Effect of nanoparticles in growth of test - bacteria

Confident effect of five magnetic composite nanoparticles (FeP@Ag, FeP@Pd, CoP, NiP, Fe2O3@AГ) on growth of test bacteria colonies (Acinetobacter baumannii, Еscherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) in five replicates each is considered. Reliable inhibitors of colonies of all five test bacteria were nanoparticles FeP@Ag. CoP nanoparticles are reliable inhibitors of growth of 4 test bacteria (except for test bacteria Escherichia сoli). NiP nanoparticles are reliable inhibitors of growth of 2 test bacteria: Escherichia сoli and Klebsiella pneumoniae. Bacteria Escherichia сoli were most sensitive to the effect of magnetic nanoparticles; and bacteria Pseudomonas aeruginosa and Staphylococcus aureus were most resistant to the effect of magnetic nanoparticles. The prospects of the method are in the possibility of multiple reuse of the magnetic particles with antimicrobial properties for bacterial decontamination of the studied sources of water and removal of magnetic nanoparticles from the treated liquids by electromagnet. The method can find use in water treatment facilities for household, Industrial and medical wastes.


Introduction
On 29 January 2018 World Health Organization (WHO) published data of epidemiological surveillance service on high resistance level to many antibiotic classes of the bacterial strain series [1,2]. This endangers efficiency of antibiotics [2]. Nanosize materials are considered new antimicrobial agents [3,4].
The aim of the study is to find bactericidal, bacteriostatic and stimulatory action of various magnetic composite nanoparticles deposited on agar nutrient medium in Petri dishes on growth of five test-bacteria in five replicates each.
All powder samples have been produced by chemical sedimentation method. FeP powders (d ~ 50 nm, C(P) ~ 5 %), NiP (d ~ 250 nm, C(P) ~ 5 %) and CoP powders (d ~ 300 nm, C(P) ~ 5 %) have been produced from solution of the following composition: salt of respective metal (sulfate), sodium citrate, sodium hypophosphite, sodium hydroxide. Ag powder was produced from solution of the following composition: silver nitrate, sodium hypophosphite, sodium hydroxide. Pd powder was produced from solution of the following composition: palladium chloride, sodium hypophosphite, sodium hydroxide. The powder of Fe2O3 nanoparticles coated with polysaccharide arabinogalactan (Fe2O3@AG) has been produced solution of the following composition: ferrous sulphate, arabinogalactan, sodium hydroxide.
The surface of FPA nutrient agar medium pre-dispensed in sterilized Parti dishes was added 0.1 ml of produced water suspension of the powder and anointed with sterile spreader. Suspension prepared from day-old test bacteria according to the Tarasevich optical turbidity standard by 10 units which was dispensed by Pasteur pipette into the replicator base wells (figure1). The replicator handle was used to make replicates up to 25 test bacteria simultaneously on the surface of FPA agar nutrient medium with particles under study in Petri dishes. The dishes with inoculated test bacteria were incubated in a thermostat at 37 °С. The growth of test bacteria depended on the action of nanoparticles.
Response of test-bacteria to the action of nanoparticles was evaluated after three days of their joint growth by the difference in the size of test-bacteria colonies in experiment and controls, when the bacterial colonies grown in control had pronounced pigmentation [6]. Controls were dishes with testbacteria not exposed to the action of nanoparticles. On figure 2 show sequence of applying bacterial suspensions into the replicator base wells. The data were statistically processed according to the method of G.F. Lakin [7]. Account was made of arithmetical mean of the diameter of bacterial colonies under study, standard error of the mean. The criterion of evaluation was standard value of normalized deviate (tst) for 95-99.9% -th level of significance.
The effect of magnetic composite nanoparticles on test bacteria was evaluated as positive (stimulating) or negative (inhibiting) when the size of test bacterial colonies in the experiment reliably increased or decreased as compared to control. If the size of colonies in the experiment did not differ reliably from control the action of magnetic composite nanoparticles was considered uncertain [6,7]. Figure 3 shows results of experiment of effect of FeP@Ag magnetic composite nanoparticles of coreshell type, on the growth of five test bacterial colonies (each test-bacterium in five replicates) after 18 hours of incubation in thermostat. In control on the nutrient medium without nanoparticles all colonies of test bacteria have grown up. In experiment no growth of all colonies of 5 test bacteria on the medium with FeP@Ag magnetic composite nanoparticles occurred. Table 1 presents experimental data on -bactericidal effect of FeP@Ag particles on the growth of colonies of five test bacteria in experiment and in control.  Tables 4 and 5 present data on antimicrobial effect of СоP nanoparticles and Fe2O3 nanoparticles coated with polysaccharide arabinogalactan (Fe2O3@AG) on the growth of the same 5 colonies of test bacteria, each in five replicates.   [8,9]. Besides, СоP nanoparticles have been found to reliably inhibit growth of colonies of test-bacteria Acinetobacter baumannii and Pseudomonas aeruginosa (p≥0.001); Staphylococcus aureus (p≥0.01); Klebsiella pneumoniae 0.05. Action of Escherichia соli only was uncertainly stimulating.

Results and discussion
The action of Fe2O3 nanoparticles coated with polysaccharide arabinogalactan uncertainly stimulated growth of test-bacteria Acinetobacter baumannii and Klebsiella pneumoniae and reliably stimulated growth of test-bacteria Escherichia соli, Pseudomonas aeruginosa, Staphylococcus aureus.
It should be noted that number 1 to inhibit the growth of colonies of all five test bacteria were FeP@Ag nanoparticles; number 2-СоP nanoparticlesreliable inhibitors of growth of 4 test bacteria (except for bacteria Escherichia соli); number 3 -NiP nanoparticlesreliable inhibitors of growth of 2 test bacteria: Escherichia соli and Klebsiella pneumoniae.
Escherichia соli was most responsive to the effect of nanoparticles, most resistant were Pseudomonas aeruginosa and Staphylococcus aureus.
One of the reasons that the nanoparticles are more efficient than the classical antibacterial agents is their high ratio of surface to the volumethis gives rise to new mechanical, chemical, electrical, optical, magnetic and other properties different from their volumetric properties [3].
The silver nanoparticles as other metal-containing particles are specified by unique properties associated with high ratio of their surface to the volume defining their high efficiency (8,9). Immense specific surface of the nanoparticles makes the "evaporation" processes more intensive to increase the concentration of metal ions. Further on the electrostatic interaction on the cell membrane makes the bacterial cell absorb the metal ions. Penetration of heavy metal ions inside the cell can trigger a cycle of chemical reactions in which the metal will act as a catalyst. The possible processes enumerated can trigger apoptosis processes of bacterial cell.
Effect of heavy metals on cytoplasmic membrane cause substantial changes. First of all, they are associated with impairment of their functions leading to the loss by the cells of amino acids, nucleotides< inhibition of transport processes and ultimately to the death of the bacterial cell [10].
The prospects of magnetic nanoparticles possessing antimicrobial properties is in their multiple use. Magnetic properties of nanoparticles at room temperature makes possible to easily remove the nanoparticles from the treated liquids by electromagnet after bacterial decontamination. Magentic nanoparticles with antimicrobial properties can find application at water treatment facilities of any liquid wastes (household, industrial, medical).