aeruginosa. As such, we hypothesise that these microalgae have the ability to produce quorum-quenching compound(s). Further characterisation of quorum-quenching compound(s) produced by microalgae may be beneficial in the bio-control of pathogenic bacteria in aquaculture and may act as candidates for novel antibiotics.

Bdellovibrio bacteriovorus, is a small mono-flagellate Gram-negative delta-proteobacterium, which has a bi-phasic lifecycle, consisting of a predatory phase; in which they invade on other Gram-negative bacteria and digest the prey cell’s content to grow and septate, or host independent phase; in which they can grow and septate in media rich in amino acids as well as vitamins and cofactors. As B. bacteriovorus can kill other Gram-negative bacteria including pathogens, they have potential to be used as a ‘living antibiotic’. I have been part of this field since 2004, a time at which the first B. bacteriovorus genome (HD100) had just been sequenced and made available, and only one study into making deletion mutants had been published. During my time in this field, the research has expanded almost exponentially, with the understanding of core pathways and systems that make B. bacteriovorus so novel being highlighted and greatly understood. In addition new techniques and methodologies never before attempted in B. bacteriovorus research have been made possible and I have been lucky to be a part of this and carried out some of the work myself. In particular I have worked on the mutation and phenotype testing of genes encoding pathways for motility, prey cell lysis, B. bacteriovorus intra-cellular signalling, and bi-phasic growth switching. These advances from my work including an animal trial into the predatory nature of B. bacteriovorus have laid the foundation for its use as a novel ‘living antibiotic’ in the future.

Bdellovibrio bacteriovorus are small, predatory bacteria that grow within the periplasmic space of a host bacterium. Bdellovibrio has a biphasic life-cycle switching from a uni-nucleoid, growth-senescent ‘attack-phase’ to a novel, multi-nucleoid filamentous ‘growth-phase’, which elongates and divides, growing saprophytically within the periplasmic space of their prey. Little is known to date about Bdellovibrio developmental processes and cell division within this periplasmic niche. Recent publications have demonstrated that bacterial cytoplasms house highly organised matrices of protein structures, called the bacterial cytoskeleton. The Bdellovibrio processes of prey-cell entry, filamentous cell growth and division coordination brings cellular morphological changes and challenges that could be coordinated by cytoskeletal elements. Green Fluorescent protein (GFP)-tagging and gene knock out approaches were used to gain insights into the function of these elements including: an Intermediate filament like protein Ccrp, which has a role in the maintenance of cell morphology; two actin homologues, which appear to function at different points in the predatory cycle, MreB1 and MreB2; and a new type of cytoskeletal element designated ‘bactofilin’, which may have a role in cell division control. Recent advances in GFP technologies have led to the development of optimised GFP variants, such as mTFP1 and mCherry. These have been used to reveal previously unseen detail of Bdellovibrio development within prey. Bdellovibrio do not follow the familiar pattern of bacterial cell division by binary fission, instead divide synchronously at multiple sites along their length, once prey resources are depleted. This yields both odd and even numbers of progeny Bdellovibrio.

To investigate the effectiveness of physical forces in destroying microorganisms a heat resistant (D7Soc=20 min) Gram-positive vegetative bacterium, Microbacterium lacticum, a Gram-negative vegetative bacterium, Escherichia coli, and vegetative cells and spores of Gram-positive bacterium, Bacillus subtilis, were subjected to high mechanical energies using gelatin and maize grits as carriers. A twin screw extruder, a piston capillary rheometer and a rotational rheometer were used. When extruded with gelatin there was a strong correlation between the destruction of M. lacticum and the die wall shear stress and specific mechanical energy (SME). Within the limit of detection, no surviving M. lacticum could be detected in gelatin at the highest die wall shear stress of 409 kPa and a SME of 390 kJ -kg" , giving at least 5.3 decimal reductions. There was no surviving M. lacticum in maize grits at 289 kPa die wall shear stress and 294 kl-kg" SME, giving a 4.6 decimal reduction. The temperature at the extruder die remained below 61°C for all the extrusion experiments indicating that the bacterial destruction was due to combined shear and thermal forces rather than thermal forces alone. It was suggested that the thermal energy supplied during extrusion weakened the bacterial cell wall, making the cells susceptible to shear forces. A maximum 3.2 decimal reduction in the number of spores of B. subtilis in maize grits was obtained at 595 kPa die wall shear stress and 844 kJ.kg-1 SME, below 43°C extruder die temperature. There was no statistically significant difference in the survival of B. subtilis PS346 and B. subtilis PS361, which is a heat sensitive strain due to lack of (l- and P-SASP proteins, under the same extrusion conditions, suggesting that the main destruction mechanism was not heat. High reduction in the number of the viable spores suggested a possible "mechanical germination" inside the dynamic environment of the extruder. A 4.2 logarithmic reduction in the number of M lacticum in 30% (wwb) moisture content gelatin was observed in an unsheared sample in the piston capillary rheometer at 192 MPa and 60°C, showing that pressure could cause major destruction at high temperatures (60-75°C). No survival of M. lacticum was detected beyond 695 kPa shear stress and 64 MPa at 60°C, suggesting that an optimum combination of shear, thermal and pressure forces can cause an important reduction in the numbers of vegetative cells. Shearing of the microorganisms in the rotational rheometer in gelatin showed that the shear resistance of the microorganisms were different. Although only three species of bacteria were tested, it appeared that Gram-positive bacteria were more resistant to shear forces than Gram-negative bacteria. The results suggested that the destruction of the microorganisms at low shear forces (-3 kPa shear stress) was due to weakening of the bacterial cell wall at temperatures above 60°C. A maximum 1.4 logarithmic reduction in the number of M. lacticum was achieved after 4 min of shearing at 804 S-1 shear rate and 75°C. Based on the heat resistance data, thermal forces were not enough to cause significant destruction in the numbers of the microorganism, however, the temperature played a significant role by weakening the bacterial cell wall making it susceptible to shear forces. In this context, it is possible that there was a synergistic relationship between the shear and thermal forces. A shear D-value concept was introduced which was used to evaluate the shear resistance of microorganisms at different temperatures. Starch conversion (determined by differential scanning calorimeter) due to low temperature extrusion of maize grit inoculated with M. lacticum and spores of B.subtilis showed that there is a positive correlation between the bacterial destruction and the starch conversion. Up to 94% starch conversion was obtained during low temperature extrusion of maize grit where the estimated degree of starch conversion due to heat alone was 3.8%. The results suggested that if the shear forces can be optimally combined with thermal forces, an acceptable sterility can be achieved at significantly lower temperatures which would help to keep the quality of food products high.

Bacteria have been isolated from virtually every environment on Earth. The Antarctic continent is no exception. In this extremely cold and dry environment bacteria have colonised various refugia and have evolved a number of strategies for coping with the extreme physico-chemical fluctuations they are exposed to within the environment. These psychrophilic adaptations include cold adapted proteins and lipids which are interest for biotechnology in areas such as frozen foods, agriculture and cryogenic storage. One type of cold adapted protein of particular interest is the antifreeze protein (AFP) for its recrystalisation inhibition and thermal hysteresis activity. It was first isolated from Antarctic fish in the 1970, but has since been found in plants, fungi, insects and bacteria. Over 800 bacterial isolates were cultured from lakes of the, Vestfold Hills, Larsemann Hills and MacRobertson Land, Antarctica. Approximately 87% were Gram negative rods. A novel AFP assay designed for high-throughput analysis in Antarctica, demonstrated putative activity in 187 isolates. Subsequent SPLAT analysis (qualification assessment of recrystalisation inhibition activity) of the putative positive isolates showed 19 isolates with significant recrystalisation inhibition activity. These 19 isolates were cultured from five separate lakes with substantial physico-chemical differences. The 19 AFP active isolates were characterised, using amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequencing, as predominantly belonging to genera from the a- and y-Proteobacteria, although they were more prominent in the gamma subdivision. One of these isolates (213, Halomonas sp.) was shown as dominant within its community by DGGE analysis, indicating a possible selective advantage for AFP active bacteria. This is the first report of the phylogenetic distribution of AFP activity within bacteria, thus providing information which could enable future bacterial AFP assessments to be aimed at specific taxonomic groups.

Ten overlapping fragments covering the entire Clostridium difficile toxin A gene were cloned and expressed in Escherichia coli. Eight fragments (a', a2, b, c, d, e, f and g) represented the first 5.55kb of the gene whereas two fragments (hl and h2) each spanned the entire C-terminal repeat region of the molecule. All activities relating to binding to carbohydrates (i. e. cold haemagglutination of rabbit erythrocytes), binding to bovine thyroglobulin and non-specific binding to a murine monoclonal antibody were restricted solely to peptides H1 (amino acids [aa] 1834-2683) and H2 (aa 1832-2683). Peptide H2 alone also displayed the ability to bind to cells and to be internalised into endosome-like compartments within the cells. Taken together with the observation that peptide H2 caused a cytopathic effect on Vero cells which was atypical of the holotoxin, these results may indicate that the repeat region of toxin A stimulates intracellular signalling pathways prior to Rho glucosylation. Peptide A2 (aa 1-536) glucosylated recombinant RhoA (rRhoA) in vitro, whereas peptides A'(aa 1-205), B (aa 542-859), C (aa 114-859), D (aa 869-1330), E (aa 542- 1161), G (aa 869-1830) and H2 (aa 1832-2683) did not. The results obtained for peptides A', A2 and C indicate that the first 536 as encompass the catalytic domain for this activity, that more than the first 205 as alone are needed for expression of enzymic activity, and that for a peptide to be active it must not lack the first 113 aa. The first 113 as of the holotoxin are probably essential for the correct folding of the catalytic domain and expression of its activity. These studies were also the first to locate the toxin A ATP binding site to a peptide spanning as 542-859 (peptide B) of the holotoxin. Antibody reaction profiles of antiserum to holotoxin A against toxin A peptides and of antiserum to the peptides against holotoxin A indicate that this region is unexposed in the native state. Also of interest was the observation that the only peptides, which contained the nucleotidebinding site (B and E), lacked the ability to glucosylate rRhoA. Further peptide A2, which possessed glucosyltransferase activity, lacked the nucleotide-binding site. These studies therefore, suggest that a nucleotide-binding site is not required for in vitro glucosylation of rRhoA by toxin A, and fail to identify a role for the toxin A nucleotide binding site. An engineered truncated form of toxin A, consisting of the first 539 as of the holotoxin (encompassing glucosyltransferase activity) fused to the 852 as C-terminal peptide H2 (repeat end binding portion) caused a conventional cytopathic effect (CPE), but was 1,400 fold less cytotoxic to Vero cells than the holotoxin. Peptide A2 (aa 1-536) alone had no effect on Vero cells or in rabbit ileal loops suggesting that peptide H2 aided delivery of the glucosyltransferase molecule into cells leading to a CPE. The truncated toxin lacked the nucleotide binding site and the putative membrane-translocating domain (internal hydrophobic region). The reduced activity of the truncated toxin suggests that although not essential for cytotoxic activity, the nucleotide-binding site and the internal hydrophobic region are important for stability and/or efficient translocation of the holotoxin into the cytosol.

In this study several components responsible for the formation and function of the unidirectional flagellum of R. sphaeroides WS8 were identified via the characterisation of motility impaired TnphoA mutants. The role of the alternative sigma factor sigma 54 in flagellar gene regulation was also examined. Mutant M18 was defective in a fliI homologue, characterisation of this mutant revealed that FliI is not essential for flagellar formation in R. sphaeroides. This differs from that reported in the literature for S. typhimurium and so highlights the importance of studying R. sphaeroides as a model for flagellar motility. Analysis of another mutant Nm7 revealed that it was defective in FliF, a rotor component around which other flagellar components assemble. Overexpression of a FliF fusion protein allowed the production of anti FliF antiserum. DNA sequencing upstream and downstream of the fliF gene, revealed several other genes encoding flagellar components and a potential flagellar gene regulator (Torf). fliE, encoding a component of the basal body of unknown function, was identified upstream of fliF, an interposon mutant was created and was unable to be complemented by the wild type gene in trans suggesting a dominant effect. This is the first dominant mutation to be isolated in any fliE . The gene encoding the motor component FliG was also identified downstream of fliF and its C-terminal motility domain was found to contain regions that are conserved between FliG proteins from unidirectional and bidirectional motors, these may play a role in motor rotation and not switching. An overexpressed poly histidine FliG fusion protein was found to form a complex with the FliF-GST fusion protein ill vitro. The torf gene encodes a protein with homology to sigma 54 enhancer binding proteins. The Torf protein lacks any obvious DNA binding motif and may represent a novel member of the sigma 54 enhancer binding protein family.

The reduction in cost and increase in throughput of whole genome sequencing (WGS) technologies, and the advent of benchtop WGS instruments such as the Illumina MiSeq, means that WGS is no longer restricted to large genome centres and consortia. The number of microbial genomes in public repositories is ever increasing due to the availability of WGS technologies to research groups, with individual genera having their own dedicated genome databases. Pseudomonas aeruginosa is an opportunistic pathogen and a major cause of healthcare associated infection in immunocompromised individuals and cystic fibrosis (CF) patients. The first complete genome sequence of a P. aeruginosa strain was that of the most commonly studied laboratory strain P. aeruginosa PAO1, sequenced in 2000. It was found in a study, published in 2010, that there were differences between the chromosomal sequences of two isolates of P. aeruginosa PAO1 originating from two different public strain collections, as well as differences compared to the reference sequence. It was therefore proposed that P. aeruginosa PAO1 exists as variable sublines in strain collections, whose differences included a 2.2~Mb chromosomal inversion and a prophage insertion compared to the reference sequence, as well as single nucleotide polymorphisms (SNPs) and short insertions and deletions (INDELs) which are unique to individual sublines. Since the current genomic reference sequence is based on one of these variable sublines, this study aims to deduce the sequence of the original P. aeruginosa Strain 1 (PAO) from which all the PAO1 sublines are derived, using sequence information from several laboratory sublines and from P. aeruginosa strains PAO2 and PAO3, which were directly derived from the PAO progenitor strain. This could be used as a more universal reference which is representative of a range of PAO1 sublines, to which they are all more closely related. Although most genetic studies of P. aeruginosa are carried out in PAO1, this strain is in no way the archetype for this diverse species. As whole genomes of other P. aeruginosa strains became available, comparative genomics revealed that PAO1 shared only 80% of its genome with other strains. P. aeruginosa is a highly ubiquitous organism which is able to adapt to a variety of environmental niches, as well as to human and animal hosts. This is thought to be related to the large genome size and genetic complexity of the organism, with 10% of its genes devoted to regulatory functions. Mutations in quorum sensing (QS) genes have been reported in multiple studies of host adapted P. aeruginosa isolates. QS is the mechanism by which a bacterium adapts from the lifestyle of an individual cell to a multicellular community via the regulation of specific target genes, and has been shown to regulate key virulence factors. A study of 49 clinical isolates, collected from various wound sites from distinct inpatients and outpatients at the Queen's Medical Centre in Nottingham within a few days of one another, revealed that this set of isolates displayed a diverse range of QS phenotypes. Two of these isolates were identified as producing high levels of N-acylhomoserine lactone (AHL) QS signal molecules (QSSMs) and low levels of alkyl quinolone (AQ) QSSMs, which had not previously been found in clinical isolates of P. aeruginosa, at the time of the study. WGS of these 49 clinical isolates was carried out in this study, where the aim was to; (1) determine the genomic diversity of these isolates, and relate this to previously sequenced P. aeruginosa strains, and (2) analyse the key QS genotypes of these isolates, and attempt to relate these to the phenotypes observed, in particular for the two isolates which were AHL-proficient and AQ-deficient.

Avian mycobacteriosis is a chronic infectious disease of poultry caused by different mycobacteria belonging to the M. avium-intracellular complex but primarily subspecies avium. Data on the interaction between M. avium and avian cells is very limited. This study describes the invasion and intracellular survival of M. avium isolates from both chicken and calf sources infecting THP-1-human-like macrophages and HD11 avian macrophage-like cells. There was strain to strain variation in initial invasion between the isolates tested but this variation was independent of the host-source of isolate. Invasion of the host cells was increased in the presence of host specific serum, both calf and poultry serum enhanced M. avium invasion of avian cells not seen with human cells and human serum increased the bacterial invasion of human cells, but not seen with avian cells. Both mannose and scavenger receptors were the dominant receptor for uptake of bacteria in human (THP-1) and avian (HD11) cells respectively. Simultaneous blocking mannose-, scavenger- and complement receptors did not result in complete inhibition of bacterial invasion in both cell lines suggesting that the phagocytic receptors are not working independently and may cooperate to interact with diverse ligands on the bacterial surface simultaneously for optimal binding and internalization. Invasion was both actin and tubulin dependent. The inhibitory effects of combinatory blocking of receptors and cell cytoskeleton are synergistic on the bacterial invasion of both THP-1 and HD11 cells. Post-invasion, nitric oxide and reactive oxygen species played different roles in the intracellular bacterial survival. Nitric oxide production was correlated with a reduction in intracellular survival of M. avium. In contrast, induction of reactive oxygen species enhanced M. avium survival and inhibition of reactive oxygen species using antioxidants led to a significant reduction in bacterial survival. Entry via specific receptors could have significant effect on the bacterial survival within the host cells. M. avium infection of HD11 can lead to disruption of tubulin and subsequent inhibition of both the phagosomal acidification and lysosome fusion so enhancing intracellular survival of the bacterial strains within the infected cells. In conclusion, the differences observed between bacterial isolates and host cells being infected suggest subtle differences in initial invasion and survival mechanisms between different isolates and hosts cells some of which are strain dependent and some are host cell dependent.

Pseudomonas aeruginosa (PA), a Gram-negative opportunistic rod with ubiquitous presence in a panoply of different environments, secretes a wide array of virulence determinants that have established it as one of the leading nosocomial pathogens. Many of these virulence factors are regulated by the quorum sensing (QS) system that responds to environmental cell density variations. PA can ultimately trigger the onset of severe acute and chronic infections, especially in immunosuppressed subjects. The QS network in PA is comprised of at least four multi-layered interconnected subsystems with hierarchical organisation. From these, three (las, rhl and pqs) play a pivotal role in the production of virulence factors (e.g., lectins and pyocyanin) with relevant participation in the development and maintenance of biofilm matrices. The QS network is divided in two major signaling pathways, the one driven by N-acylhomoserine lactone signals and the one driven by 2-alkyl-4-quinolone molecules. Two alkyl quinolones of core importance exist in PA, the 2-heptyl-3-hydroxy-4(1H)quinolone, typically recognised as the “Pseudomonas quinolone signal” (PQS) and its precursor 2-heptyl-4(1H)-quinolone (HHQ). In addition to the regulatory involvement of the las and rhl quorum sensing systems, the biosynthesis of PQS production is also positively regulated by PqsR-dependent transcription of the pqsABCDE operon (a multivirulence factor regulator also known as MvfR). For that reason, the alkyl quinolone (AQ) signalling pathway, and more specifically its major regulator PqsR, are widely seen as promising targets for novel antimicrobial approaches. Because of the growing presence of multidrug resistant PA in the clinical setting, representing both an immediate menace to immunocompromised patients and a heavy burden on hospital budgets, the development of more rapid and affordable screening strategies for detection of the pathogen are required. Thus, new screening strategies adapted to clinical samples and successful novel synthetic small PqsR antagonists can lead the way to a new Era in the battle against hyper-virulent/-resistant PA strains. Primarily focusing on the AQ system, this research project investigated three inter-related areas, namely: (a) the highthroughput screening of novel synthetic small molecule antagonists of the PqsR protein, designed for suppression of virulence-associated phenotypes, (b) the development of a luminescent PQS-based screening bioreporter to be applied in the clinical setting, and finally (c) the optimisation of a methodology combining liquid extraction surface analysis (LESA) and mass spectrometry (MS) for screening PQS-related AQs from in vivo bacterial extracts, and also intended for future screening of a variety of clinical samples (e.g., blood plasma, urine and saliva). A large number of synthetic small molecules with putative PqsR antagonism were obtained from our French partner GreenPharma, and studied for their capacity to interfere with expression of the key player of the pqs system, PqsR. By applying a rational selection strategy, based on the inhibitory effects on pqsA expression, assessment of metabolical exertion, and assay studies on the ultimate repression of key virulence-associated phenotypes (lectins LecA and LecB, pyocyanin, PQS-associated AQs) and impact on biolfilm formation, a final selection comprised of the four best antagonists was obtained. Compounds GPZ002966, GPZ004927, GPZ824390 and GPZ273902 had their cytotoxicity subsequently studied keeping in mind their applicability in pre-clinical studies. Overall, these PqsR antagonists promoted very strong inhibition of pqsA and lecA expression, strongly reduced production of pyocyanin and PQS-related AQs (HHQ, HQNO and C7-PQS itself), showed a strong degree of biofilm inhibition, with IC50 scores sitting at the nanomolar level, and no signs of metabolical arrest was reported by the test strains used. Some explanations, focusing on the functional and structural organisation/composition of these compounds are also offered based on a comparative analysis against a number of the most prolific PqsR antagonists recently developed. The bioluminescent PQS-based biosensor for the detection of PA was engineered to respond to the presence of exogenous PQS that forms a complex with the regulatory PqsR protein, ultimately stimulating the expression of a luxCDABE-fused pqsA promoter. The biosensor was subsequently inserted in a non-pathogenic E. coli recipient by means of chromosomal integration, devoid of the sdiA LuxR homolog that could potentially interfere with the recognition of PqsR. A silent reporter was observed when in E. coli, but further assessments to its genetic integrity did not reveal any single nucleotide polymorphisms (SNP). In addition, after further testings to its activity in different established PA mutants, devoid of genes that constituted the bioreporting system or to it directly associated, a fully functional bioreporter was confirmed. Finally, a few possible explanations as to what might be in the origin of a defective bioreporter in E. coli are discussed. Lastly, a new LESA-MS protocol based on the surface sampling of dried bacterial extracts, envisaging its potentialities as a rapid and cheap screening method for detection of AQs, was designed and optimised. Even though the method has been widely used in a variety of research scenarios, this is the first time LESA-MS is applied as a screening methodology in the context of bacterial extract screening. Overall, the optimisation process showed that LESA-MS is an approach with numerous potentialities and immediate advantages, where one emphasises sampling simplicity, fast delivering of results, sensitivity to AQs at the nanomolar level (especially for C7-PQS and the precursor HHQ). But simultaneously, this methodology also revealed limitations inherent to its setting up that constrain an effective screening. The most emphatic ones being the volatility of the preparations to intra-sampling variability, and to a certain degree, an unexpected insensitivity to important QS N-acyl homoserine lactones (AHLs), namely C4-HSL and 3-oxo-C12-HSL. Nevertheless, such limitations do not present themselves as an insurmountable barrier, and based on results from available studies making use of the LESA-MS a number of possibilities to work around these are also presented.