N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas

Some members of the moderately halophilic genus Halomonas, such as H. eurihalina, H. maura, H. ventosae and H. anticariensis, produce exopolysaccharides with applications in many industrial fields. We report here that these four species also produce autoinducer molecules that are involved in the cell-to-cell signaling process known as quorum sensing. By using the N-acyl homoserine lactone (AHL) indicator strains Agrobacterium tumefaciens NTL4 (pZRL4) and Chromobacterium violaceum CV026, we discovered that all the Halomonas strains examined synthesize detectable AHL signal molecules. The synthesis of these compounds was growth-phase dependent and maximal activity was reached during the late exponential to stationary phases. One of these AHLs seems to be synthesized only in the stationary phase. Some of the AHLs produced by H. anticariens FP35^T were identified by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry as N-butanoyl homoserine lactone (C₄-HL), N-hexanoyl homoserine lactone (C₆-HL), N-octanoyl homoserine lactone (C₈-HL) and N-dodecanoyl homoserine lactone (C₁₂-HL). This study suggests that quorum sensing may also play an important role in extreme environments.     

Targeting Acyl Homoserine Lactones (AHLs) by the quorum quenching bacterial strains to control biofilm formation in Pseudomonas aeruginosa

Navigating novel biological strategies to mitigate bacterial biofilms have great worth to combat bacterial infections. Bacterial infections caused by the biofilm forming bacteria are 1000 times more resistant to antibiotics than the planktonic bacteria. Among the known bacterial infections, more than 70% involve biofilms which severely complicates treatment options. Biofilm formation is mainly regulated by the Quorum sensing (QS) mechanism. Interference with the QS system by the quorum quenching (QQ) enzyme is a potent strategy to mitigate biofilm. In this study, bacterial strains with QQ activity were identified and their anti-biofilm potential was investigated against the Multidrug Resistant (MDR) Pseudomonas aeruginosa. A Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136-based bioassays were used to confirm the degradation of different Acyl Homoserine Lactones (AHLs) by QQ isolates. The 16S rRNA gene sequencing of the isolated strains identified them as Bacillus cereus strain QSP03, B. subtilis strain QSP10, Pseudomonas putida strain QQ3 and P. aeruginosa strain QSP01. Biofilm mitigation potential of QQ isolates was tested against MDR P. aeruginosa and the results suggested that 50% biofilm reduction was observed by QQ3 and QSP01 strains, and around 60% reduction by QSP10 and QSP03 bacterial isolates. The presence of AHL degrading enzymes, lactonases and acylases, was confirmed by PCR based screening and sequencing of the already annotated genes aiiA, pvdQ and quiP. Altogether, these results exhibit that QQ bacterial strains or their products could be useful to control biofilm formation in P.aeruginosa.     

Quorum sensing inhibitors from the red alga,<Emphasis Type="Italic">Ahnfeltiopsis flabelliformis</Emphasis>

Three new acylhomoserine lactone (AHL) inhibitors have been isolated from the Korean red alga.Ahnfeltiopsis flabelliformis, via bioactivity-guided fractionation using the recombinantAgrobacterium tumefaciens liquid culture bioassay. Unlike the majority of AHL inhibitors reported to date, these compounds were α-d-galactopyranosyl-(1→2)-glycerol (floridoside) (1), betonicine (2), and isethionic acid (3), all of which are structurally unrelated to AHLs.     

Anti‐quorum sensing activity of Psidium guajava L. flavonoids against Chromobacterium violaceum and Pseudomonas aeruginosa PAO1

Psidium guajava L., which has been used traditionally as a medicinal plant, was explored for anti‐quorum sensing (QS) activity. The anti‐QS activity of the flavonoid (FL) fraction of P. guajava leaves was determined using a biosensor bioassay with Chromobacterium violaceum CV026. Detailed investigation of the effects of the FL‐fraction on QS‐regulated violacein production in C. violaceum ATCC12472 and pyocyanin production, proteolytic, elastolytic activities, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 was performed using standard methods. Possible mechanisms of QS‐inhibition were studied by assessing violacein production in response to N‐acyl homoserine lactone (AHL) synthesis in the presence of the FL‐fraction in C. violaceum ATCC31532 and by evaluating the induction of violacein in the mutant C. violaceum CV026 by AHL extracted from the culture supernatants of C. violaceum 31532. Active compounds in the FL‐fraction were identified by liquid chromatography–mass spectrometry (LC–MS). Inhibition of violacein production by the FL‐fraction in a C. violaceum CV026 biosensor bioassay indicated possible anti‐QS activity. The FL‐fraction showed concentration‐dependent decreases in violacein production in C. violaceum 12472 and inhibited pyocyanin production, proteolytic and elastolytic activities, swarming motility and biofilm formation in P. aeruginosa PAO1. Interestingly, the FL‐fraction did not inhibit AHL synthesis; AHL extracted from cultures of C. violaceum 31532 grown in the presence of the FL‐fraction induced violacein in the mutant C. violaceum CV026. LC–MS analysis revealed the presence of quercetin and quercetin‐3‐O‐arabinoside in the FL‐fraction. Both quercetin and quercetin‐3‐O‐arabinoside inhibited violacein production in C. violaceum 12472, at 50 and 100 μg/mL, respectively. Results of this study provide scope for further research to exploit these active molecules as anti‐QS agents.     

N-acyl homoserine lactone mediated interspecies interactions between A. baumannii and P. aeruginosa

Acinetobacter baumannii and Pseudomonas aeruginosa are pathogens capable of colonizing the same infection sites and employing N-acyl homoserine lactone (AHL) based quorum-sensing systems to co-ordinate biofilm formation. Hence, the effect of P. aeruginosa AHLs on biofilm formation by A. baumannii and vice versa were investigated using the biofilm impaired quorum sensing mutants, A. baumannii M2 (abaI::Km) and P. aeruginosa PAO-JP2. Complementing the mutants with heterologous, extracted and pure AHLs increased biofilm mass significantly. The surface area coverage and biovolume also increased significantly as observed by confocal scanning laser microscopy which corroborated scanning electron microscope analysis. Autoinducer synthase gene promoters of A. baumannii, P _abaI-lacZ, and P. aeruginosa, P _lasI-lacZ, were induced (p < 0.05) by heterologous AHLs. Growth of A. baumannii was not inhibited by pyocyanin of P. aeruginosa which may allow their co-existence and interaction in the clinical setting, thereby affecting the severity of combined infections and therapeutic measures to control them.     

N-acylhomoserine lactone-degrading bacteria isolated from hatchery bivalve larval cultures

Quorum sensing (QS) systems, which depend on N-acylhomoserine lactone (AHL) signal molecules, mediate the production of virulence factors in many pathogenic microorganisms. One hundred and forty-six bacterial strains, isolated from a bivalve hatchery, were screened for their capacity to degrade five synthetic AHLs [N-butyryl-dl-homoserine lactone (C₄-HSL), N-hexanoyl-dl-homoserine lactone (C₆-HSL), N-octanoyl-dl-homoserine lactone (C₈-HSL), N-decanoyl-dl-homoserine lactone (C₁₀-HSL) and N-dodecanoyl-dl-homoserine lactone (C₁₂-HSL)] using well diffusion agar-plate assays with three biosensors, Chromobacterium violaceum CV026, C. violaceum VIR07 and Agrobacterium tumefaciens NTL4 (pZLR4). The results of these assays led to our choosing four strains (PP2-67, PP2-459, PP2-644 and PP2-663) that were able to degrade all five synthetic AHLs, thus showing a wide spectrum of quorum quenching (QQ) activity. We subsequently confirmed and measured the QQ activity of the four strains by high-performance liquid chromatography plus mass-spectrometry analysis (HPLC–MS). One of the strains which showed the highest AHL-degrading activity, PP2-459, identified as being a member of the genus Thalassomonas was chosen for further study. Finally, using thin-layer chromatography (TLC), we went on to confirm this strain's capacity to degrade the AHLs produced by other non-pathogenic and pathogenic bacteria not taxonomically related.     

Quorum quenching by Bacillus cereus U92: a double-edged sword in biological control of plant diseases

In the present survey, quorum quenching activity was examined from a biocontrol point of view. Acyl-homoserine lactone (AHL) degrading bacteria were isolated from tomato rhizosphere using two standard bioreporter strains and different synthetic AHLs and then identified according to 16S rDNA sequences. Five isolates capable of inactivating both short and long 3oxo-substituted AHLs showed high similarity with the genera Bacillus, Microbacterium and Arthrobacter, and thereby Bacillus cereus U92 was determined as the most efficient quorum quencher strain. In the quantitative experiments, this strain remarkably inactivated all synthetic AHLs up to 80%. In the laboratory co-cultures, B. cereus U92 efficiently quenched QS-regulated phenotypes in Agrobacterium tumefaciens, Pseudomonas aeruginosa, Pseudomonas chlororaphis and Chromobacterium violaceum. The strain successfully reduced the frequency of Ti-plasmid conjugal transfer in A. tumefaciens by about 99% in the binary cultures. Meanwhile, in a more natural environment, this strain acted as a biocontrol agent, efficient in alleviating QS-regulated crown gall incidence on tomato roots (up to 90%) as well as attenuating Pectobacterium soft rot on potato tubers (up to 60%). On the other hand, reducing phenazine production in P. chlororaphis operated as a suppressor of its QS-regulated biocontrol activity and also inhibited pyocyanin production in P. aeruginosa, a plant growth-promoting bacterium, by 75%. In general, B. cereus U92 seems very promising in the biological control of pathogenic bacteria; however, its broad AHL-degrading activity has a detrimental role on beneficial microbes which should not be neglected.     

Production of acylated homoserine lactones by Aeromonas and Pseudomonas strains isolated from municipal activated sludge

Up to now, the production and role of N-acyl homoserine lactones (AHLs) in activated sludge have been poorly understood. In this study, cross-feeding assays with the reporter strains Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 were used to investigate AHL signal production by municipal activated sludge samples. AHL signal production was consistently detected from municipal activated sludge when different samples were incubated on nutrient media. From one municipal activated sludge sample, 10 strains producing AHL-like auto inducers were isolated by an overlay technique. 16S rDNA-based phylogenetic analysis showed that eight of the isolates belonged to Aeromonas spp. and two to Pseudomonas spp. Box-PCR indicated that six of these Aeromonas isolates were different strains and the two Pseudomonas strains were identical. The production of AHL or AHL-like compounds by these strains was confirmed by thin layer chromatography and biosensor overlays. The six different Aeromonas strains were found to produce the same set of AHLs, including N-hexanoyl-L-homoserine lactone. These results may indicate the possible presence of AHLs in municipal activated sludge. The potential roles of AHL in this eco system are briefly discussed.     

Detection of acylated homoserine lactones produced by Vibrio spp. and related species isolated from water and aquatic organisms

Aims: To assess the diversity in production of acylated homoserine lactones (AHLs) among Vibrio spp and related species. Methods and Results: A total of 106 isolates, with representatives of 28 Vibrio spp and related species, were investigated for the production of AHLs. For this, a rapid method for the screening of AHLs was developed based on the use of bacterial biosensors using a double‐layer microplate assay. At least one bacterial biosensor was activated in 20 species, Agrobacterium tumefaciens being the most frequently activated biosensor. One isolate of Vibrio anguillarum, Vibrio rotiferianus and Vibrio metschnikovii activated the Chromobacterium violaceum biosensor, which is not common among the Vibrionaceae family. For those species with more than one isolate, the biosensor activation profile was the same except for two species, V. anguillarum and V. metschnikovii, which varied among the different isolates. Conclusions: AHL production was observed in the majority of the studied species, with a diverse biosensor activation profile. Significance and Impact of the Study: The high diversity in AHL production is in consistence with the high diversity in ecological niches of the Vibrionaceae family. The absence of AHL detection in eight species warrants further work on their quorum‐sensing systems.     

Biofilms on Indwelling Urethral Catheters Produce Quorum-Sensing Signal Molecules In Situ and In Vitro

Acylated homoserine lactones (AHLs) are chemical signals that mediate population density-dependent (quorum-sensing) gene expression in numerous gram-negative bacteria. In this study, gram-negative bacilli isolated from catheters were screened for AHL production by a cross-feeding assay utilizing an AHL-responsive Agrobacterium tumefaciens reporter strain. Positive reactions were obtained from 14 isolates of Pseudomonas aeruginosa; negative or weakly positive reactions were recorded for isolates of five other species. P. aeruginosa biofilms were then produced on catheters in a physical model of the bladder. Sections of colonized all-silicone catheters gave positive reactions for the quorum-sensing signal molecules as did sections that had been cleaned of biofilm and autoclaved. Control sections of unused catheters were negative in the tests. Sections from four of nine catheters that had been freshly removed from patients gave positive reactions for AHLs. Cleaned autoclaved sections of three of these catheters also gave strongly positive reactions for AHLs. These results demonstrate that AHLs are produced by biofilms as they develop on the catheters both in vitro in the model and in vivo in the patient’s bladder. They represent the first demonstration of AHL production by biofilms in a clinical setting.     

Production and Degradation of N-Acylhomoserine Lactone Quorum Sensing Signal Molecules in Bacteria Isolated from Activated Sludge

N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n=107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n=46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.     

Nonbioluminescent Strains of Photobacterium phosphoreum Produce the Cell-to-Cell Communication Signal N-(3-Hydroxyoctanoyl)homoserine Lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (R_f value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.     

Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout

Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains. The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V. anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V. anguillarum indicating autoinduction. AHLs were produced by all serotypes (O1 to O10) and by non-typable strains. During infection with V. anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram. Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL. No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM. Our data show that AHLs are produced by almost all V. anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.     

Overexpressed recombinant quorum quenching lactonase reduces the virulence,motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min⁻¹ for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48–75.9%), and rhamnolipids (52.7–63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.

     

Analysis of N-acyl homoserine-lactone quorum-sensing molecules made by different strains and biovars of Rhizobium leguminosarum containing different symbiotic plasmids

Strains of Rhizobium leguminosarum use a cell density-dependent gene regulatory system to assess their population density. This is achieved by the accumulation of N-acyl-homoserine lactones (AHLs) in the environment during growth of the bacteria and these AHLs stimulate the induction of various bacterial genes that are up-regulated in the late-exponential and stationary phases of growth. A genetically well-characterised strain of R. leguminosarum biovar viciae was found to have four genes, whose products synthesise different AHLs. We have analysed AHL production by four genetically distinct isolates of R. leguminosarum, three of bv. viciae and one of bv. phaseoli. Distinct differences were seen in the pattern of AHLs produced by the bv. viciae strains compared with bv. phaseoli and the increased levels and diversity of AHLs found in bv. viciae strains can be attributed to the rhiI gene, which is located on the symbiotic (Sym) plasmid and is up-regulated when the bacteria are grown in the rhizosphere. Additional complexity to the profile of AHLs is found to be associated with highly transmissible plasmid pRL1JI of R. leguminosarum bv. viciae, but this is not observed with some other strains, including those carrying different transmissible plasmids. In addition to AHLs produced by the products of genes on the symbiotic plasmid, there is clear evidence for the presence of other AHL production loci. Expression levels and patterns of AHLs can change markedly in different growth media. These results indicate that there is a network of quorum-sensing loci in different strains of R. leguminosarum and these loci may play a role in adapting to rhizosphere growth and plasmid transfer.     

Development of a sensitive bioassay method for quorum sensing inhibitor screening using a recombinantAgrobacterium tumefaciens

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the β-galactosidase activity (β-GAL) of a recombinantAgrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing β-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration-dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of β-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.     

Heterologous overexpression of quorum-sensing regulators to study cell-density-dependent phenotypes in a symbiotic plant bacterium <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis>

Quorum-sensing is widespread among many prokaryotic lineages. In order to investigate quorum regulation in the plant bacterium Mesorhizobium huakuii which produces an N-acyl homoserine lactone (AHL) quorum signal, the Agrobacterium quorum-sensing regulator TraR was heterologously expressed in this bacterium. The resulting strains showed reduced AHL production in the supernatant compared to wild-type, but similar intracellular levels of AHLs were detected, suggesting that M. huakuii AHLs can be bound to intracellular TraR proteins and thus become unavailable for its own quorum systems. M. huakuii overexpressing TraR formed thinner biofilms than the wild-type, suggesting a role played by quorum-sensing in biofilm formation.Hui Wang and Zengtao Zhong contributed equally to this work.     

Presence of Quorum-Sensing-Mediated Gene Regulation in Pathogenic Ice-Nucleation-Active (INA) Bacteria

Nine out of a total of 20 pathogenic ice-nucleation-active bacteria, with different levels of inducible INA, were tested and found positive for their ability to synthesize quorum-sensing (QS) signals. The bacteria were isolated from willow plants and belonged to the genera Bacillus, Erwinia, Pseudomonas and Sphingomonas. As reporter bacteria, to detect the homoserine lactone (HSL) autoinducer, Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeroginosa, Aeromonas hydrophila and Vibrio fischeri strains were used. We thus provide evidence that pathogenic ice-nucleation bacteria with inducible INA produce QS signals that in other bacteria have been shown to be in the control of genes of importance for pathogenicity.     

Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.