z

Vol. 2, Issue 5, pp 29-35, Winter 2018

ORIGINAL ARTICLE

Preparation of nanohybrid antibiotic from levofloxacin and

determination its inhibitory efficacy against Staphylococcus aureus

isolated from diabetic foot ulcer

Sara M. AL-Hussaini1 , Ali Abdul Kadhim Al-Ghanimi1 and Hesham Ibrahim Kadhim2

1 Department of Biology, College of Science, University of Kerbala, Kerbala, Iraq. 2 Babil Health Directorate, Hilla, Iraq.

ARTICLE INFORMATIONS Article History:

Submitted: 29 December 2017 Revised version received:

15 January 2018

Accepted: 20 January 2018 Published online: 1 March 2018

Key words: Nanohybrid Layered double hydroxide

Levofloxacin

S.aureus

Corresponding author: Sara M. AL-Hussaini

Email: saraalhussaini333@gmail.com Department of Biology

College of Science

University of Kerbala

Kerbala

Iraq.

ABSTRACT Objective: This study aimed to Prepare nano hybrid from levofloxacin antibiotic and evaluation of its inhibitory effect against Staphylococcus aureus from diabetic foot ulcer. Methods: A total 43 swab samples were collected from diabetic foot ulcer

patients hospitalized in AL-Hussein Teaching Hospital in Sacred Kerbala province, it has been obtained 9 isolates were Staphylococcus aureus. A nanohybrid antibiotic LEV-LDH was prepared using direct ion exchange between antibiotic levofloxacin (LEV) and layered double hydroxide (LDH). The new nano antibiotic was identified. FT-IR spectroscopy revealed the appearance of new groups in the LEV-LDH spectrum, which indicates the formation of a new compound. The X-Ray Diffraction (XRD) spectrum revealed the appearance of new diffraction planes in the nanohybrid antibiotic spectrum in compare with spectrum of layered double hydroxide. The two and three-dimensional image of the above compound in atomic force microscope confirmed the formation of new antibiotic with nano dimensions. The antimicrobial activity of the nanohybrid antibiotic was studied against 9 isolates of S. aureus. Results: The results showed that the highest inhibition was obtained

against isolate 3 with average inhibition zone of 34.642 mm. The combination between the antibiotics LEV-LDH and teicoplanin was studied against S. aureus bacteria, the results showed a synergistic inhibition effect against some isolates while others gave antagonist inhibition effect. Conclusion: The efficacy of the nanohybrid levofloxacin antibiotic in inhibition of isolated S. aureus.

Copyright©2018, Sara M. AL-Hussaini This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

INTRODUCTIONA global statistics refer to that 15% of people with

diabetes globally exposed to diabetic foot ulcer that is a

source of many diseases. Different types of

microorganisms contribute in an incidence of infections

of foot ulcer and bacteria stands in front of it by

possessing of different virulence firstly, and their

resistance to antibiotics secondly. Acute infections

almost limited to single type of Gram positive and

negative spherical bacteria, while chronic infections

characterized by presence of 3-5 types of coagulase-

negative Staphylococcus bacteria and Corynebacterium

species in colonization of soft tissue, as well as foot

ulcer may allow to the presence of some types of

anaerobic bacteria and fungi1.

Available online at http://www.sjomr.org

SCIENTIFIC JOURNAL

OF MEDICAL RESEARCH Vo. x, Issue x, pp xx - xx, Winter

2018

Citation: AL-Hussaini S.M., Al-Ghanimi A.A. and Kadhim H.I. “Preparation of nanohybrid antibiotic from levofloxacin and determination

its inhibitory efficacy against Staphylococcus aureus isolated from diabetic foot ulcer”. Sci. J. Med. Res. 2018, 2 (5): 29-35.

ISSN: 2520-5234

Sci. J. Med. Res., Vol. 2, Issue 5, pp 29- 35, Winter 2018 29 .

Several studies have indicated the dominance of S.

aureus in the diabetic foot ulcers infections, as the

surface components of these bacteria adhere to the host

and then release many of the virulence factors that

invade the immune system such as α-hemolysin and

phenol soluble modulins and Panton-valentine

Leukocidin These factors can analyze host cells as well

as secretion Several factors inhibit the function of

neutrophils such as Chemotaxis inhibitory protein of

staphylococci These bacteria also have many antigens

(which activate T cells) such as (SE: enterotoxins , SEI:

enterotoxin –like protein and TSST: toxic shock

syndrome toxin) In addition, exfoliative toxins facilitates

the invasion of bacteria to host skin2.

Levofloxacin antibiotic belong to the third generation of

fluoroquinolones and characterized by wide-spectrum

effect against bacteria. Its inhibitory effect affects both

gram-negative and gram-positive bacteria3. In recent

years, nanotechnology has tended to find solutions that

can effectively delivery the therapeutic compound to the

target site. The drugs have been loaded onto

nanocarriers. The latter are easily received by cells

compared to the larger molecules, so they have been

successfully used as delivery tools of vital active

compounds because the method of combining the drug

with a nanocarrier and the strategy of reaching the target

is very important for its use in treatment4.

MATERIALS AND METHODS

Preparation of the nanohybrid antibiotic from

layared magnesium/ aluminum double hydroxide

(Mg / Al-NO3LDH) with the levofloxacin antibiotic

by direct ion exchange method:

The nanohybrid antibiotic was prepared according to the

method described by Bashi et al 5

, with some

modifications. Briefly, 50 ml of the levofloxacin were

added to layered double-hydroxide solution (resulting

from dissolving 1 g of the layered double-hydroxide

precipitate in 50 ml of deionized water) and the

mixture was magnetically stirred at room temperature

for 2 hours. The mixture was placed in shaking

incubator at 37 °C for 18 hours followed by incubation

in an incubator at 40° C for 24 hrs. The mixture was

centrifuged at 5000 rpm for 20 minutes, washed several

times with deionized water and dried at 50°C . It should

be noted that the tested nanohybrid antibiotic is given

the Mg / Al-LEV-LDH or LEV-LDH symbol.

Characterization of the nanohybrid antibiotic: The nanohybrid antibiotic under study was characterized

by using several methods including Fourier transform

infrared spectroscopy (FT-IR); X-ray diffraction (XRD);

)Atomic Force Microscope, AFM) and precise analysis

of C, H and N elements. I. FT-IR

The infrared spectrum for each of nanohybrid

levofloxacin and levofloxacin free as well as the layered

double hydroxide (LDH) was assessed, by making disk

from the compound under study with potassium bromide

(KBr) after grinded well, and measuring the infrared

spectrum in a wave number range (400-4000)cm-1

.

II. XRD

X-ray diffraction spectrum was used to characterize the

nanohybrid levofloxacin. XRD explains the difference in

the thickness of the layer before and after the

intercalation process for levofloxacin antibiotic by using

Brack's low ( nλ = 2dSinθ).

III. Atomic Force Microscope (AFM)

In order to measure the diameters, sizes and aggregation

of the nanoparticles, The samples of the nanohybid

levofloxacin were characterized by AFM . Samples were

assessed by Dr. Abdul Karim Al Samarrai, College of

Science, University of Baghdad. IV. Precise analysis of C, H and N elements C, H and N percentages in the nanohybrid levofloxacin

and levofloxacin free were analyzed.

The inhibitory efficacy test of LEV-LDH and free

levofloxacin against S. aureus The inhibitory efficacy of nano antibiotic LEV-LDH and

free levofloxacin against S. aureus was tested according

to the Well Diffusion Assay method6. The minimum

inhibitory rate of the nano antibiotic against the bacteria

under study was also determined by using

concentrations antibiotic (0.005-3) mg / ml.

Statistical analysis

The results were analyzed statistically in order to find

out the significant differences among the rate of

inhibition factors studied against S. aureus isolates that

were isolated from diabetic foot ulcer these factors

resembled by (1) both levofloxacin antibiotic types free

and nanohybrid as well as the Teicoplanin free, (2)

bacterial isolates, (3) the concentrations of used

antibiotics and may select the significant differences at

the possibility level of 0.01 as included a statistical

analysis of experiments in two duplicates as has been

tested significant differences among averages using the

least significant difference test (LSD) and on the

possibility level of 0.017.

RESULTS AND DISCUSSION Characterization of nanohybrid levofloxacin

antibiotic 1- Characterization by using infrared spectrum FT-IR :

A- Infrared spectrum for free levofloxacin antibiotic

(LEV):

Figure 1 shows many of the unique bands of free

levofloxacin appeared. The two bands at frequencies

3416 and 3271 cm-1

indicate a stretch vibration of the

hydroxyl group (OH). The appearance of the bands at

frequencies (2929, 2848 and 2804) cm-1

, indicate to

presence of aliphatic ( C-H ) stretch. The appearance of

the band at 1724 cm-1

is due to stretch of the strong

carbonic acidic group (C=O), while the band appearance

at 1620 cm-1

indicates the stretch of ketone. It is also

evident that there is a structural stretch of benzene at the

frequencies (1527 and 1446) cm-1

. Finally, there is an

aromatic ( C-H ) flex at the frequency of 802 cm-1

8.

B- Infrared spectrum for magnesium / aluminum layered

double hydroxide:

As shown in Figure 2 the magnesium/aluminum layered

double hydroxide showed a number of specific bands at

certain frequencies. The band at 3527 cm-1

is due to the

OH bond stretch vibration, which is a transverse band

30 AL-Hussaini S.M. et.al., 2018 .

due to interference from different types of hydroxyl,

such as layered double hydroxide and physically

adsorbed water Hydroxyl9,10

. The distinctive band at

1383 cm-1

is attributed to the nitrates group (NO3)

between the layers11,12

, while the bands between 400-

600 cm-1

return to vibration of Mg-O and Al-O bond13

. C- Infrared Spectroscopy for nanohybrid levofloxacin

Mg/Al - LEV -LDH:

Figure 3 shows that the nano hybrid antibiotic shows

several new characteristic bands indicate the success of

antibiotic levofloxacin intercalation between the layered

double hydroxide. The appearance of the two bands at

frequencies (3477 and 3358) cm-1

refers to stretch of the

hydroxyl group (O-H) and had a shift towards a higher

frequency. The appearance of the band at 2897 cm-1

indicates the presence of the aliphatic (C-H) stretch. The

band appearance at 1672 cm-1

is due to stretch of the

Carbonyl Group (C=O) and has a shift to the lower

frequency. While stretch shift of the ketone bands was

observed at a lower frequency through the appearance of

a band at 1589 cm-1

. The structural stretch of benzene is

observed at 1481 cm-1

. The band at 1361 cm-1

frequency

returns to the stretch of nitrate group NO3, and the band

appearance at 765 cm-1

indicates a C-H aromatic flex8.

2- Characterization by using X-ray diffraction spectrum

(XRD( :

The X-ray diffraction spectrum of Mg/Al-LEV-LDH

and Mg/Al-NO3-LDH was studied to obtain the

differences in thickness of the layer before and after the

Levofloxacin intercalation between layered double

hydroxide by using the Brack's law.

Figure 4 shows the X-ray diffraction spectroscopy of

Mg/Al-NO3-LDH, observing the crystalline levels (003),

(006) and (009). The level (003) appears at the angle of

10.47 ° with crystalline distance of 0.84 nm. The level

(006) appears at 22.03 ° with crystalline distance is 0.41

nm, while the level (009) is at 34.44 ° with crystalline is

0.26 nm.

By observing the X-ray diffraction spectrum of the

Mg/Al-LEV-LDH in Figure 5 the intercalation process

appears to have been successful. The level (003) appears

at approximately 2° angle with a crystalline distance of

1.85 nm, while the level (006) at the angle 9.7 ° with a

crystalline distance of 0. 92 nm. The level (009) appears

to interfere with the level (006) of the layered double

hydroxide due to its proximity to the site, which cannot

be separated very clearly.

Figure 1: Infrared spectrum for magnesium/Aluminum layered

double hydroxide

Figure 2: Infrared spectrum for levofloxacin free (LEV free)

Figure 3: Infrared spectrum for nanohybrid levofloxacin antibiotic

(LEV-LDH)

Figure 4 : X-ray diffraction spectrum (XRD) for the

magnesium/Aluminum layered ( NO3-LDH Mg/Al-)double

hydroxide)

Figure 5 : X-ray diffraction spectroscopy of nano hybrid

levofloxacin Mg/Al-LEV-LDH

0

100

200

300

400

500

10 20 30 40 50

(009)0.28nm

(006)0.92nm

(003)1,85nm

2/degree

Inte

nsity

Sci. J. Med. Res., Vol. 2, Issue 5, pp 29- 35, Winter 2018 31 .

3- Characterization by using Atomic Force Microscope

(AFM): The outer surface of the Mg/Al-LEV-LDH was studied

by using an atomic force microscope. Figure 6 a shows a

two-dimensional image of the nano antibiotic showing

molecular aggregation of spherical forms. Figure 6 b

shows a three-dimensional image of a section of the

nanohybrid antibiotic surface, where the elevation of

molecular assemblies of up to 5.15 nanometers indicates

the manufacture of a nanohybrid antibiotic from free

antibiotic and layered double hydroxide.

Table 1 shows that the means of particle size of the

Mg/Al-LEV-LDH is 94.83 nanometer. The preparation

process of this hybrid antibiotic resulted in 56.67%

nanoparticles of the total number of particles with a

diameters of (60-100) nanometer. The highest

percentage of these nanoparticles was 11.11% for 85 nm

and lowest was 1.11% for 60 nm, respectively.

The results of the current study are in agreement with

those found by Beherei et al 14

found. The results of

antibacterial nanoparticle were obtained at the rate of

diameters ranging between 95.5- 196 nanometer, while

the results of the current study are not in agreement with

AL-Hamdani15

and Al-Jubouri16

, where the means of

particles of Oxytetracyclin and Vancomycin loaded

between layered double hydroxide averaged 76.16 and

85.81 nm, respectively.

a

b Figure 6 : (a) Two-dimensional, (b) three-dimensional, image of the

nano hybrid antibiotic Mg\Al-LEV-LDH

Table1: Diameters, sizes, and assemblies of the nanocompound molecules Mg / Al-LEV-LDH after testing with a force microscope.

Diameter

(nm)<

Volume

(%)

Cumulation

(%)

Diameter

(nm)<

Volume

(%)

Cumulation

(%)

Diameter

(nm)<

Volume

(%)

Cumulation

(%)

60.00 1.11 1.11 85.00 11.11 31.11 110.00 12.22 77.78

65.00 5.56 6.67 90.00 10.00 41.11 115.00 8.89 86.67

70.00 2.22 8.89 95.00 7.78 48.89 120.00 6.67 93.33

75.00 7.78 16.67 100.00 7.78 56.67 125.00 2.22 95.56

80.00 3.33 20.00 105.00 8.89 65.56 130.00 4.44 100.00

Precise analysis of elements in levofloxacin antibiotic

The results of precise analysis of the elements shown in

Table 2 with the nanohybrid levofloxacin antibiotic and

levofloxacin free on the percentages of carbon, hydrogen

and nitrogen were (21.152, 3.168, 4.421)% and (48.141,

5.882, 8.985)% , respectively. Through these results

show that the percentage of levofloxacin that loaded

between layered double hydroxide was 43.93 %.

Table2: Precise analysis of elements carbon, hydrogen and

nitrogen in levofloxacin antibiotic.

Sample C% H% N%

Mg/Al-LEV-LDH 21.152 3.168 4.421

LEV Free 48.141 5.882 8.985

The inhibitory efficiency for nanohybrid LEV-LDH

against bacterial isolates of S.aureus

Table 3 indicates that the bacterial isolates 3 and 12

were the most affected by the inhibitory effect of the

free LEV with the inhibition diameters of (43 and 40.5)

mm, respectively, while the bacterial isolates 2 and 3

were the most affected by the inhibitory effect of the

nanohybrid antibiotic with 36 mm inhibition diameters,

for both of them.

The results of the statistical analysis confirm these

results, as the isolates were the most sensitive to the

studied subjects, given that they had the highest

inhibitory rate of mentioned isolates (34.642 and

32.785) mm, respectively, while (25.385 and 29.5) mm,

for the nanohybrid antibiotic, respectively.

Bisht et al17

tested the inhibitory efficacy of levofloxacin

loaded on a group of polymers against S. aureus

bacteria. The results showed that the NF1 polymer-

loaded antibiotic was the most effective against the

bacteria with an inhibitor diameter 25.9 mm.

In another study by Bagga et al18

to study the inhibitory

efficacy of the nanohybrid antibiotic Levofloxacin

against S. aureus bacteria showed that the minimum

inhibitory concentration of this antibiotic was 0.128

μg/ml.

The synergistic inhibitory action of nanohybrid

antibiotic against S. aureus

The synergistic inhibitory activity of nanohybrid

antibiotic prepared in this study was tested against S.

32 AL-Hussaini S.M. et.al., 2018 .

aureus isolated from diabetic foot ulcer in the sacred

Kerbala province.

Antimicrobial combination therapy is usually used to

reduce treatment time and prevent the appearance of

microorganisms resistance to drugs as well as to expand

the drugs therapeutic spectrum19, 20, 21, 22, 23

.

Table1: The inhibitory efficacy of nanohybrid antibiotic LEV-LDH and free LEV against S. aureus isolated from diabetic foot ulcer.

The synergistic inhibitory activity between the nano

hybrid antibiotic LEV-LDH and free antibiotic

Teicoplanin against S. aureus isolates was studied. The

results in Table 4 has already shown a synergistic effect

against some isolates while actually showing antagonist

effect against others. The synergistic effect was against

isolates 3, 12, 14c, 20a, 20b, 20c, 21a and 21b,

respectively, with inhibition diameters of (37.5, 28,

23.25, 24. 5, 31, 29, 22.75 and 23) mm, respectively by

using 3 mg/ml concentration. While the antagonism

effect was against the bacterial isolate 2 with an

inhibition diameter of 27.5 mm by using the same

concentration of the antibiotics.

The results of the statistical analysis are identical to the

results above, as the isolates referred to it above are the

most sensitive to the combination of studied antibiotics.

In a study by Xu et al 24

to identify the synergistic effect

of nano-loaded antibiotics on silver against S. aureus

bacteria, the nano antibiotic LEV had the highest

An

tibio

tic

Bacte

rial

isola

te

Antibiotic concentration(mg/ml)

LSD0.01 for

antibiotic con. 3 1 0.5 0.25 0.1 0.01 0.005

Diameter of inhibition in mm

LE

V-F

ree

2 37 35 34 31.25 26.5 17 15

0.205

3 43 40.25 38.5 37.25 35 26 22.5

12 40.5 38 37 34.5 33 26 20.5

14c 29.5 22.5 17 0 0 0 0

20a 25 20.5 15.25 10 0 0 0

20b 28 26.75 25 22.5 17.5 0 0

20c 27.5 25 21 17.5 16 0 0

21a 27.5 22.5 17 12.25 0 0 0

21b 29 26.75 24 21.5 20 19.25 0

LE

V-L

DH

2 36 35 29 26.5 24 14.5 12.5

3 36 34.5 34 31.5 30 21 19

12 20 18.5 17 15.5 0 0 0

14c 14 12.25 0 0 0 0 0

20a 20 18.5 17 16.25 0 0 0

20b 21 19 17.5 15 0 0 0

20c 19.5 18 15.5 11 0 0 0

21a 13.5 12 11 0 0 0 0

21b 13 12 11.75 0 0 0 0

LSD0.01 for

Isolates

0.109

LSD0.01 For

isolates 21b 21a 20c 20b 20a 14c 12 3 2 Isolate

0.232

20.071 11.321 15.285 17.107 10.107 9.857 32.785 34.642 27.964 Rate for free

5.25

5.214 9.142 10.357 10.25 3.75 10.142 29.5 25.285 Rate for nano

Sci. J. Med. Res., Vol. 2, Issue 5, pp 29- 35, Winter 2018 33 .

inhibitory effect of the minimum inhibitory

concentration (8-16) μg/ml.

The mechanism of action of the antibiotic Teicoplanin is

shown by inhibiting the synthesis of peptidoglycan in

the bacterial cell wall via the non-specialized link and

the saturation of the outer layers of the bacterial

peptidoglycan. Teicoplanin is bound to the end-D-Ala-

D-Ala terminals of the peptidoglycan precursors via the

cleft found in the Teicoplanin molecule25

.

The inhibitory effect of drug combination can be greater

or smaller than expected from their individual effects,

according to synergistic or antagonistic interactions

between drugs, respectively26

.

Table4: The synergistic inhibitory activity of the antibiotic LEV-LDH against S. aureus isolated from the diabetic foot ulcer.

Conclusions The efficacy of the nanohybrid levofloxacin antibiotic in

inhibition of isolated S. aureus. The combination

between nanohybrid levofloxacin and free teicoplanin

antibiotics has shown a synergistic inhibition effect

against some isolates, while showing antagonistic

inhibition effect against others.

REFERENCES 1. Lipsky B.A., Berendt A.R., Deery H.G., Embil J.M., Joseph W.S.,

Karchmer A.W., Lefrock J.L., Lew D.P., Marder J.T., Norden

C.and Tan J.S. "Diagnosis and treatment of diabetic foot

infection". Clinical Infectious Diseases. 2006; 117(7): 212S-238S.

DOI:10.1097/01.prs.0000222737.09322.77.

2. Dunyach-Remy C., Essebe N.C., Sotto A. and Lavigne J.P.

"Staphylococcus aureus Toxins and Diabetic Foot Ulcers: Role in

Pathogenesis and Interest in Diagnosis". Toxins (Basel). 2016;

8(7): 1-20. DOI:10.3390/toxins8070209.

3. Sarisaltik D. and Teksin Z.S. "Bioavailability File: Levofloxacin".

FABAD J. Pharm. Sci. 2007; (32) : 197-208.

4. Suri S.S., Fenniri H. and Singh B. "Nanotechnology – based drug

delivery systems". J. Occup. Med. Toxicol. 2007; 2 :16.

doi:10.1186/1745-6673-2-16.

5. Bashi A.M., Haddawi S.M. and Mezaal, M.A. "Layered Double

Hydroxide Nanohybrid Intercalation with Folic Acid Used as

Delivery System their Controlled Release Properties". Arab J Sci

Eng . 2013; 38(7): 1663-1680.

6. Egorove N.S. "Antibiotics ascientific approach". Mir Publishers,

Moscow. 1985.

7. AL-Imam M.M. "Design and analysis of experiments". Dar AL

Marikh Publishing . Kingdom of Saudi Arabia . 2007 .

8. Silverstein R.M., Webster F.X. and Kiemle D.J. "Spectrometric

Identification of organic compounds". 7th Ed. John Wiley and

Sons . INC. Printed in the United States of America. 2005.

9. Cheng D.R., Zhu S.Y., Yin Z.H., Ding W.W., Mu Z.X., Su Z.R.

and Sun H.C. "Prevalence of bacterial infection responsible for

bovine mastitis". African Journal of Microbiology Research. 2010;

4(11) : 1110-1116.

10. Parida K.M., Sahoo M. and Singha S. "Synthesis and

characterization of a Fe (III)- Schiff base complex in a Zn-Al LDH

host for cyclohexane oxidation". Journal of Molecular Catalysis A

: Chemical . 2010; 329 : 7-12 .

11. Arizaga G.G., Gardolinski J.E., Schreiner W.H. and Wypych F.

"Intercalation of an oxalatooxoniobate complex into layered

double hydroxide and layered zinc hydroxide nitrate". Journal of

Colloid and Interface Science. 2009; 330(2): 352–358.

DOI:10.1016/j.jcis.2008.10.025 .

12. Chai H., Xu X., Lin Y., Evans D.G. and Li D. "Synthesis and UV

absorption properties of 2,3-dihydroxynaphthalene-6-sulfonate

anion-intercalated Zn–Al layered double hydroxides". Polymer

Degradation and Stability . 2009; 94(4):744–749.

doi.org/10.1016/j.polymdegradstab.2008.09.007 .

13. Feng Y., Li D., Wang Y., Evans D.G. and Duan X. "Synthesis and

characterization of a UV absorbent-intercalated Zn–Al layered

double hydroxide". Polymer Degradation and Stability. 2006;

91(4):789–794. doi.org/10.1016/j.polymdegradstab.2005.06.006 .

14. Beherei H.H., El-Magharby A. and Abdel-Aal M.S. "Preparation

and characterization of novel antibacterial nano-ceramic-

Antibiotic LEV – LDH/ Teico Free LSD0.01

For antibiotic

con.

Con.Antibiotic

(mg/ml) 3 1 0.5 0.25 0.1 0.01 0.005

Bacterial isolate Diameter of inhibition in mm

2 27.5 25 22 20 18 0 0

0.515

3 37.5 35.5 32.5 30 27 12.25 0

12 28 26.5 21.5 13.25 0 0 0

14c 23.25 21 19.5 17 14 0 0

20a 24.5 23 19 16.75 14.5 0 0

20b 31 27.5 25.5 24 21 15 0

20c 29 25.5 24 23.5 20.5 16.5 0

21a 22.75 19.5 18 16 14 0 0

21b 23 20 17.5 15.25 13.5 0 0

LSD0.01

For isolates 0.616

34 AL-Hussaini S.M. et.al., 2018 .

composites for bone grafting". Der Pharma Chemica. 2011;

3(6):10-27.

15. AL-Hamdani M.S. "Inhibitory efficiency determination of Nano

prepared Oxytetracyclin and Tylosin antibiotics against bacterial

cows mastitis in sacred Karbala province". MSc. Thesis. 2015.

16. Al-Jubory N.A. "Evaluation of inhibitory efficiency of some free

and nano antibiotics against Staphylococcus aureus locally

isolated". MSc. Thesis. 2016.

17. Bisht N., Goswami L. and Kothiyal P. "Preparation and

evaluation of in-situ oral topical gel of levofloxacin by using

combination of polymers". Indian Journal of Drugs. 2014; 2(4):

142-151.

18. Bagga P., Ansari T.M., Siddiqui H.H., Syed A., Bahkali A.H.,

Rahman M.A. and Khan M.S. "Bromelain capped gold

nanoparticles as the novel drug delivery carriers to aggrandize

effect of the antibiotic levofloxacin". EXCLI Journal. 2016;

(15):772-780. DOI:10.17179/excli2016-710 .

19. Dancey J.E. and Chen H.X. "Strategies for optimizing

combinations of molecularly targeted anticancer agents". Nat Rev

Drug Discovery. 2006; 5(8): 649 – 659. DOI:10.1038/nrd2089 .

20. Hopkins A.L. "Network pharmacology: the next paradigm in drug

discovery". Nat. Chem. Biol. 2008; 4(11): 682 – 690.

DOI:10.1038/nchembio.118 .

21. Fischbach M.A. and Walsh C.T. "Antibiotics for emerging

pathogens". Science. 2009; 325(5944): 1089 – 1093.

DOI:10.1126/science.1176667 .

22. Bush K., Courvalin P., Dantas, G., Davies J., Eisenstein B.,

Huovinen P., Jacoby G.A., Kishony R., Kreiswirth B.N., Kutter

E., Lerner S.A., Levy S., Lewis K., Lomovskaya O., Miller J.H.,

Mobashery S., Piddock L.J., Projan S., Thomas C.M. and

Tomasz A. "Tackling antibiotic resistance". Nat. Rev. Microbiol.

2011; 9(12): 894-896. DOI:10.1038/nrmicro2693 .

23. Worthington R.J. and Melander C. "Combination approaches to

combat multidrug-resistant bacteria". Trends Biotechnol. 2013;

31(3): 177 – 184. DOI:10.1016/j.tibtech.2012.12.006 .

24. Xu N., Cheng H., Xu J., Li F., Gao B., Li Z., Gao C., Huo K., Fu

J. and Xiong W. "Silver-loaded nanotubular structures enhanced

bactericidal efficiency of antibiotics with synergistic effect in vitro

and in vivo". International Journal of Nanomedicine. 2017; 12

(1): 731-743. DOI:10.2147/IJN.S123648 .

25. Reynolds P.E. "Structure, biochemistry and mechanism of action

of glycopeptide antibiotics". Eur J Clin Microbiol Infect Dis. 1989;

8(11):943-50.

26. Keith C.T., Borisy A.A. and Stockwell B.R. "Multicomponent

therapeutics for networked systems". Nat. Rev. Drug Discov.

2005; 4(1): 71–78. DOI:10.1038/nrd1609 .

Sci. J. Med. Res., Vol. 2, Issue 5, pp 29- 35, Winter 2018 35 .