Impact of Different Magnetic Nanoparticles Fe3O4, Co and Ni on P.arguinosa Bacterial Growth

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Impact of Different Magnetic Nanoparticles Fe

3

O

4

,

Co and Ni on

P.arguinosa

Bacterial Growth

Sahar E. Abo-Neima

1

, Hussein A. Motaweh

2 1, 2

Physics Department, Faculty of Science, Damanhur University, Egypt.

Abstract - In this experimental study we focused on the effects of different types of magnetic nanoparticles (MNPs) impact on pathogenic bacteria. A strain of P.arguinosa(ATCC 27853) was chosen for this investigation. Heavy metals such as Fe3O4, Co and Ni exist in trace amounts as essential elements in biological systems and play important roles in biochemical reactions of living systems. We study the effects of different concentrations of magnetic nanoparticles such as Fe3O4, Co and Ni with low (30µg/ml), medium (300µg/ml) and high (3mg/ml) concentrations, the inhibition % of P.arguinosa bacteria growth as a function of different Fe3O4, Co and Ni-NPs at the highest used concentrations (3mg/ml) increased to 57%, 81.25% and 93.75% respectively. By studding the antimicrobial activity for different modes of antibiotics action which are inhibitors to cell wall, protein and DNA, the results indicated that the AMC and C antibiotics becomes more sensitive to P.arguinosa bacteria after treated the bacteria with 3mg/ml of Fe3O4-NPs as compared to control bacteria. Also the KZ, C and E antibiotics becomes more sensitive to P.arguinosa bacteria after treated the bacteria with 3mg/ml of Co-NPs, also the CFR, C, S and NOR antibiotics becomes more sensitive to P.arguinosa bacteria after treated the bacteria with 3mg/ml of Ni-NPs but becomes more resistance to the other used antibiotics. The effect of Fe3O4, Co and Ni levels on P.arguinosabiofilm formation was determined. The inhibitory effect of Fe3O4, Co and Ni-NPs onbacterial cells increased as the concentrations of MNPs increased also the number of colony forming unit (cfu) of P.arguinosa after overnight incubation at different concentrations of MNPs was decreased as the concentration increased. In our study we indicated the better utilization of MNPs for specific application and find an alternative treatment with or without the use of antibiotics. The microbial effect of MNPs was compared based on diameter of inhibition zone in diffusion tests. TEM image of treated bacteria observed that MNPs were capable to penetrate the thick cell wall with release of intracellular material and subsequent cell wall deformation and cell wall thickening. The cell wall will be damage and the content of the cell leakage. The density of cytoplasmic components in treated cells was obviously decreased compared with the control. Plasma membranes of treated cells were detached from the cell wall, leaving open spaces between the membrane and cell wall. Furthermore, DNA was condensed. From our results it was concluded that Fe3O4, Co and Ni-NPs at concentrations (3mg/ml) reduce the growth of P. aeruginosa by inhibition % 57%, 81.25% and 93.75% respectively. This types of MNPs can be used in biomedical application, Drugs which developed to diagnose and treat diseases, antimicrobial activity and toxicity for bacterial cells.

Index Term-- P.arguinosa

, nanoparticles, Heavy metals,

inhibition zone, antibiotics, inhibition percentage.

1. INTRODUCTION

In recent days, hospital injuries have become a serious problem in health, as Pseudomonas aeruginosa (P. aeruginosa) which are one of the main causes of hospital infection, as they have a high ability to resist many antibiotics, which lose their effectiveness day after day. P.aeruginosa is gram–negative pathogen bacteria which is one of the major global health concerns at present as the increase of bacterial resistance to antibiotics and difficult to cure owing to their failure to respond to most antibiotic. P. aeruginosa is a perfect model organism for the study of biofilms. These biofilms are found on both biotic and abiotic surfaces; can withstand phagocytic environments and exhibit 100-1000 fold greater resistance to antibiotics (Hoiby et al., 2010).

P. aeruginosa forms biofilms that have a minimum inhibitory concentration (MIC) for antibiotics up to 1,000-fold higher than that of planktonic bacteria. P. aeruginosa is resistant to common antimicrobial agents because of its ability to transfer genetic materials to the other bacteria (Morita et al., 2014), as there are many microbes that have the ability to produce and form biofilms, which inhibit the work of antibiotics. The biofilm is a pool of bacterial cells attached to each other, forming a living membrane,this membrane creates a narrow barrier against anti-bacterial agents. In biofilm; P. aeruginosa remains on vital and non-alkaline surfaces such as catheters and medical devices. Biofilm cells are not killed by antibiotic treatments which are effective against planktonic cells (Costerton et al., 1999). Magnetic nanoparticles (MNPs) have proven to be one of the most effective methods against bacterial infections. We need new strategies to identify and develop drugs to control bacterial infections. The interaction of MNPs with microorganisms and nanotechnology has found a special position in medical science. The production of nanoparticles has started an evolution in various medical treatments; diagnosis and drug deliveries (Lellouche & Friedman., 2012).

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Magnetic materials have different applications in medical science where magnetic particles are oxidized by magnetic transition metals such as iron, cobalt and nickel,

iron oxide like magnetite (Fe3O4) is more stable against oxidation and ferromagnetic in nature, and have attracted much interest because they belong to the class of materials having non toxicity and biological compatibility by the presence of Fe ions. The magnetic properties of the materials depend on the size of the nanoparticles and the presence of ionic species in their proportions. The average particle diameter can be obtained between 5-10nm by changing experimental conditions (Dove & kalaniya., 2012).

Fe3O4-MNP is one of the important particles that can be used in biomedical applications; many studies have shown that they are toxic to many pathogenic bacteria by weakening mitochondrial functions, nuclei and DNA. Fe3O4 have been used in biomedical areas such as magnetic resonance imaging (MRI), drug delivery and hyperthermia because of its biocompatibility and magnetic properties .Many studies have shown the size-dependent antimicrobial and anti-biofilm effects of these nanoparticles and their physico-chemical properties.

Hence, biofilm must be combated by some anti-biofilm strategies that bind to different surfaces in some situations, these anti-biofilm methods reduce catheter -associated infections but more techniques are required in vivo to improve local and lasting delivery of their antimicrobials effects

MATERIALS AND METHODS Bacterial strain and growth conditions

The used strain of P. aeruginosa in this study was ATCC (27853), colonies were grown overnight under aerobic conditions and inoculate on afresh Luria-Bertani (LB) agar plate at 37oC overnight. The overnight cultures were used to inoculate fresh LB media to an initial OD600 of 0.05 and cultures were grown at rotation 300rpm in an orbital shaker at 37oC, (UV/VIS spectrometer, AC 115/230V, 50/60Hz) were used for all spectrophotometric assays.

Preparation of magnetite Fe3O4 -NPs

In a typical procedure, 1.5g of Fe (NO3)2,179.8548 g/mol molar mass was dissolved in 50ml of PVP (Polyvinylpyrrolidone from Sigma Aldrich USA) and Re-distilled water was used throughout the experiment. The solution was added into a round bottom flask with stirring. The colour of the mixture was dark yellow. About 15ml of NaOH (1M) was rapidly added to the mixture, and a Nano powder suspension was formed. The suspension was kept at 75 °C for 1h. After cooling to room temperature, the particles were separated by centrifugation and were washed with distilled water to remove any contaminations (Ahmed et al., 2016). Co and Ni -NPs were purchased from US Research Nanomaterials, Inc with its characterization.

Characterization of MNPs

X-Ray Diffraction (XRD)

XRD experiments of the crystalline phase of a powder sample of MNPs were performed using an X-ray diffractometer (STADI-MP, STOE Company, with monochromatized Cu Kα radiation). The parameters of the measurements were: 40 kV and 30 mA, angular variation range 5 to 100° in steps of 0.05° for each 10 s (geometry θ −2θ) (khorramdoust et al., 2017). Transmission Electron Microscopy (TEM)

The morphology of the MNPs in the colloidal suspension was observed by TEM microscope at 100kV. A drop of the colloidal suspension (100μg Fe3O4/mL) was dispersed and dried on a copper grid covered with collodion and carbon prior to the experiment (khorramdoust et al., 2017).

Biofilm formation and assay

To evaluate the biofilm inhibition or biofilm dispersal capacity of substances in 96 well plate. 1st step: Grow bacteria overnight on Agar plates at 37C. 2nd step: Inoculate bacteria from overnight plate into 10 ml of LB broth in 50 ml Falcon tube and incubate overnight.3rd step: Measure the OD600 and adjust to 0.01 in culture medium.4thstep:Take 200µl of adjusted cell suspension in to wells of a 96 plate. 5th step: Incubate at 37C for 24 or 48hrs in a moist chamber (normally a plastic box with wet tissue in it).6th step: Biofilm takes one or two days to develop. For inhibition of biofilm, the treatment should be added after platting. For dispersal, treatment should be given after the static biofilm has developed.7thstep: carefully remove the culture with a multichannel pipette.8th step:Wash the wells two times with 250 µl phosphate buffer saline.9thstep:to the well wall or the bottom if observed.11thstep:dry the plate at room temperature.12thstep: Dissolve the biofilm with 200 µl 30% acetic acid (alternative can be DMSO, however note that the biofilm has to be completely removed from the wall of well for quantitative assessment).13thstep:Measure optical density at 600 nm with incubation time every hour after treated bacteria with different concentrations of MNPs.

Antibiotic susceptibility test (AST)

The antibiotic sensitivity test was carried out using Mueller-Hinton agar (Oxide) medium as described by (Ebie et

al., 2001)and in the Manual of Antimicrobial Susceptibility

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antibiotics using the agar diffusion method. The antibiotics used in this study were chosen to represent different modes of action. These discs were Amicacin [AK(30µg)], Ampicillin [AMP (10µg)], Ceftriaxone [CRO(30µg)], Cefuroxime [CXM(30µg)],Ceftazidime[CAZ(30µg)],Amoxicillin/Clavula nic acid [AMC(30µg)], Streptomycin [S(10µg)] which inhibit cell wall synthesis. Also Ciprofloxacin [CIP (5µg)], Ofloxacin [OFX (5µg)], and Norfloxacin [NOR(10µg)] which are inhibitors for bacterial DNA, in addition to Erythromycin [E(15µg)], Cefadroxil [CFR (30µg)], and Chloramphenicol [C(30µg)] which are inhibitors for proteins. After plate inoculation and incubation at 37oC for 24 h, the diameters of the inhibition or stimulation zone of exposed and unexposed cells were measured in mm. The method used to measure the sensitivity of the bacterial cells toward different antibiotics was disc method by Bauer-Kirby technique (Amin et al., 2013).Using 150 × 15 mm petri dishes plates, about 60 ml of sterile Mueller-Hinton agar was poured into each plate, to give a uniform agar layer of depth 4 mm. The plates were allowed to dry at 37 ºC for 30 min. using a sterile cotton-wool swab; the micro-organisms were equally distributed over the agar surface. As soon as possible as and not later than 15 min, the antibiotic discs were applied in order that diffusion and growth proceed simultaneously. Discs were arranged at least 15 mm from the edge of the plate and 20 mm apart of each other. Plates were incubated at 37ºC for 24 hr. The diameters of the inhibition or stimulation zones were measured in mm after 24h using ruler stick.

TEM for bacterial sample

Bacterial suspensions P. aeruginosa, were treated by Fe3O4, Co and Ni-NPs at concentration (3mg/ml). The control was prepared from the same batch of bacterial suspension at the same condition as the treated one. The plate count method confirmed no bacteria growth after NPs treatment. The control and the treated bacteria suspension were centrifuged at 2600 rpm for 5min and washed with a buffer solution 0.1 M sodium cacodylate, pH 7.2-7.4. Primary fixation of the bacteria consisted of a 1:1 mixture of a 1%solution of OsO4 containing 7.5 mg of potassium ferricyanide per ml and a buffer solution containing 5% glutaraldehyde (Hasty & Hay, 1978). Five milliliter of the primary fixation solution was added to the bacteria. They were fixed for 1hr, followed by centrifugation and washing with the buffer 2 times and 3 times with deionized water for 10 min. An ethanol graded dehydration series (25%-100%) was used to dehydrate the sample for 20 min, followed by 100% acetone for 15 min. The samples were infiltrated and embedded in Embed 812 epoxy resin (Yenugu et al. 2004).Ultra-thin sections (70 nm) were obtained by microtome and placed on 400 mesh uncoated copper grids. Post staining was done with 2% uranyl acetate and Reynold’s lead citrate. The samples were examined and photographed at an accelerating voltage of 75 kV using a Hitachi H-7000 transmission electron microscopy at the Electron Microscopy Core Laboratory.

Statistical analysis

The significance of the results was determined using the two-tailed Student’s t-test and a P-value less than 0.05 was considered significant.

Results & Discussion

Nanotechnology is a promising field for production of new types of nanomaterial with medical applications. MNPs are clusters of molecules, ions, or atoms with diameters in the range of 1–100 nm. This size range is so interesting, which gives the opportunity to the particle to penetrate the gap between small molecules and bulk materials. NPs are formed through most periodic table elements. Metallic elements form a large group of NPs. MNPs have properties that make them suitable for use in medical applications. They are considered as efficient bacterial agents. Many MNPs show anti-bacterial, anti-viral, anti-angiogenesis, and anti-cancer properties, some of which are useful in treating arthritis as well as previous research has shown

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in different areas of applications, such as catalysis (Gawande & Varma ., 2013, Sharad., 2014), magnetic storage media (Terris & Thomson., 2005), biosensors (Kavitha et al.,2013), magnetic resonance imaging (MRI) (Qiao et al., 2009 ,Haw et al., 2010), and targeted drug delivery (Salem et al., 2015– Wani et al., 2014). Iron also appears to play an important role in the biofilm matrix. This research started as an exploration of whether P. aeruginosa could obtain and use Fe3O4, Ni and Co-NPs to satisfy their requirement or if the

potential reactive oxygen species would create a toxic environment which would be toxic to bacteria.

Phase purity and crystallinity of the synthesized Fe3O4 -NPs can be identified via XRD analysis. Fig. 1 shows the XRD pattern of the synthesized MNPs, which was quite identical to Fe3O4- MNPs and indicating that the sample has a cubic crystal system. Also, we can see that no characteristic peaks of impurities were observed. Sharp peaks also suggest that the Fe3O4 nanoparticles have good crystallize structure

Fig. 1. XRD pattern of Fe3O4- NPs

TEM analysis for Fe3O4-MNPs

The size and morphology of the synthesized Fe3O4-NPs were analysed by using TEM. TEM image of synthesized Fe3O4-NPs (Fig.2) seen that all nanoparticles exhibit spherical, cubic shapes, Fe3O4-NPs is small in size and possess magnetic characteristics. Thus, the Fe3O4-MNPs were well prepared in Nano size.

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Nanoparticles requirements for growth and biofilm formation

The influence of Fe3O4, Co and Ni -NPs on P. aeruginosa growth was determined in LB media supplemented with different concentrations (0, 30µg/ml, 300µg/ml, 3mg /ml) the level of growth decreased as the iron, cobalt and nickel concentration increased respectively (Fig.3, 4 and 5).

Fig. 3. The effect of different Fe3O4-NPs concentrations on P.arguinosa bacteria.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0 10 20 30

Op

ti

cal

d

e

n

si

ty (

600

n

m

)

Incubation time (hour)

Co-NPs

control 30µg/ml 300µg/ml 3mg/ml

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Fig. 5. The effect of different Ni-NPs concentrations on P.arguinosa bacteria.

The effect of iron Fe3O4, Co and Ni levels on P.arguinosa strain (ATCC 27853) biofilm formation was determined. The inhibitory effect of MNPs on bacterial cells increased as the concentrations of NPs increased (Fig.6.). The number of colony forming unit (CFU) of P.arguinosa after overnight incubation at the presence of different concentrations of NPs was decreased as the concentration increased.

Fig . 6. The inhibitory effect of Fe3O4,Co and Ni nanoparticles on P.arguinosa bacteria.

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Fig. 7. The inhibition % of P.arguinosa bacteria as a function of different Fe3O4-NPs concentrations.

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Fig. 9. The inhibition % of P.arguinosa bacteria as a function of different Ni-NPs concentrations.

The presence of an inhibition zone clearly the antibacterial effect of NPs at high concentration (3 mgm/ml), which indicated that the AMC and C antibiotics becomes more sensitive to P.arguinosa bacteria after treated the bacteria with concentration of Fe3O4 -NPs but becomes more resistance to the other antibiotics as shown in table I. Also the KZ,C and E antibiotics becomes more sensitive to P.arguinosa bacteria after treated the bacteria with concentration (3mgm/ml) of Co-NPs but becomes more resistance to the other antibiotics as shown in table II, also the CFR,C,S and NOR antibiotics becomes more sensitive to P.arguinosa bacteria after treated the bacteria with concentration(3mgm/ml) of Ni-NPs but becomes more resistance to the other antibiotics as shown in table III. Fig.10.indicated inhibition zones for bacterial sample unexposed (a) and treated with concentration 3mgm/ml of Fe3O4-NPs (b) Co-NPs (c) and Ni-NPs (d).

Table I

Antibiotic sensitivity of P.arguinosa before and after treated with concentration 3 mgm/ml of Fe3O4-Nps (Mean inhibition zone diameter in mm)

After treated with Fe3O4-NPs Before treated with Fe3O4-NPs

Antibiotics

Cell wall inhibitors

19 25

AMP

20 24

CRO

22 13

AMC

26 40

AK

24 26

SAM

--- ---

KZ

18 22

CFR

9 14

CAZ

Proteins inhibitors

16 12

C

12 26

E

--- 30

S

DNA inhibitors

23 26

NOR

26 38

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Table II

Antibiotic sensitivity of P.arguinosa before and after treated with concentration 3 mgm/ml of Co-Nps (Mean inhibition zone diameter in mm)

After treated with Co –NPs Before treated with Co -NPs

Antibiotics

Cell wall inhibitors

14 25 AMP 15 24 CRO 20 34 AMC 19 40 AK 17 26 SAM 8 --- KZ 16 22 CFR 18 14 CAZ Proteins inhibitors 26 12 C 35 26 E 16 30 S DNA inhibitors 22 26 NOR 22 38 CIP Table III

Antibiotic sensitivity of P.arguinosa before and after treated with concentration 3 mgm/ml of Ni-Nps (Mean inhibition zone diameter in mm)

After treated with Ni -NPs Before treated with Ni -NPs

Antibiotics

Cell wall inhibitors

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Fig. 10.Inhibition zones for bacterial sample unexposed (a) and treated with concentration 3mgm/ml of Fe3O4-NPs (b) Co-NPs (c) and Ni-NPs (d).

TEM used to study the morphological shape of bacteria treated with Fe3O4, Co and Ni-NPs.

Fig. 11.indicated TEM images of P. aeruginosa (A) the normal P. aeruginosa without NPs treatment, P. aeruginosa treated with (B,C) Fe3O4-NPs (D) Co -NPs and (E,F) Ni-NPs ,the cell membrane is smooth and appears a dark area where

the sample had a high electron density this means that normal

structure of cell membrane (Fig.11.A). TEM image of treated bacteria observed that Fe3O4, Co Ni-NPs were capable to penetrate the thick cell wall. The density of cytoplasmic components in treated cells was obviously decreased compared with the control. Plasma membranes of treated cells were detached from the cell wall, leaving open spaces between the membrane and cell wall. Furthermore, DNA was condensed. Several cells with a roughness of the cell surface can be observed (Fig.11. from B to F). The increase in roughness and amorphous mass could be associated with the perforation of the cell wall with release of intracellular material and subsequent cell wall deformation and cell wall thickening. The cell wall will be damage and the content of the cell leakage.

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Fig. 11. TEM images of P. aeruginosa (A) the normal P. aeruginosa without nanoparticles treatment, P. aeruginosa treated with (B,C) Fe3O4-NPs (D) Co-NPs

and (E,F) Ni-NPs.

Conclusion

From our results it was concluded that

Fe3O4, Co and Ni- NPs at concentrations (3mg/ml) reduce the growth of P. aeruginosa by inhibition percentage 57%, 81.25% and 93.75% respectively.The presence of an inhibition zone clearly the antibacterial effect of NPs at the

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(3mgm/ml) of Ni-NPs. From TEM image of treated bacteria observed that Fe3O4, Co Ni-NPs were capable to penetrate the thick cell wall with release of intracellular material and subsequent cell wall deformation and cell wall thickening. The cell wall will be damage and the content of the cell leakage.

MNPs can be used in biomedical application, Drugs which developed to diagnose and treat diseases, antimicrobial activity and toxicity for bacterial cells. The physical investigations with XRD and TEM have been carried out in order to understand the interesting structural changes involved in the system which may find important in biomedical applications.

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Figure

Fig. 2. TEM of Fe3O4- NPs.
Fig. 2. TEM of Fe3O4- NPs. p.4
Fig. 1. XRD pattern of Fe3O4- NPs
Fig. 1. XRD pattern of Fe3O4- NPs p.4
Fig. 3. The effect of different Fe3O4-NPs concentrations on P.arguinosa bacteria.
Fig. 3. The effect of different Fe3O4-NPs concentrations on P.arguinosa bacteria. p.5
Fig. 4. The effect of different Co-NPs concentrations on P.arguinosa bacteria.
Fig. 4. The effect of different Co-NPs concentrations on P.arguinosa bacteria. p.5
Fig. 5. The effect of different Ni-NPs concentrations on P.arguinosa bacteria.
Fig. 5. The effect of different Ni-NPs concentrations on P.arguinosa bacteria. p.6
Fig . 6. The inhibitory effect of Fe3O4,Co and Ni nanoparticles on P.arguinosa bacteria

Fig .

6. The inhibitory effect of Fe3O4,Co and Ni nanoparticles on P.arguinosa bacteria p.6
Fig. 7. The inhibition % of P.arguinosa bacteria  as a function of different Fe3O4-NPs concentrations
Fig. 7. The inhibition % of P.arguinosa bacteria as a function of different Fe3O4-NPs concentrations p.7
Fig. 8. The inhibition % of P.arguinosa bacteria as a function of different Co-NPs concentrations
Fig. 8. The inhibition % of P.arguinosa bacteria as a function of different Co-NPs concentrations p.7
Table I before and after treated with concentration 3 mgm/ml of  Fe

Table I

before and after treated with concentration 3 mgm/ml of Fe p.8
Fig. 9. The inhibition % of P.arguinosa bacteria as a function of different Ni-NPs concentrations
Fig. 9. The inhibition % of P.arguinosa bacteria as a function of different Ni-NPs concentrations p.8
Fig. 10. Inhibition zones for bacterial sample unexposed (a) and treated with concentration 3mgm/ml of Fe3O4-NPs (b) Co-NPs (c) and Ni-NPs (d)
Fig. 10. Inhibition zones for bacterial sample unexposed (a) and treated with concentration 3mgm/ml of Fe3O4-NPs (b) Co-NPs (c) and Ni-NPs (d) p.10
Fig. 11. TEM images of P. aeruginosa (A) the normal P. aeruginosa without nanoparticles treatment, P
Fig. 11. TEM images of P. aeruginosa (A) the normal P. aeruginosa without nanoparticles treatment, P p.11

References

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