David C. Rowley

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

ABSTRACT 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.

Antagonistic interactions among marine bacteria impede the proliferation of Vibrio cholerae.

ABSTRACT Changes in global climate have raised concerns about the emergence and resurgence of infectious diseases. Vibrio cholerae is a reemerging pathogen that proliferates and is transported on marine particles. Patterns of cholera outbreaks correlate with sea surface temperature increases, but the underlying mechanisms for rapid proliferation of V. cholerae during ocean warming events have yet to be fully elucidated. In this study, we tested the hypothesis that autochthonous marine bacteria impede the spread of V. cholerae in the marine environment. It was found that some marine bacteria are capable of inhibiting the growth of V. cholerae on surfaces and that bacterial isolates derived from pelagic particles show a greater frequency of V. cholerae inhibition than free-living bacteria. Vibrio cholerae was less susceptible to antagonism at higher temperatures, such as those measured during El Niño-Southern Oscilliation and monsoonal events. Using a model system employing green fluorescent protein-labeled bacteria, we found that marine bacteria can directly inhibit V. cholerae colonization of particles. The mechanism of inhibition in our model system was linked to the biosynthesis of andrimid, an antibacterial agent. Antibiotic production by the model antagonistic strain decreased at higher temperatures, thereby explaining the increased competitiveness of V. cholerae under warmer conditions. These findings suggest that bacterium-bacterium antagonism is a contributing mechanism in regulating the proliferation of V. cholerae on marine particles.

Natural products chemistry and taxonomy of the marine cyanobacterium Blennothrix cantharidosmum.

A Papua New Guinea field collection of the marine cyanobacterium Blennothrix cantharidosmum was investigated for its cytotoxic constituents. Bioassay-guided isolation defined the cytotoxic components as the known compounds lyngbyastatins 1 and 3. However, six new acyl proline derivatives, tumonoic acids D-I, plus the known tumonoic acid A were also isolated. Their planar structures were defined from NMR and MS data, while their stereostructures followed from a series of chiral chromatographies, degradation sequences, and synthetic approaches. The new compounds were tested in an array of assays, but showed only modest antimalarial and inhibition of quorum sensing activities. Nevertheless, these are the first natural products to be reported from this genus, and this inspired a detailed morphologic and 16S rDNA-based phylogenetic analysis of the producing organism.

Honaucins A−C, Potent Inhibitors of Inflammation and Bacterial Quorum Sensing: Synthetic Derivatives and Structure-Activity Relationships

Honaucins A-C were isolated from the cyanobacterium Leptolyngbya crossbyana which was found overgrowing corals on the Hawaiian coast. Honaucin A consists of (S)-3-hydroxy-γ-butyrolactone and 4-chlorocrotonic acid, which are connected via an ester linkage. Honaucin A and its two natural analogs exhibit potent inhibition of both bioluminescence, a quorum-sensing-dependent phenotype, in Vibrio harveyi BB120 and lipopolysaccharide-stimulated nitric oxide production in the murine macrophage cell line RAW264.7. The decrease in nitric oxide production was accompanied by a decrease in the transcripts of several proinflammatory cytokines, most dramatically interleukin-1β. Synthesis of honaucin A, as well as a number of analogs, and subsequent evaluation in anti-inflammation and quorum-sensing inhibition bioassays revealed the essential structural features for activity in this chemical class and provided analogs with greater potency in both assays.

Effects of Cranberry Extracts on Growth and Biofilm Production of Escherichia coli and Staphylococcus species

Biofilm producing bacteria such as Staphylococcus species and Escherichia coli are the most common cause of catheter related urinary tract infections (UTIs). The American cranberry (Vaccinium macrocarpon) is utilized widely as a prophylaxis for UTIs due to its prevention of microbial adhesion. Cranberry contains proanthocyanidins (PACs), which have been implicated as active constituents responsible for its bacterial antiadhesive properties. Despite overwhelming data supporting cranberrys beneficial effects against human pathogenic bacteria, there is limited information regarding its effects on biofilm formation. This study evaluated the effects of three proprietary PAC‐standardized cranberry extracts on the inhibition of bacterial growth and biofilm production against a panel of clinically relevant pathogens: Staphylococcus epidermidis, Staphylococcus aureus, clinical methicillin‐resistant S. aureus (MRSA), Staphylococcus saprophyticus and Escherichia coli. The extracts inhibited the growth of the Gram‐positive bacteria (Staphylococcus spp.) but not the Gram‐negative species (E. coli) with minimum inhibitory concentrations in the range 0.02–5 mg/mL. The extracts also inhibited biofilm production by the Gram‐positive bacteria but did not eradicate their established biofilm. These results suggest that cranberry may have beneficial effects against the growth and biofilm producing capability of Gram‐positive bacteria pathogens. Copyright

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.

Probiotic Strains for Shellfish Aquaculture: Protection of Eastern Oyster, Crassostrea virginica, Larvae and Juveniles Againsl Bacterial Challenge

ABSTRACT Bacterial pathogens, including several Vibrio spp. and Roseovarius crassostreae, cause severe mortality of larval and juvenile eastern oysters. The introduction of beneficial bacterial isolates in oyster hatcheries and nurseries for the biocontrol of bacterial diseases is a good alternative to the use of antibiotics. The goal of this study was to screen and characterize marine bacterial isolates as potential agents to prevent larval and juvenile mortality by the oyster pathogens Vibrio tubiashii and R. crassostreae. Screening of bacterial isolates from Rhode Island marine organisms and environment using agar-based assay methods for detection of antimicrobial activity against oyster pathogens led to the isolation of candidate probionts Phaeobacter sp. S4 and Bacillus pumilus RI06-95. Pretreatment of larval and juvenile oysters for 24 h with 102–106 cfu/mL Phaeobacter sp. S4 or B. pumilus RI06-95 protected larval oysters against mortality resulting from challenge with R. crassostreae and V. tubiashii (relative percent survival (RPS) range, 9%–56%). These probiotics also protected juvenile oysters against challenge with V. tubiashii (RPS, 37%–50%). Probiotic isolates had no negative impact on oyster survival. Protection conferred to larvae against bacterial challenge was short-lived, lasting for only 24 h after removal of the probiotics from the incubation water. These results suggest the potential of marine bacterial isolates Phaeobacter sp. S4 and B. pumilus RI06-95 to serve as biocontrol agents to reduce the impact of bacterial pathogens in the culture of Crassostrea virginica.

Synthesis and biological activity of P2–P4 azapeptidomimetic P1-argininal and P1-ketoargininamide derivatives: a novel class of serine protease inhibitors

Abstract Molecular modeling and topographic considerations of the thrombin-specific sequences Boc-Asp-Pro-Arg-TS or Ac-d-Phe-Pro-Arg-TS (TS = transition state analog electrophilic center) and related scaffolds led to the design of novel P 2 –P 4 -azapeptidomimetic P 1 -argininal and P 1 -ketoargininamide derivatives ( 3a-j ). The synthesis and biological activity of these potential serine protease inhibitors are presented.

Structure–activity studies of echinomycin antibiotics against drug-resistant and biofilm-forming Staphylococcus aureus and Enterococcus faecalis

Four echinomycin antibiotics were isolated from the culture broth of a marine streptomycete, and their structures were determined by a combination of chemical and spectroscopic analyses. Antibiotic activities were measured against drug-resistant and biofilm-forming strains of Staphylococcus aureus and Enterococcus faecalis. Minimum inhibitory concentrations ranging from 0.01 microM to greater than 14 microM clearly defined structure-activity relationships for antibiotic potency. Echinomycin was the most active compound with a MIC of 0.03 microM against methicillin-resistant S. aureus and 0.01 microM against biofilm-forming E. faecalis.

Vibrio cholerae exploits sub-lethal concentrations of a competitor-produced antibiotic to avoid toxic interactions

Vibrio cholerae is a human pathogenic marine bacterium inhabiting coastal regions and is vectored into human food and water supplies via attachment to particles including detritus, phytoplankton, and zooplankton. Particle colonization by the pathogen is inhibited by an antagonistic interaction with the particle-associated Vibrionales bacterium SWAT3, a producer of the antibiotic andrimid. By analyzing the individual movement behaviors of V. cholerae exposed to a gradient of andrimid in a microfluidics device, we show that the pathogen has a concentration dependent avoidance response to sub-lethal concentrations of the pure antibiotic and to the metabolites produced by a growing colony of SWAT3-wild-type. This avoidance behavior includes a 25% increase in swimming speeds, 30% increase in run lengths, and a shift in the direction of the bacteria away from the andrimid source. Consequently, these behavioral shifts at low concentrations of andrimid would lead to higher diffusivity and result in the dispersion of bacteria away from the competitor and source of the antibiotic. Such alterations in motility were not elicited in response to a non-andrimid-producing SWAT3 mutant, suggesting andrimid may be a negative effector of chemotaxis for V. cholerae. The behavioral response of colonizing bacteria to sub-inhibitory concentrations of competitor-produced antibiotics is one mechanism that can influence microbial diversity and interspecific competition on particles, potentially affecting human health in coastal communities and element cycling in the ocean.