NPN Uptake Research Articles

Application of Floating TiO2 Photocatalyst for Methylene Blue Decomposition and Salmonella typhimurium Inactivation

The growing level of wastewater as well as pollution of freshwater by various bacteria are essential worldwide issues which have to be solved. In this contribution, nanocrystalline anatase TiO2 films deposited by magnetron sputtering on high-density polystyrene (HDPE) beads were applied as floating photocatalysts for Salmonella typhimurium bacterial inactivation in water for the first time. Additionally, the photocatalytic degradation of methylene blue dye in the presence of HDPE beads with TiO2 film under UV-B irradiation was investigated. The suitability to adopt such floating photocatalyst structures for practical applications was tested in cycling experiments. The detailed surface morphology, crystal structure, elemental mapping, surface chemical composition and bond analysis of deposited TiO2 films were investigated by scanning electron microscope, X-ray diffractometer and X-ray photoelectron spectroscope techniques. The bacterial viability as well as MB decomposition experiments showed promising results by demonstrating that 6% of bacterial colonies were formed after the first run and only about 1% after the next four runs, which is an appropriate outcome for practical applications. NPN uptake results showed that the permeability of the outer membrane was significantly increased as well.

Synergistic Generation of Reactive Oxygen Species by Visible Light Activated TiO2 and S. Enterica Interaction

Abstract A lot of human activities have negative impact on water quality and sometimes result in the biological water contamination. Currently used chemical (chlorine, ozone, and etc.) and physical (UV) water disinfection methods have strong environmental disadvantages or suffers from limited efficiency. To overcome these problems, scientists suggest to use photocatalyst activated advanced oxidation processes. One of the most studied photocatalysts which attracts a lot of research interest is titanium dioxide. TiO2 application for the disinfection of water, air or surfaces is increasingly encouraged by researchers. However, to unlock its full potential it is highly desirable to make it suitable for the visible light activation. In the current study the effect of visible light assisted photocatalytic treatment to the outer membrane permeability of Salmonella enterica bacteria and how it changes under different titanium dioxide concentrations was analysed. The results from the treatment of relatively complex Salmonella enterica bacteria organism were compared to the visible light activated TiO2 ability to oxidise considerably simpler objects like methylene blue molecules. The efficiency of TiO2 photocatalytic disinfection process was evaluated using spread plate technique. Membrane permeability of the treated Salmonella enterica bacteria was determined by NPN uptake factor assay. Generation of intracellular reactive oxygen species was evaluated by Dichlorodihydrofluorescein diacetate fluorescence measurements. The key finding of this study was that intense wide spectrum visible light irradiation and TiO2 powder synergistically inactivate S. enterica bacteria and halt its potential to form colonies. High amounts of intracellular reactive oxygen species could be seen as the main suspects for the observed inactivation of S. enterica.

Bacterial biofilm formation on ion exchange membranes

Ion exchange membranes (IEMs) often suffer from biofouling, which reduces ion exchange rates and increases energy consumption in water treatment processes, such as electrodialysis, reverse electrodialysis, membrane capacitive deionization, and Donnan dialysis, and in energy devices, such as microbial fuel cells. In the present study, microbial biofilm formation was studied on anion exchange membranes (AEMs) and a cation exchange membranes (CEMs) of the homogeneous and heterogeneous types. Biofilm formation of Pseudomonas aeruginosa PAO1 on the IEMs was higher on the CEMs than on the AEMs, although more dead cells were found on the AEMs, likely due to the presence of quaternary ammonium moieties on the AEM surface, which are bactericidal. An XTT assay and NPN uptake tests confirmed the antimicrobial properties of the AEM surface. The results also suggested that the surface roughness of the membranes affected interactions between bacteria and the IEMs, being more pronounced on the heterogeneous IEMs than on the homogeneous IEMs. Counter-ion transport properties were studied under the Donnan exchange regime for both pristine and biofouled IEMs. The reduction of counter-ion transport due to biofouling was more pronounced for heterogeneous CEMs and AEMs than for their homogeneous counterparts, while it was more noticeable for the AEMs than for the CEMs. The latter result is explained based on the preferential adsorption of the negatively charged EPS components to the positively charged AEMs.

Antimicrobial potential of myristic acid against Listeria monocytogenes in milk.

Listeria monocytogenes (L. monocytogenes), an important food-borne pathogenic microorganism, has resistance immune function to many commonly used drugs. Myristic acid is a traditional Chinese herbal medicine, but it has been rarely used as a food additive, limiting the development of natural food preservatives. In this study, the antibacterial activity and mechanism of myristic acid against L. monocytogenes were studied. The minimum inhibitory concentration (MIC) of myristic acid against 13 L. monocytogenes strains ranged from 64 to 256 μg ml-1. The time-kill assay demonstrated that when myristic acid was added to dairy products, flow cytometry confirmed that myristic acid influenced cell death and inhibited the growth of L. monocytogenes. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and NPN uptake studies illustrated that myristic acid changed the bacterial morphology and membrane structure of L. monocytogenes, which led to rapid cell death. Myristic acid could bind to DNA and lead to changes in DNA conformation and structure, as identified by fluorescence spectroscopy. Our studies provide additional evidence to support myristic acid being used as a natural antibacterial agent and also further fundamental understanding of the modes of antibacterial action.

Conessine as a novel inhibitor of multidrug efflux pump systems in Pseudomonas aeruginosa

BackgroundHolarrhena antidysenterica has been employed as an ethnobotanical plant for the treatment of dysentery, diarrhoea, fever, and bacterial infections. Biological activities of the principle compound, conessine including anti-diarrhoea and anti-plasmodial effects were documented. Our previous study reported potency of Holarrhena antidysenterica extract and conessine as resistance modifying agents against extensively drug-resistant Acinetobacter baumannii. This study aimed to investigate (i) whether conessine, a steroidal alkaloid compound, could act as a resistance modifying agent against multidrug-resistant Pseudomonas aeruginosa, and (ii) whether MexAB-OprM efflux pump involved in the mechanism.MethodsConessine combined with various antibiotics were determined for synergistic activity against P. aeruginosa PAO1 strain K767 (wild-type), K1455 (MexAB-OprM overexpressed), and K1523 (MexB deletion). H33342 accumulation assay was used to evaluate efflux pump inhibition while NPN uptake assay was assessed membrane permeabilization.ResultsConessine significantly reduced MICs of all antibiotics by at least 8-fold in MexAB-OprM overexpressed strain. The levels were comparable to those obtained in wild-type strain for cefotaxime, levofloxacin, and tetracycline. With erythromycin, novobiocin, and rifampicin, MICs were 4- to 8-fold less than MICs of the wild-type strain. Loss of MexAB-OprM due to deletion of mexB affected susceptibility to almost all antibiotics, except novobiocin. Synergistic activities between other antibiotics (except novobiocin) and conessine observed in MexB deletion strain suggested that conessine might inhibit other efflux systems present in P. aeruginosa. Inhibition of H33342 efflux in the tested strains clearly demonstrated that conessine inhibited MexAB-OprM pump. In contrast, the mode of action as a membrane permeabilizer was not observed after treatment with conessine as evidenced by no accumulation of 1-N-phenylnaphthylamine.ConclusionsThe results suggested that conessine could be applied as a novel efflux pump inhibitor to restore antibiotic activity by inhibiting efflux pump systems in P. aeruginosa. The findings speculated that conessine may also have a potential to be active against homologous resistance–nodulation–division (RND) family in other Gram-negative pathogens.

Chemical composition, antibacterial activity of Cyperus rotundus rhizomes essential oil against Staphylococcus aureus via membrane disruption and apoptosis pathway

The chemical composition, antibacterial activity and mechanism of essential oil from Cyperus rotundus rhizomes against Staphylococcus aureus were investigated in this study. Results showed that α-cyperone, cyperene and α-selinene were the major components of the essential oil. The essential oil exhibited strong antibacterial activity against S. aureus with the minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) were 10 and 20 mg/mL respectively, and the antibacterial effects increased with increasing essential oil concentrations and treatment time. The electric conductivity, cell membrane integrity, NPN uptake, and membrane potential assays demonstrated that essential oil disrupted the membrane integrity of S. aureus. Electron microscope observations further confirmed that essential oil destroyed cell membrane. Moreover, we found that essential oil could induce cells death of S. aureus through apoptosis pathway based on apoptosis analysis. These findings suggested that essential oil mainly exerted antibacterial activity by damaging cell membrane and membrane-mediated apoptosis pathway.

Antibacterial activity and a membrane damage mechanism of Lachnum YM30 melanin against Vibrio parahaemolyticus and Staphylococcus aureus

This study aimed to investigate the antibacterial activity of Lachnum YM30 intracellular melanin (LIM). The dilution method was used to measure the MIC and MBC of Gram-negative bacteria Escherichia coli, Salmonella typhi, Vibrio parahaemolyticus and Gram-positive bacteria Listeria monocytogenes, Bacillus megaterium, Staphylococcus aureus. MIC values for each bacterial strain were 0.4, 2.4, 0.2, 0.6, 0.8, 0.3 mg/mL, and the MBC values were 0.4, 3.2, 0.3, 0.8, 0.8, 0.4 mg/mL, respectively. Bacteriostasis of LIM against S. aureus (G+) and V. parahaemolyticus (G−) was further revealed by SEM observation, testing integrity of cell membrane, NPN uptake and membrane potential. MTT experiment was used to estimate the cell cytotoxicity of LIM. The results showed that LIM significantly damaged the integrity of cell membrane, increased the leakage of cell contents, raised the quantity of NPN uptake and reduced the membrane potential. LIM had no obvious cell cytotoxicity at low concentrations. These findings indicated that LIM had antibacterial activity and could be applied as a potential natural bacteriostatic agent in the food or pharmaceutical industry.

3,6-O-[N-(2-Aminoethyl)-acetamide-yl]-chitosan exerts antibacterial activity by a membrane damage mechanism

A novel chitosan derivative, 3,6-O-[N-(2-aminoethyl)-acetamide-yl]-chitosan (AACS), was successfully prepared to improve water solubility and antibacterial activity of chitosan. AACS had good antibacterial activity, with minimum inhibitory concentrations of 0.25mg/mL, against Escherichia coli and Staphylococcus aureus. Cell membrane integrity, electric conductivity and NPN uptake tests showed that AACS caused quickly increasing the release of intracellular nucleic acids, the uptake of NPN, and the electric conductivity by damaging membrane integrity. On the other hand, hydrophobicity, cell viability and SDS-PAGE experiments indicated that AACS was able to reduce the surface hydrophobicity, the cell viability and the intracellular proteins through increasing membrane permeability. SEM observation further confirmed that AACS could kill bacteria via disrupting their membranes. All results above verified that AACS mainly exerted antibacterial activity by a membrane damage mechanism, and it was expected to be a new food preservative.

The antimicrobial effects of wood-associated polyphenols on food pathogens and spoilage organisms

The antimicrobial effects of the wood-associated polyphenolic compounds pinosylvin, pinosylvin monomethyl ether, astringin, piceatannol, isorhapontin, isorhapontigenin, cycloXMe, dHIMP, ArX, and ArXOH were assessed against both Gram-negative (Salmonella) and Gram-positive bacteria (Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus) and yeasts (Candida tropicalis, Saccharomyces cerevisiae). Particularly the stilbenes pinosylvin, its monomethyl ether and piceatannol demonstrated a clear antimicrobial activity, which in the case of pinosylvin was present also in food matrices like sauerkraut, gravlax and berry jam, but not in milk. The destabilization of the outer membrane of Gram-negative microorganisms, as well as interactions with the cell membrane, as indicated by the NPN uptake and LIVE/DEAD viability staining experiments, can be one of the specific mechanisms behind the antibacterial action. L. monocytogenes was particularly sensitive to pinosylvin, and this effect was also seen in L. monocytogenes internalized in intestinal Caco2 cells at non-cytotoxic pinosylvin concentrations. In general, the antimicrobial effects of pinosylvin were even more prominent than those of a related stilbene, resveratrol, well known for its various bioactivities. According to our results, pinosylvin could have potential as a natural disinfectant or biocide in some targeted applications.

Activities of muscadine grape skin and polyphenolic constituents againstHelicobacter pylori

To identify active phenolic constituents in muscadine grape skin (MGS) extracts and determine interactions among compounds while further exploring their anti-Helicobacter pylori potential in vitro. The inhibitory effects of quercetin and resveratrol, active polyphenols identified in MGS extracts, against H. pylori were investigated. Quercetin and resveratrol significantly (P < 0.05) reduced H. pylori counts regardless of pH with minimal bactericidal concentrations of 256 and 128 μg ml(-1), respectively. MGS extracts displayed the highest efficacy, suggesting additional unidentified compounds not determined in this study. Time-course viability experiments showed a dose-dependent anti-H. pylori response to quercetin and resveratrol. Interestingly, neither quercetin nor resveratrol affected H. pylori outer membrane (OM) integrity as determined by 1-N-phenylnaphthylamine (NPN) uptake assays. However, treatment with MGS extract did increase NPN uptake, indicating OM destabilization possibly by additional unknown components. Furthermore, quercetin was found to enter H. pylori as measured by HPLC supporting intracellular drug accumulation. Quercetin and resveratrol possess strong anti-H. pylori activity in vitro and are independent of pH. Our results also suggest that these compounds do not affect H. pylori OM integrity as previously hypothesized and that the primary antimicrobial activity of quercetin may be linked to interactions with intracellular components. The anti-H. pylori effects of quercetin and resveratrol suggest that these compounds may be useful in the dietary prevention and/or treatment of H. pylori infection.

Antibacterial mechanism and application of ε-polylysine conjugation with dextran.

e-Polylysine-dextran conjugate is a compound conjugated with e-polylysine (PL) and dextran through the Maillard reaction. The antimicrobial activity of PL-dextran conjugate was investigated using six Gram-positive and Gram-negative bacteria. The integrity of the cell membranes of the representative bacteria was determined by the amount of intracellular components released from the cells. It was found that the PL-dextran conjugate retained most of the original antimicrobial activity of PL. When treated with PL or the PL-dextran conjugate, the amount of releasing materials at absorbance of 260 nm quickly increased in both Gram-negative and positive bacteria, but the absorbance values were different due to the difference in cell structures. Furthermore, an outer membrane (OM) permeability assay was performed by fluorescent probe 1-N-phenylnaphthylamine (NPN). The results showed that PL or PL-dextran conjugate rapidly increased the NPN uptake. In addition, the preservative effect and the emulsifying properties of the PL-dextran conjugate were also observed when it is applied in coffee whitner. These findings indicate that the PL-dextran conjugate can be applied in the food processing industry.

Weakening of Salmonella with Selected Microbial Metabolites of Berry-Derived Phenolic Compounds and Organic Acids

Gram-negative bacteria are important food spoilage and pathogenic bacteria. Their unique outer membrane (OM) provides them with a hydrophilic surface structure, which makes them inherently resistant to many antimicrobial agents, thus hindering their control. However, with permeabilizers, compounds that disintegrate and weaken the OM, Gram-negative cells can be sensitized to several external agents. Although antimicrobial activity of plant-derived phenolic compounds has been widely reported, their mechanisms of action have not yet been well demonstrated. The aim of our study was to elucidate the role of selected colonic microbial metabolites of berry-derived phenolic compounds in the weakening of the Gram-negative OM. The effect of the agents on the OM permeability of Salmonella was studied utilizing a fluorescence probe uptake assay, sensitization to hydrophobic antibiotics, and lipopolysaccharide (LPS) release. Our results show that 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid (3,4-diHPP), 3-(4-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, and 3-(3-hydroxyphenyl)propionic acid efficiently destabilized the OM of Salmonella enterica subsp. enterica serovar Typhimurium and S. enterica subsp. enterica serovar Infantis as indicated by an increase in the uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN). The OM-destabilizing activity of the compounds was partially abolished by MgCl2 addition, indicating that part of their activity is based on removal of OM-stabilizing divalent cations. Furthermore, 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3,4-diHPP increased the susceptibility of S. enterica subsp. enterica serovar Typhimurium strains for novobiocin. In addition, organic acids present in berries, such as malic acid, sorbic acid, and benzoic acid, were shown to be efficient permeabilizers of Salmonella as shown by an increase in the NPN uptake assay and by LPS release.

Berry Phenolics: Antimicrobial Properties and Mechanisms of Action Against Severe Human Pathogens

: Antimicrobial activity and mechanisms of phenolic extracts of 12 Nordic berries were studied against selected human pathogenic microbes. The most sensitive bacteria on berry phenolics were Helicobacter pylori and Bacillus cereus. Campylobacter jejuni and Candida albicans were inhibited only with phenolic extracts of cloudberry, raspberry, and strawberry, which all were rich in ellagitannins. Cloudberry extract gave strong microbicidic effects on the basis of plate count with all studied strains. However, fluorescence staining of liquid cultures of virulent Salmonella showed viable cells not detectable by plate count adhering to cloudberry extract, whereas Staphylococcus aureus cells adhered to berry extracts were dead on the basis of their fluorescence and plate count. Phenolic extracts of cloudberry and raspberry disintegrated the outer membrane of examined Salmonella strains as indicated by 1-N-phenylnaphthylamine (NPN) uptake increase and analysis of liberation of [14C]galactose- lipopolysaccharide. Gallic acid effectively permeabilized the tested Salmonella strains, and significant increase in the NPN uptake was recorded. The stability of berry phenolics and their antimicrobial activity in berries stored frozen for a year were examined using Escherichia coli and nonvirulent Salmonella enterica sv. Typhimurium. The amount of phenolic compounds decreased in all berries, but their antimicrobial activity was not influenced accordingly. Cloudberry, in particular, showed constantly strong antimicrobial activity during the storage.

Temperature shock, injury and transient sensitivity to nisin in Gram negatives.

The effect of thermal stresses on survival, injury and nisin sensitivity was investigated in Salmonella Enteritidis PT4, PT7 and Pseudomonas aeruginosa. Heating at 55 degrees C, rapid chilling to 0.5 degrees C or freezing at -20 degrees C produced transient sensitivity to nisin. Cells were only sensitive if nisin was present during stress. Resistance recovered rapidly afterwards, though some cells displayed residual injury. Injury was assessed by SDS sensitivity, hydrophobicity changes, lipopolysaccharide release and NPN uptake. LPS release and hydrophobicity were not always associated with transient nisin sensitivity. Uptake of NPN correlated better but persisted longer after treatment. Thermal shocks produce transient injury to the outer membrane, allowing nisin access. After treatment, the permeability barrier is rapidly restored by a process apparently involving reorganization rather than biosynthetic repair. Inclusion of nisin during food treatments that impose sub-lethal stress on Gram negatives could increase process lethality, enhancing microbiological safety and stability.

Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro.

We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 microg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 microg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic beta-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, and hefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed in E. coli, P. aeruginosa, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics.

Fluorometric assessment of gram-negative bacterial permeabilization.

Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria.

Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas aeruginosa.

The interaction of the polycationic rabbit alveolar macrophage cationic proteins MCP-1 and MCP-2 (or their identical neutrophil equivalents NP-1 and NP-2) with the surface of Pseudomonas aeruginosa was investigated. Both proteins bound avidly to purified lipopolysaccharide, as judged by their ability to competitively displace the probe dansyl polymyxin with 50% inhibition (I50) values of 2 to 3 microM. Similar I50 were measured with dansyl polymyxin as a probe for cell surface binding, suggesting that the initial binding site for MCP-1 and MCP-2 on the surface of cells was lipopolysaccharide. Both MCP-1 and MCP-2 permeabilized outer membranes to the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The initial rate of NPN uptake plotted against the concentration of MCP-1 or MCP-2 gave sigmoidal curves, suggesting cooperative permeabilization of the outer membrane. Replotting the data as a Hill plot gave an affinity parameter, S0.5, the concentration of MCP giving a half-maximal increase in the rate of NPN uptake, of 5 and 25 microM for MCP-1 and MCP-2, respectively, and thus subsequent studies concentrated on the more active permeabilizer MCP-1. Permeabilization of outer membranes to NPN was a function of buffer pH, with lower pH considerably favoring the permeabilizing effects of MCP-1. Thin-section electron microscopic visualization of MCP-1-treated cells showed production of extended blebs. Further evidence of an altered cell surface after MCP-1 treatment was obtained by demonstrating that treated unopsonized cells were more efficiently phagocytosed by unelicited rabbit alveolar macrophages. The data overall suggest that macrophage cationic proteins interact with the P. aeruginosa outer membrane in a manner typical of other polycations and suggest that one of their major functions may be to permeabilize the outer membrane.

Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa.

The mode of interaction of the polycationic aminoglycoside antibiotics with the surface of Pseudomonas aeruginosa cells was studied with the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The addition of the aminoglycoside gentamicin to intact cells in the presence of NPN led to a shift in the fluorescence emission maximum from 460 to 420 nm. At the same time the NPN fluorescence intensity increased fourfold. Gentamicin caused no such effects when added to outer membrane vesicles, suggesting that the increased fluorescence resulted from the interaction of gentamicin with intact cells. Gentamicin-promoted NPN uptake was inhibited by the divalent cations Mg2+ and Ca2+, but occurred in the absence of gentamicin transport across the inner membrane. Low concentrations of gentamicin (2 micrograms/ml) caused NPN fluorescence to increase over a period of 4 min in a sigmoidal fashion. At higher concentrations (50 micrograms/ml) the increase occurred within a few seconds. The final fluorescence intensity was almost independent of the gentamicin concentration. A centrifugation technique was used to demonstrate that gentamicin caused actual uptake of NPN from the supernatant. The initial rate of NPN uptake varied according to the gentamicin concentration in a sigmoidal fashion. Similar data were obtained for seven other aminoglycoside antibiotics. The data, when reanalyzed as a Hill plot, gave a series of lines with a mean slope (the Hill number) of 2.26 +/- 0.26, suggesting that the interaction of aminoglycosides with the cell surface to permeabilize it to NPN involved at least three sites and demonstrated positive cooperativity. There was a statistically significant relationship between the pseudoassociation constant K, from the Hill plots and the minimal inhibitory concentrations for the eight antibiotics. These results are consistent with the concept that aminoglycosides interact as a divalent cation binding site on the P. aeruginosa outer membrane and permeabilize it to the hydrophobic prove NPN.