Nanoparticle Interactions with Bacteria: Toxicity and Chemotaxis

Two projects are presented in this report. First, a silver resistant strain of Pseudomonas sp. (closest relatives Pseudomonas putida and Pseudomonas plecoglossicidaI) was isolated from Town Neck Beach in Sandwich, MA. This strain could grow in 2ppm AgNO3 and at silver nanoparticle concentrations of 2000ppm. The lack of a toxic effect of high silver nanoparticle concentrations and the formation of precipitates in the colonies indicates that the strain may be tolerant of solid silver. Attempts were made to extract and sequence a silver resistance plasmid. Second, chemotaxis experiments using nanoparticles as chemoattractants/repellants were performed using the silver resistant Pseudomonas, Vibrio gazogenes, and swamp water from Cedar Swamp. It was demonstrated that there was no chemoattractant effect of reactive iron nanoparticles in Cedar Swamp water despite the generation of hydrogen, which could be a potentially very strong chemoattractant. Additionally it was shown that while V. gazogenes did not see silver as a chemorepellant, the silver resistant Pseudomonas sp. was repelled by 50 nm silver nanoparticles. Silver resistance and the toxicity of silver nanoparticles The amount of nanoscale silver is increasing rapidly in consumer products the ability to synthesize these particles on a large scale improves. Silver nanoparticles (NPs) are toxic to bacteria, and are currently used in everything from medical devices to sport socks and washing machines to deter microbial growth. Silver is a particularly toxic heavy metal as it interferes with the electron transport chain and binds to DNA (Thurman, 1989)The existence of silver resistant bacteria however is well known, and these bacteria may be less impacted by the presence of silver nanoparticles. Certain bacteria have been shown to accumulate or precipitate silver into silver metal or other insoluble form. To date, studies of silver nanoparticle toxicity have focused on strains of bacteria that lack silver resistance. Therefore it is interesting to study the toxicity of silver nanoparticles to silver resistant bacteria. Materials and Methods Isolation of silver resistant bacteria Samples of sand and water were obtained from Town Neck Beach in Sandwich, MA. Sea water was concentrated by centrifuging 50 ml of seawater for 20 minutes at 4100 rpm. The supernatant was removed and the cells were resuspended in 1 ml of seawater. Concentrated seawater (100 microliters) was plated on nutrient agar and R2A plates containing 0.2, 2, and 20 ppm silver (in the form of silver nitrate). Plates were incubated overnight at 30C and 6 colonies appearing on the 20 ppm NA plates were streaked for isolation on 20 ppm silver nutrient agar plates. Isolates were identified by colony PCR of the 16S rRNA gene. Briefly, colonies were picked and placed in 20 microliters of 1% Nonidet P-40, which was boiled for 5 minutes at 100C. A fraction (0.5 microliters) of this extract was added to a PCR reaction using primers 8F and 1492R (Lane, 1991). PCR product was treated with ExoSAP and directly sequenced. Sequences were uploaded and compared against the ribosomal database project for identification. Plasmid extraction and attempted sequencing 500 ml of silver resistant Pseudemonas strain 3 was grown in nutrient broth with 20 g/L silver (in the form of AgNO3). Cells were pelleted by centrifugation, and plasmid DNA was extracted using a Qiagen Midiprep large scale plasmid purification kit. DNA was quantified using the nanodrop spectrophotometer. 50 microliters of DNA was sheared by repeatedly pipetting up and down using a 20 microliter pipette. These 50 microliters were concentrated via ethanol precipitation, and resuspended in 1 l sterile molecular grade water. One microliter of 2x master mix was added to the concentrated DNA, and incubated at 72C for 10 minutes to add A overhangs to the ends of the sheared DNA. This mixture was ligated into the TOPO cloning vector (Invitrogen) and cloned into One Shot chemical competent E. coli. Growth was slow, and after 30 hours approximately 70 clones were picked for sequencing. Growth curves A 1:1000 dilution of an overnight culture of silver resistant Pseudomonas was aliquoted in 200 l volumes into 96 well clear polystyrene culture plate. AgNO3 or 10 nanometer PVP modified silver nanoparticles were added to the wells in increasing concentrations (triplicate samples for each concentration). The absorbance at 600 nm was monitored approximately hourly for 14 hours. Toxicity assay An overnight culture of silver resistant Pseudomonas was pelleted by centrifugation and resuspended in an equal volume of sea water base. This suspension was aliquoted in 100l volumes into a black costar 96 well plate. Increasing concentrations of AgNO3 or 10 nanometer PVP modified silver nanoparticles were added to each well (in triplicate), and the plates were shaken at room temperature for 20, 40, or 60 minutes, at which point 100 l of 2x BacLight assay (propidium iodide and SYTO 9, Invitrogen) was added to each well. The samples were incubated for 15 minutes at which point fluorescence was quantified with an excitation of 485nm and an emission of both 530 and 630 nanometers. Calibration samples were made with equal concentration live and killed (with isopropanol) suspensions, and a calibration curve was made using the ratio of fluorescence at 530 nm to 630 nm according to manufacturers protocol. PCR and quantitative PCR amplification of silver resistance genes An attempt was made to quantify environmental silver resistance genes from Eel Pond sediment using quantitative PCR. The silver resistance primers described by Woods et. al. were used with BioRad SYBR green 2X master mix. Primers for each gene were as follows: silE forward 5’-aggggaaacggtctgacttc3’, silE reverse 5’-atatccatgagcgggtcaac-3’, silRS forward 5’-ggcaatcgcaatcagatttt-3’, silRS reverse 5’gtggaggatactgcgagagc-3’, silCBA forward 5’-cgggaaacgctgaaaaatta-3’, silCBA reverse 5’gtacgttcccagcaccagtt-3’, silF forward 5’-cgatatgaatgctgccagtg-3’, silF reverse 5’attgccctgctgaataaacg-3’, silB forward 5’-caaagaacagcgcgtgatta-3’, silB reverse 5’-gctcagacattgctggcata-3’, silA forward 5’cttgagcatgccaacaagaa-3’, silA reverse 5’-cctgccagtacaggaaccat-3’, silP forward 5’-cctgggtttacagcgtcatt-3’, and silP reverse 5’-atggcacctgaggtttgttc-3’. Standard PCR cycles were 3 min at 95 °C followed by 35 cycles of 95 °C for 40 s, 55°C for 40 s and 72 °C for 40 s with a final elongation step was performed at 72 °C for 10 min (Woods, 2009). Quantitative PCR conditions for the SilA and SilE genes with the same primers. Cycling parameters were an initial activation at 95C for 10 minutes followed by 50 cycles of 1 minute at 55C followed by 15 seconds of 95C. Detection was at the end of the extension. Calibration curves were constructed using serial dilutions of silver resistant Pseudomonas strain 3 DNA. Results and Discussion Isolation and characterization of a silver resistant Pseudomonas strain Six colonies were chosen for further investigation from the initial plating. Of the six chosen isolates, four had a closest relative as an Acinetobacter sp. and two were members of the genus Pseudomonas. Of the six strains, the two Pseudomonas sp. strains grew more effectively on silver amended plates. Silver resistant strain three, a Pseudomonas sp. most closely related to Pseudomonas putida, a common soil bacterium, and Pseudomonas plecoglossicidaI, a fish pathogen, was the easiest to grow on both silver plates, and could also be grown in liquid media in the presence of silver nitrate, and was therefore chosen for further studies. Figure 1. Six silver resistant isolates and their nearest neighbors as determined by the Weighbor weighted neighbor-joining tree building algorithm. Silver resistant Pseudomonas strain 3 were motile gram negative rods which form smooth sticky colonies on nutrient agar. When strain 3 was plated on nutrient agar with increasing concentrations of silver, from 20mg/l to 100mg/L to 200mg/L, the underside of the colonies became significantly darker, and the agar surrounding the colonies turned brown with increasing silver concentrations. In early streaks, the formation of precipitates in the center underside of the colonies was visible. This is consistent with the results of Haefeli et. al. showing that Pseudomonas sp. can precipitate silver ions although further characterization is needed to identify the precipitate in this study (Haefeli, 1989). Likely as it increases with silver concentration it is a silver compound. Figure 2. Phase contrast micrograph of silver resistance Pseudomonas sp. surrounding a precipitate from 200 mg/L silver nutrient agar plate Figure 3. Colony morphology (A) of silver resistant Pseudomonas sp. on a nutrient agar plate amended with 200mg/L silver. Darkening of colonies with increase in silver concentration (B), clockwise from top left 20mg/L Ag, 100 mg/L Ag, 200 mg/L Ag. A B Pseudomonas sp. silver resistant strain 3 was able to be transferred to liquid culture and grew in both the presence of silver nitrate and silver nanoparticles. The measured minimum inhibitory concentration of AgNO3 was 20ppm Ag+, and the strain was still able to grow in the presence of 2000ppm 10 nanometer silver nanoparticles. Figure 4. Growth of silver resistant Pseudomonas sp. in the presence of silver nitrate. Figure 5. Growth of silver resistant Pseudomonas sp. in the presence of 10 nm PVP coated silver nanoparticles Silver nanoparticles were less toxic to stationary phase bacteria at similar silver concentrations than the dissolved ion. The toxicity of silver nanoparticles has been proposed to be a combination of three methods: dissolution of Ag+ ions, the stimulation or reactive oxygen species production, and the disruption of membrane integrity (Thurman, 1989). Concentrations of 20 ppm silver nitrate was enough to see a decrease in the percent live bacteria after a 20 minute exposure, whereas live bacteria did not decrease significantly over the course of an hour with nanoparticle concentrations up to 1000ppm. The solubility of silver increases with pH and although the dissolved silver concentrations in the nanoparticle suspensions could not be measured, it can be imagined that it would be close to 1% of the silver nanoparticle concentration (Fabrega, 2009 ). This would result in the 10ppm dissolved ion concentration in the 1000 ppm nanoparticle suspensions which would be close to the MIC. Therefore since no toxic effect is seen at a 1000 ppm nanoparticle concentration, it can be imagined that there is not a nanoparticle effect observed in this case. This could be the result of the PVP stabilizing coating on the nanoparticles which electrosterically repels the bacteria, or it can be imagined that since the bacteria appears to be precipitating silver, it is tolerant to living in close relation to silver particles, rendering them less toxic than to an unadapted organism. Figure 6. Toxicity of silver nitrate on suspensions of silver resistant Pseudemonas strain 3 over a one hour exposure period. 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 P e ce n t liv e b ac te ri a Time (minutes) Control 0.2 ppm AgNO3 2 ppm Ag NO3 20 ppm Ag NO3 200 ppm Ag NO3 1000 ppm AgNO3 Figure 7. Toxicity of 10 nanometer silver on suspensions of silver resistant Pseudemonas strain 3 over a one hour exposure period. Attempted sequencing of plasmid DNA A reasonably large number of metal resistance genes are plasmid mediated. Because of this it was attempted to extract, clone and sequence plasmid DNA from a large culture of the silver resistant Pseudomonas. A total of 5.4g of DNA was extracted and the sheared DNA had a size of approximately 1.5 kbp according to agarose gel electrophoresis. The clones from the Invitrogen TOPO cloning kit grew particularly slowly. Sequencing failed, however PCR using M13 primers revealed that the vector had inserts between about 1.5 and 8 kpb. It was eventually determined that low copy numbers of targets. In the future sequencing may be attempted on this clone library by first amplifying the insert by PCR and then direct sequencing. PCR amplification of silver resistance genes and qPCR of silver resistance in the environment Positive amplification of SilA, SilE, Sil B and SilF were obtained using template DNA from the silver resistant Pseudomonas isolate, however multiple products were obtained for the SilE and SilF 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 P e rc e n t liv e b ac te ri a Time (minutes) Control 0.2 ppm Ag NPs 2 ppm Ag NPs 20 ppm Ag NPs 200 ppm Ag NPs 1000 ppm Ag NPs genes, possibly because of a low annealing temperature, as Primer BLAST did not indicate additional PCR products from known genome sequences. Because of the strong amplification, SilA and SilE were used in quantitative PCR. Quantitative PCR results showed DNA extracted from Eel Pond sediment and Eel pond sediment exposed to silver nanoparticles had larger copy numbers of SilA and SilE genes than the total DNA of the silver resistant isolate. This larger than expected result could indicate that the quality of the standard DNA is not satisfactory, or that the primers are not targeting the desired genes. Studies of silver resistant isolates from medical environments indicate that the sil genes are only present in 5-10% of isolates (Fabrega, 2009). This number could increase in sediment, but it still appears that the assay must be optimized prior to quantification of genes from the environment. Figure 8. Amplification of silver resistant genes from silver resistant Pseudomonas strain 3. SilCBA, and SilP primers gave negative results. In conclusion, a silver resistant Pseudomonas strain was isolated, and its silver resistance was characterized. The strain was able to grow in the presence of 20ppm silver nitrate, and 1000 ppm silver nanoparticles. The lack of a toxic effect at these high nanoparticle concentrations implies that there may not be specific nanoparticle toxicity either because of nanoparticle coating, or because of the ability of the bacteria to survive in the presence of colloidal silver. Silver resistance genes SilA, SilB, SilE and SilF were amplified from the Pseudomonas sp. Optimizing the PCR and quantitative PCR assay would be interesting as there has been little work on detecting silver resistance genes in the environment which could give an idea of how affected an area has been from a release of silver or silver nanoparticles. Chemotaxis towards nanomaterials Nanoparticles often have interesting characteristics, including toxicity, that may attract or deter motile bacteria from encountering them. Nanomaterials could potentially act as chemoattractants by creating favorable environments for hydrogenotrophic microorganisms, or could be chemorepellents because of the release of toxic ions. One interesting nanomaterial that could potentially act as a chemoattractant is nanoscale zero-valent iron. These 50-100 nm particles have a zero-valent iron core and a magnetite shell. When in water, they react rapidly to not only reduce the environment, but also to produce hydrogen. This could attract sulfate reducers or methanogens in anaerobic environments, or other organisms that like hydrogen in aerobic environments. A possible chemorepellent would be silver nanoparticles, as silver ion is released from these. In this project, RNIP was studied as a possible chemoattractant, using natural samples in anoxic conditions. Silver nanoparticles were studied as a possible chemorepellent using pure cultures of Vibrio gazogenes and Pseudomonas putida. Materials and Methods Sampling site Water and sediment samples were obtained for chemotaxis experiments from Cedar Swamp. Nalgene sampling bottles were capped underwater to minimize exposure to oxygen. Nanoparticles Reactive nano-iron particles (RNIP 10 DS) were obtained from Toda Kogoya company (Japan) and stored in an inert atmosphere. Silver nanoparticles (10 and 50 nm, coated with polyvinylpyrrolidone) (NanoAmor) were dispersed at a concentration of 10 mg/ml in sterile DI water using a 1/8 inch ultrasonic probe (Heat Systems Ultrasonics, now Misonix), at power 3 for 10 minutes on ice. Particle suspensions were then diluted appropriately in filter sterilized swamp water, or sea water base, and under Nitrogen in the case of RNIP Chemotaxis apparatus Chemotaxis experiments were performed in sterilized modified Meplats bottles (see figure x) according to the procedures of Jörg Overmann{Overmann, 2005 #1}. Bottles were filled with either water from Cedar Swamp or a 1:10 dilution of an overnight culture in the case of V. gazogenes and the silver resistant Pseudomonas sp. Eight capillaries of each test substance were filled with nanoparticle suspensions. RNIP and Cedar Swamp Capillaries were loaded with three different concentrations of RNIP. In addition, 8 capillaries were filled with filter sterilized Cedar Swamp water as a control. The chemotaxis bottle was filled under a steady stream of nitrogen, and was incubated overnight in an anaerobic jar with a nitrogen atmosphere. After the incubation the capillaries were pooled into microcentrifuge tubes. Volumes of 0.5 ml of the pooled samples were filtered onto a 0.2 micron Isopore filter. The filters were allowed to dry, and were stained with 1 g/ml DAPI. A total of 8 fields were counted on each filter under 100x magnification. Figure 9. Apparatus for the in-situ measurement of chemotaxis to nanomaterials. Silver and pure culture chemotaxis experiments Vibrio gazogenes was obtained from Alex Petroff who isolated the strain from Stoney Beach. A silver resistant Pseudomonas pudita was isolated from the Cape Cod Bay and the details of isolation and characterization are above in the first project. Silver nanoparticles (50nm diameter) were added to capillaries at concentrations of 0.005, 0.05, and 0.5 mg/ml. Eight capillaries of each suspension were incubated for 30 minutes to 1 hour after which they were emptied into microcentrifuge tubes and quantified by a bulk fluorescence assay described below. Quantification of Chemotaxis Calibration curves were constructed by diluting the 1:10 overnight dilution to five different concentrations. Two hundred microliters of sample or standard (in triplicate) were added to wells on a costar black 96 well plate. Each sample was fixed with a final concentration of 1% paraformaldehyde. Samples were incubated at 37C for 15 minutes. After fixing the cells, 1 microliter of 1000x SYBR green was added to each well. The plate was incubated for another 15 minutes at 37C. After the incubation the fluorescence was measured with an excitation of 494nm and an emission of 521nm. Calibration curves were linear as shown in Figure 9. Figure 10. Calibration curve for the SYBR green quantification of Vibrio gazogenes. Results RNIP and Natural Samples Cedar Swamp was chosen as an appropriate natural sample for the RNIP chemotaxis experiments. The swamp sediment is anaerobic, and the water has a pH of approximately 4.5. This pH would likely give the RNIP a half life of less than two weeks, and would result in the largest hydrogen y = 1634.5x + 15.033 R2 = 1 0 100 200 300 400 500 600 700 800 900 0.00 0.10 0.20 0.30 0.40 0.50 0.60 R F U