Contribution of quorum‐sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1

Aims: To investigate roles of quorum‐sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin‐resistant variant (hereinafter DR1R). Methods and Results: The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane‐degradation and biofilm‐formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm‐formation and hexadecane‐biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild‐type cell supernatant and exogenous C₁₂‐AHL. Conclusions: The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. Significance and Impact of the Study: This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.     

A quorum sensing‐defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post‐harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall‐degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild‐type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild‐type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild‐type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI‐inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild‐type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down‐regulated in the mutant compared with the wild‐type strain.     

The global regulator Crc modulates metabolism,susceptibility to antibiotics and virulence in Pseudomonas aeruginosa

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross‐talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high‐throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing‐regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta‐lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.     

Physiological and metabolic responses for hexadecane degradation in <Emphasis Type="Italic">Acinetobacter oleivorans</Emphasis> DR1

The hexadecane degradation of Acinetobacter oleivorans DR1 was evaluated with changes in temperature and ionic salt contents. Hexadecane degradation of strain DR1 was reduced markedly by the presence of sodium chloride (but not potassium chloride). High temperature (37°C) was also shown to inhibit the motility, biofilm formation, and hexadecane biodégradation. The biofilm formation of strain DR1 on the oil-water interface might prove to be a critical physiological feature for the degradation of hexadecane. The positive relationship between biofilm formation and hexadecane degradation could be observed at 30° C, but not at low temperatures (25°C). Alterations in cell hydrophobicity and EPS production by temperature and salts were not correlated with biofilm formation and hexadecane degradation. Our proteomic analyses have demonstrated that metabolic changes through the glyoxylate pathway are important for efficient degradation of hexadecane. Proteins involved in fatty acid metabolism, gluconeogenesis, and oxidative stress defense proteins appear to be highly expressed during biodégradation of hexadecane. These results suggested that biofilm formation and oxidative stress defense are important physiological responses for hexadecane degradation along with metabolic switch to glyoxylate pathway in strain DR1.     

Secondary Metabolites Produced by the Marine Bacterium Halobacillus salinus That Inhibit Quorum Sensing-Controlled Phenotypes in Gram-Negative Bacteria

Certain bacteria use cell-to-cell chemical communication to coordinate community-wide phenotypic expression, including swarming motility, antibiotic biosynthesis, and biofilm production. Here we present a marine gram-positive bacterium that secretes secondary metabolites capable of quenching quorum sensing-controlled behaviors in several gram-negative reporter strains. Isolate C42, a Halobacillus salinus strain obtained from a sea grass sample, inhibits bioluminescence production by Vibrio harveyi in cocultivation experiments. With the use of bioassay-guided fractionation, two phenethylamide metabolites were identified as the active agents. The compounds additionally inhibit quorum sensing-regulated violacein biosynthesis by Chromobacterium violaceum CV026 and green fluorescent protein production by Escherichia coli JB525. Bacterial growth was unaffected at concentrations below 200 μg/ml. Evidence is presented that these nontoxic metabolites may act as antagonists of bacterial quorum sensing by competing with N-acyl homoserine lactones for receptor binding.     

Influence of <Emphasis Type="Italic">Pseudomonas aeruginosa pvdQ</Emphasis> Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required for up-regulated during swarming motility, which is triggered by high cell densities. As high-density bacterial populations also display elevated antibiotic resistance, studies have demonstrated that swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study is to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions, and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduced PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a two to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.     

Indole and 7‐hydroxyindole diminish Pseudomonas aeruginosa virulence

Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa.     

A Quadruple Knockout of lasIR and rhlIR of Pseudomonas aeruginosa PAO1 That Retains Wild-Type Twitching Motility Has Equivalent Infectivity and Persistence to PAO1 in a Mouse Model of Lung Infection

It has been widely reported that quorum-sensing incapable strains of Pseudomonas aeruginosa are less virulent than wild type strains. However, quorum sensing mutants of P. aeruginosa have been shown to develop other spontaneous mutations under prolonged culture conditions, and one of the phenotypes of P. aeruginosa that is frequently affected by this phenomenon is type IV pili-dependent motility, referred to as twitching motility. As twitching motility has been reported to be important for adhesion and colonisation, we aimed to generate a quorum-sensing knockout for which the heritage was recorded and the virulence factor production in areas unrelated to quorum sensing was known to be intact. We created a lasIRrhlIR quadruple knockout in PAO1 using a published technique that allows for the deletion of antibiotic resistance cartridges following mutagenesis, to create an unmarked QS knockout of PAO1, thereby avoiding the need for use of antibiotics in culturing, which can have subtle effects on bacterial phenotype. We phenotyped this mutant demonstrating that it produced reduced levels of protease and elastase, barely detectable levels of pyoverdin and undetectable levels of the quorum sensing signal molecules N-3-oxododecanoly-L-homoserine lactone and N-butyryl homoserine lactone, but retained full twitching motility. We then used a mouse model of acute lung infection with P. aeruginosa to demonstrate that the lasIRrhlIR knockout strain showed equal persistence to wild type parental PAO1, induced equal or greater neutrophil infiltration to the lungs, and induced similar levels of expression of inflammatory cytokines in the lungs and similar antibody responses, both in terms of magnitude and isotype. Our results suggest, in contrast to previous reports, that lack of quorum sensing alone does not significantly affect the immunogenicity, infectiveness and persistence of P. aeruginosa in a mouse model of acute lung infection.     

Gram-Positive Marine Bacteria as a Potential Resource for the Discovery of Quorum Sensing Inhibitors

Inhibitors of bacterial quorum sensing have been proposed as potentially novel therapeutics for the treatment of certain bacterial diseases. We recently reported a marine Halobacillus salinus isolate that secretes secondary metabolites capable of quenching quorum sensing phenotypes in several Gram-negative reporter strains. To investigate how widespread the production of such compounds may be in the marine bacterial environment, 332 Gram-positive isolates from diverse habitats were tested for their ability to interfere with Vibrio harveyi bioluminescence, a cell signaling-regulated phenotype. Rapid assay methods were employed where environmental isolates were propagated alongside the reporter strain. “Actives” were defined as bacteria that interfered with bioluminescence without visible cell-killing effects (antibiotic activity). A total of 49 bacterial isolates interfered with bioluminescence production in the assays. Metabolite extracts were generated from cultures of the active isolates, and 28 reproduced the bioluminescence inhibition against V. harveyi. Of those 28, five extracts additionally inhibited violacein production by Chromobacterium violaceum. Chemical investigations revealed that phenethylamides and a cyclic dipeptide are two types of secondary metabolites responsible for the observed activities. The active bacterial isolates belonged primarily to either the genus Bacillus or Halobacillus. The results suggest that Gram-positive marine bacteria are worthy of further investigation for the discovery of quorum sensing antagonists.     

Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue‐cultured plantlets grown in vitro. Six mutants of strain XaFL07‐1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly‐β‐hydroxybutyrate than the wild‐type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non‐ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild‐type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.     

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

Autonomous bacterial localization and gene expression based on nearby cell receptor density

Escherichia coli were genetically modified to enable programmed motility, sensing, and actuation based on the density of features on nearby surfaces. Then, based on calculated feature density, these cells expressed marker proteins to indicate phenotypic response. Specifically, site‐specific synthesis of bacterial quorum sensing autoinducer‐2 (AI‐2) is used to initiate and recruit motile cells. In our model system, we rewired E. coli's AI‐2 signaling pathway to direct bacteria to a squamous cancer cell line of head and neck (SCCHN), where they initiate synthesis of a reporter (drug surrogate) based on a threshold density of epidermal growth factor receptor (EGFR). This represents a new type of controller for targeted drug delivery as actuation (synthesis and delivery) depends on a receptor density marking the diseased cell. The ability to survey local surfaces and initiate gene expression based on feature density represents a new area‐based switch in synthetic biology that will find use beyond the proposed cancer model here.     

Quorum quenching is an antivirulence strategy employed by endophytic bacteria

Bacteria predominantly use quorum sensing to regulate a plethora of physiological activities such as cell-cell crosstalk, mutualism, virulence, competence, biofilm formation, and antibiotic resistance. In this study, we investigated how certain potent endophytic bacteria harbored in Cannabis sativa L. plants use quorum quenching as an antivirulence strategy to disrupt the cell-to-cell quorum sensing signals in the biosensor strain, Chromobacterium violaceum. We used a combination of high-performance liquid chromatography high-resolution mass spectrometry (HPLC-ESI-HRMSⁿ) and matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) to first quantify and visualize the spatial distribution of the quorum sensing molecules in the biosensor strain, C. violaceum. We then showed, both quantitatively and visually in high spatial resolution, how selected endophytic bacteria of C. sativa can selectively and differentially quench the quorum sensing molecules of C. violaceum. This study provides fundamental insights into the antivirulence strategies used by endophytes in order to survive in their ecological niches. Such defense mechanisms are evolved in order to thwart the plethora of pathogens invading associated host plants in a manner that prevents the pathogens from developing resistance against the plant/endophyte bioactive secondary metabolites. This work also provides evidence towards utilizing endophytes as tools for biological control of bacterial phytopathogens. In continuation, such insights would even afford new concepts and strategies in the future for combating drug resistant bacteria by quorum-inhibiting clinical therapies.     

Genotype‐by‐environment interactions due to antibiotic resistance and adaptation in Escherichia coli

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype‐by‐environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic‐free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic‐free conditions (LB medium), and did the same for the antibiotic‐sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant‐evolved genotypes were more phenotypically different from the ancestor and from each other than resistant‐nonevolved and sensitive‐evolved genotypes. This suggests genotype‐by‐environment interactions generated by antibiotic resistance mutations, observed previously in short‐term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long‐term selection on resistance mutations from fitness effects in a single environment.     

Inhibition of quorum sensing in Chromobacterium violaceum by pigments extracted from Auricularia auricular

Aims: This study aimed to search for a novel quorum‐sensing inhibitor from some fungi and analyse its inhibitory activity. Methods and Results: Chromobacterium violaceum CV026, a double mini‐Tn5 mutant, was used as an indicator to monitor quorum‐sensing inhibition. Auricularia auricular pigments from fruiting bodies were extracted using hydrochloric acid as an infusion, dissolved in alkaline dimethylsulfoxide (DMSO), sterilized by filtration through a 0·22‐μm membrane filter and added to C. violaceum CV026 cultures. Inhibitory activity was measured by quantifying violacein production using a microplate reader. The results have revealed that the alkaline DMSO‐soluble pigments significantly reduced violacein production in a concentration‐dependent manner, a quorum‐sensing‐regulated behaviour in C. violaceum. Conclusions: Auricularia auricular pigments can inhibit bacterial quorum sensing. Significance and Impact of the Study: The results suggest the bioactive constituents from edible and medicinal fungi could interfere with bacterial quorum‐sensing system, regulate its associate functions and prevent bacterial pathogenesis. Further studies were in process in our laboratory to isolate specific compounds from A. auricular pigments, evaluate them as quorum‐sensing inhibitors and analyse the exact mechanism of action.