Silver Nanoparticles as Antibacterial Action against Pseudomonas Fluorescens Isolated from Burn Infection.
Haider Qassim Raheem 1 Yasser H.Almawla2
1,2DNA Research Center,University of Babylon, Hilla, Iraq.
e-mail: [email protected] [email protected] Abstract:
Ten specimens were collected from burn patients who visited (Al-Hilla Teaching Hospital) in Babylon Province.The models remained mostly grownup on (nuterint ,MacConkey) agar dishes for separation then incubated at 37 °C for (24to48 hrs).
Bacterial isolates obtained as pure from clinical samples were identified using morphological properties and the Vitek2 system.
This work examines the (anti-bacterial) activity of silver nanoparticles (AgNps) against) (Pseudomonas fluorescens). Antimicrobial activity of Ag Nps was tested using dilutions of (600, 300, 150,75,37,5 and 18.5) and the (MIC and MBC) of each isolate is determined AgNps) shows a wide spectrum (anti-bacterial activity) against tested bacteria with an increase in inhibition zone of right proportion to the increase in (nanoparticle concentration).The (MIC of AgNps) ranged from 150μg/ml to 300μg/ml and the MBC ranged from (300)μg/ml to (600)μg/ml.
Key Words:
Silver Nanoparticles,CLSI, Pseudomonas fluorescens,Antibacterial activity.
Introduction
(Pseudomonas fluorescens exist (g-ve aerobic bacilli) by cells ranging in dimension since 2-3μm,frequently occurring in uninhabited areas of discarded-water then burning. Happen now polluted environments by establishment, then there are nope marks of infection[1]. P.fluorescens sort composite remain (G-ve) bacilli motile that stay mainly aerobic incapable to confusion of glucose (chemoorganotrophic) besides mature at pH among (4–8) similar to the majority of the memberships genus of Pseudomonas, P.fluorescens composite strains that mature the largest in a amusing peptide comprising media by a(0.1-1.0 % wt/vol) vitality basis models of these elementary media contain (nutrient/ tryptic soya broth,agar)) [2].Selective media that lack iron permits the discovery of normal fluorescence formed thru bacteria that stands enriched by better making of fluorescent media King's A/B Pseudosel media and Pseudomonas F agar media are wholly dye patterns that improve medium [3].These (media) also have other compounds, e.g. potassium, magnesium and cetrimide0, which are advanced to allow the careful growing of P. fluorescens.
Cetrimide: has a specific benefit hip preventing growing of non-Pseudomonas flora besides permits the proper pigmentation of Pseudomonas aeruginosa [4,5].Single of the complications hip separation of specific the Pseudomonas that they portion
various of the similar phenotype characters besides mature below the similar gardening circumstances.(Though) it is probable toward usage dye making which varies thru types cluster to obviously isolates differentiate since diverse sets. The (green)/blue dye (pyocyanin) freely made through P.aeruginosa is not usually formed through the species complex of P.fluorescens [5].Various types of metal and metal oxide nanoparticles (NPs) displaying antimicrobial activity have been synthesized over modern advances in nanotechnology.Metal nanoparticles containing oxides (magnesium,silver,copper, zinc iron and nickel) have been reported to have antimicrobial properties [6,7].NPs refer to spherical particles with a diameter of 1–
100 nm[8].They have a high surface-to-volume ratio as opposed to particles of similar material, but not nanoscale.Therefore,NPs are more responsive[9].The union of nanotechnology and biology is used to solve a number of biomedical difficulties and can be effectively and safely practiced in the health arena as specific NPs have been extensively investigated and have been shown to be capable of doing so (antibacterial activity), [10]. .Among several metal oxide,AgNPs concerned specific care for the reason that it's the most humble participant of the silver compound intimate and displays a variety of beneficial mental chattels,for example great superconductivity for heat,electron connection,then rotation subtleties [11].Microbial pollution of air,soil then water owing to various kinds of (microorganisms) presents difficulties in alive circumstances besides is a severe healthiness concern.Owing to the supper of strains resistant to antibiotic that cause serious infections,attention has been paid to other antimicrobial mediators ,for example mild antibiotic,polymers of cationic,metallic NPs,besides peptides.There have been a lot of considerations[12].
Materials and Methods:
(Ag Nps) (40nm) was acquired from (Zhengzhou Dongyao Nano Materials Co., LTD, China).Standardized media of(Nutrient Blood,Muller hinton and MacConkey's, MacConkey'sagar )were obtained from (HIMEDIA, India).
Isolation and Characterization:
Bacterial isolates remained in patients staying at the Teaching Hospital in Hillah, Babylon Province,Iraq.Typical bacteriological procedures for growing blood and MacConkey's agar plates were performed for 24-48 hours at 37oC for isolation and purification. Total isolates and antibiotic profile tests were confirmed by the Viteck 2 compact system (Biomérieux) [13].
Antibacterial activity of Ag Nps:
(Antimicrobial activity (AgNps) was tested against Pseudomonas fluorescence. The antibacterial action stayed passed ready as described through the (Clinical , Laboratory Standards Institute2020).Bacterial sensitivity to AgNps is tested using a disk diffusion assay.AgNps triplicates were used in dilutions (600,300,150,75,37.5 and 18.5) In sterile deionized waterThe isolates were first incubated at 4oC for 15min and then incubated at 37oC overnight.Positive test results were recorded when the
inhibition zone was detected around the disk after the incubation period and the inhibition zone diameter was measured using the digital veneer caliper[14].
Detection of Minimum inhibitory concentration and Minimum bactericidal concentration:
Bacterial isolates were incubated) at 37oC overnight,which was used to make 0.5 McFarland. Whole of 10 ml tube nutrient broth medium was prepared after each sample was inoculated (aseptic) with 1 ml of its own bacterial suspension (about 108 CFU/mL). Six dilutions of Ag Nps (200, 100, 50, 25,and 12.5μg/ml) were determined in (sterile deionized water) and negative control (without Ag Nps) was used.Tests were conducted in triplicates for each isolate.The inoculated groups were incubated at 37oC overnight.The visible turbidity in all tubes was examined after (incubation period).The lowest concentration without turbidity is the MIC for the strain tested.
Tubes with no turbidity were cultured on nutrient agar plates and incubated at 37oC overnight.Growth of bacterial colonies have been checked and the concentration that shows no growth is represented as the MBC [15,16].
Results and Discussion : Antibiotic Sensitivity Tests:
Ten isolates of P. fluorescens stayed acquired as of burn infection and recognized by straight devices then furthermore verification thru Viteck 2 compact system.
Sensitivity test results by Viteck 2 compact system showed resistant for antibioticAmpicillin,Ampicillin/Sulbactam,Cefazolin,Cefoxitin,Ceftriaxone,Cefepime ,Meropenem,Imipenem but sensitive to Gentamicin,Ceftazidime, Amikacin, Ciprofloxacin and showed intermediate to Levofloxacin as show in table (1) and figure (1).
Table (1) Antibiotic Sensitivity Tests by Viteck 2 system.
Interpretation MIC
Antimicrobial
<=32 R Ampicillin
<=32 R Ampicillin/Sulbactam
<=32 R Cefoxitin
<=32 R Cefazolin
<=4 S Ceftazidime
<=32 R Ceftriaxone
R 8
Cefepime
<=16 R Meropenem
<=8 R Imipenem
<=4 S Gentamicin
<=4 S Amikacin
<=0.25 S Ciprofloxacin
I 4
Levofloxacin
S
<=0.5 Tigecycline
Figure (1): Antibiotic Resistance Percentage for 20 P. fluorescens by Vitek 2 System.
AM,Ampicillin; A/S, Ampicillin/Sulbactam; CAZ,Ceftazidime;CARCeftriaxone;
FEP,Cefepime; CFZ ,Cefazolin; CFT,Cefotxin; MEM,Meropenem; IMP,Imipenem;;
AK,Amikacin; GN,Gantamicin;; CIP, Ciprofloxacin; LEV,Levofloxacin; TIG, Tigecycline.
Antibacterial activity of Ag Nps:
Silver nanoparticles shows powerful broad spectrum anti-bacterial activity against tested bacteria.The result showed in table (2), Ag Nps showed increase in inhibition zone diameter with the increase in nanoparticle concentration.600μg/ml concentration showed highest zone of inhibition against the test organisms.Ag Nps cause sudden decline in bacterial cell membrane integrity in addition to the release of reactive oxygen species (ROS) where superoxide species is generated and contributing in the degradation of biomolecules [15].
Table (2) Anti-bacterial activity of Ag Nps aganist P. fluorescens.
AgNPs
concentration
Inhibition zone (mm) Iso
(1)
ISO (2)
ISO (3)
ISO (4)
ISO (5)
ISO (6)
ISO (7)
ISO (8) ISO (9)
ISO (10)
600μg/ml 20 19 20 18 19.5 20 19 21 20 21
300μg/ml 17 16 15.5 16 17 18 17 18 19 18
0 10 20 30 40 50 60 70 80 90 100
AM A/S GN CFT FEP CAZ CAR CFZ AK MEM CIP LEV TIG
Resistant 100 100 0 0 100 100 100 100 0 100 0 100 0
P er centa g e
Antibiotics
Minimal Inhibitory and Minimal bactericidal concentration:
Table (3) shows that the MIC of Ag Nps ranged from 100μg/ml to 200μg/ml and the MBC ranged from 200μg/ml to 400μg/ml .
Table (3): MIC and MBC of Ag Nps for P. fluorescens .
Isolate NO MIC MBC
1 150μg/ml 300μg/ml
2 150μg/ml 300μg/ml
3 150μg/ml 300μg/ml
4 300μg/ml 600μg/ml
5 150μg/ml 600μg/ml
6 150μg/ml 300μg/ml
7 300μg/ml 300μg/ml
8 300μg/ml 600μg/ml
9 150μg/ml 300μg/ml
10 300μg/ml 600μg/ml
Conclusion:
The results of this study showed that AgNps had a significant inhibitory and antibacterial effect on P.fluorescens,which is acceptable[15].It is highly recommend ed that AgNps be used as an alternative antibacterial agent, particularly in the treatment of ectopic infections without taking the risk of developing antibiotic resistant bacterial strains.
Acknowledgements :
I am actual thankful to the Staff of DNA Research Center / University of Babylon for providing that wholly the essential aptitudes that are essential to the fruitful achievement of the present work.
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