Inhibition of Biofilm formation of Imipenem-resistant Acinetobacter baumannii using Curcuma longa extracts, silver nanoparticles and Azithromycin

 

Maysam Helan Kareem*, Dr. Abbas Yaseen Hasan

Department of Biology, College of Science, University of Diyala, Iraq

 

ABSTRACT:

Background: Acinetobacter baumannii is opportunistic pathogenic bacteria, as  gram-negative aerobic bacteria short rod-shaped (coccobacillus) widely distributed in the water and soil of natural environments which has emerged in recent decades as a global cause of nosocomial infections with high morbidity and mortality.  Material and methods: In current study, were collected different clinical specimens (150) individuals (males and females) at different ages including wounds, burns, blood, and urine. The study was conducted in Baquba Teaching Hospital in Diyala Governorate. Depending on bacteriological characteristics and biochemical tests, in addition to using the API -20E and VITEK-2 Compact kits,14 (9.3%) isolates of Acinetobacter baumannii were identified. Results: Of infection rates with Acinetobacter baumannii showed no significant difference between male (54.7%) and female patients (45.3%). The isolates were tested for sensitivity to (16) antibiotics using the Kirby-Bauer method, which showed that all isolates were (100%) resistant to Cefotaxime, Augmentin, Ceftriaxone, Cefepime, Ceftazidime, and Piperacillin. The isolates also showed a high resistance rate (92.9%) to Tetracycline and Ciprofloxacin and (85.7%) to Gentamicin and Co-Trimoxazole, while isolates showed a low resistance rate (42.8%) to Imipenem and a high sensitivity rate (85.7%) to Colistin, also our study showed that the (cold and hot) aqueous extracts and the alcoholic extract of Curcuma longa roots had a clear effect on the imipenem-resistant Acinetobacter baumannii at (12.5, 25, 50, 100 and 200)mg/ml concentrations, with inhibition diameters of (0.9, 14, 17 and 22) mm, (0, 8, 12, 15 and 19) mm, (0.9, 12, 13 and 15)mm respectively.  The results of determination of MIC and Sub MIC values regarding the inhibitory effect of  some  substances on bacterial biofilms showed that MIC value for cold aqueous extract of curcuma roots was 6.25mg/ml and the Sub MIC value was 3.13mg/ml, while using the hot aqueous and alcoholic extracts of curcuma roots showed that MIC value for each of them was 12.5mg/ml and that Sub MIC value for each of them was 6.25mg/ml, as well as results also revealed MIC values for silver nanoparticles (AgNPs) against bacterial isolates was 20000μg/ml,  and the Sub MIC value was 10,000μg/ml, whereas the MIC value for Azithromycin against bacteria was 512μg/ ml, and the Sub MIC value was 256μg/ml. Results of ELISA demonstrated that the Sub MIC of curcuma aqueous and alcoholic extracts and Azithromycin had no effect on biofilm inhibition of A. baumannii bacteria, while using the 20 nm silver nanoparticles (AgNPs) showed biofilm inhibition.

 

 

 

INTRODUCTION:

Acinetobacter baumannii is a gram negative opportunistic pathogenic bacteria, which has emerged in recent decades as a global cause of nosocomial infections with high morbidity and mortality (1). These bacteria cause infections affecting immunocompromised patients, and highly vulnerable patients are those patients in the intensive care units. These bacteria have the ability to survive in dry environments for a long time and can spread through water, air and on the skin of infected patients as well as on the hands of hospital staff (2). These bacteria cause many infections including bacteremia, meningitis, wound infections, pneumonia and urinary tract infections (3).

 

This bacteria is the second pathogen that has been isolated from the hospital environment after Pseudomonas aeruginosa (4). A. baumannii bacteria can grow at (44°C) which distinguishes it from other species of the same genera (5). The interest has recently increased in the species A. baumannii due to the US army infections, and according to reports that these bacteria caused many infections among members of the US army who served in Iraq and Afghanistan (6).

 

These bacteria are characterized by their multiple antibiotic resistance as they have the ability to develop multiple mechanisms against major antibiotic groups such as Cephalosporin, Aminoglycoside, Carbapenem and Quinolone (7). Carbapenem is the most effective treatment against infections caused by these bacteria. However, in the last years, the resistance to this antibiotic has increased, and this may be due to the production of Carbapenemase enzymes such as Oxacillinase, as well as the change of target sites represented by Penicillin Binding Proteins, Efflux pump, as well as the low permeability of the outer membrane due to reduced expression of the outer membrane proteins (8).

 

The clinical isolates of this bacteria show defense mechanisms, including the ability to produce biofilm. The biofilm is defined as a group of bacterial cells attached to both living and non-living surfaces. The biofilm is built up in three-dimensional structures where the cells are interconnected and encapsulated in an extracellular polymeric substrate (EPS) matrix, including exopolysaccharides, nucleic acids, proteins and macromolecules (9). The biofilm formation consists of four stages: the adhesion of bacteria to the surface, the formation of microcolonies, the maturity stage and the detachment stage (10). The process of the formation of the bacterial biofilm makes it capable to tolerate several factors including nutrient deficiency, pH reduction, as well as providing necessary protection to bacterial aggregations against the host defenses, thus prolonging the bacterial infection period to the host (11).

 

Recent studies have indicated the effectiveness of medicinal plants and their secondary metabolites as effective antimicrobial agents, and this interest was due to repeated and random use of antibiotics, which led to the emergence of high resistance to synthetic antibiotics by bacteria, especially when taken for prolonged periods of time and due to their side effects, which encouraged researchers and many people to use the natural extracts as therapeutic substances (12).

 

Among the plants used in the study was Curcuma longa, also known as Turmeric, which is anevergreen plant widely distributed in Asian countries. It belongs to the Zingi beraceae family. Its name Curcuma has been derived from the Arabic word (Curcum), and used since ancient times as a kind of spices, then it has been introduced into medicinal uses after discovering its therapeutic efficacy (13).

 

The nanoparticles have an antimicrobial effect against various microbes such as bacteria and fungi, especially silver nanoparticles, which have a broad spectrum anti-bacterial effect and have anti-fungal and anti-viral effects. Silver ions (Ag+) are released and interfere with the biofilm components, causing damage to the cell membrane, nucleic acid and cellular proteins (14).

 

Azithromycin is an antibiotic of the Macrolide group that is used to treat many bacterial infections such as respiratory tract infections and sexually transmitted diseases such as gonorrhea and early syphilis (15).

 

MATERIALS AND METHODS:

Isolation and diagnosis of bacterial isolates:

A total of (150) clinical specimens (sputum, wounds, burns, urine and blood) were collected from both sexes and different ages of patients who visited BaqubaTeaching Hospital from the beginning of September 2017 to the end of January 2018. The swabs were directly cultured on Blood agar and MacConkey agar and incubated at 37°C for 24 hours. The isolates were identified depending on bacteriological and biochemical characteristics (16).                                                                                                                   

 

Antibiotic susceptibility test:

The susceptibility test of bacteria to antibiotics was done depending on the Kirby-Bauer Method on the Muller-Hinton agar, where a bacterial suspension from the isolates to be tested was prepared by transferring a single 24-hour old colony grown on Mac Conkey solid medium to 5 ml of normal saline, then, the turbidity of the suspension was compared with a standard turbidity solution that gives approximately (1.5 × 10⁸) cells/ml. An amount of (0.1) ml of bacterial suspension was spread with a sterile cotton swab to the surface of solid Muller-Hinton medium and then left to dry at room temperature for 5 minutes. The antibiotic discs listed in table (1) were transferred by means of a sterile forceps to the dishes by (5-6) discs per a dish. The dishes were incubated at 37°C for (18-24) hours. Results were read by observing the inhibition are as formed around the antibiotic discs and the bacteria were considered as sensitive or resistant according to the CLSI          standards (17).

Table (1) Antibiotics used in the study

 

Preparation of hot aqueous extract:

The Kawasaki method (18) was followed, where (50)g of dried powder was dissolved in 500ml of distilled water after cooling to 60°C and putting it in a 1000ml flask and then placing it on the magnetic sterile hot plate for 2 hours. After that, the mixture was filtered by using a medical gauze, then by Whatman No.1 filter paper. The filtrate was centrifuged at 3000 RPM for 10 minutes. The filtrate was then evaporated by the rotary evaporator under low pressure and temperature ranging from 40-50°C. The remainder was dried by using large surface area dishes in the oven at 40°C until it was completely evaporated and thus a dry powder was obtained from the aqueous extract, then it was placed in tightly-closed sterile glass tubes, and after labeling them, they were kept in the refrigerator at 4°C until using them in chemical tests.

 

Preparation of cold aqueous extract:

The method of Rangasamy and Krishnaveni (19) was used to prepare cold aqueous extracts by placing 500ml distilled water in a clean glass flask and adding 50 g of the plant powder. Then the solution was left in the shaking incubator for 24 hours at 37°C. The solution was filtered by using the Whatman No.1filter paper, and the filtrate was centrifuged for 10 minutes at 3000 RPM, and concentrated by the rotary evaporator and then put in an electric oven at 40°C for the water to be completely evaporated, and therefore obtaining a dry powder of the cold aqueous extract, which in turn,was placed in closed sterile bottles and kept in the refrigerator at a temperature of 4°C until use.

 

Preparation of alcoholic extract:

The method of Jameela (20) was followed by weighing (50)g of the plant powder, and placing in a 1000 ml glass flask, then adding 500 ml of 70% ethyl alcohol to it, then left in the shaking incubator for 24 hours at 35°C. Then the mixture was filtered by a medical gauze and then by using the Whatman No.1 filter papers. The filtrate was centrifuged at 3000 RPM for 10 minutes. The filtrate was then evaporated by the rotary evaporator under low pressure and temperature ranging from 40-50°C. The remainder was dried by using large surface area dishes in the oven at 40°C until alcohol was completely evaporated and thus a semi-dry powder was obtained from the alcoholic extract, then it was placed in tightly-closed sterile glass tubes, and they were kept in the refrigerator at 4°C until use. The process was repeated many times to obtain sufficient amounts of the extract.

 

Determination of inhibitory effectiveness of Curcuma longa root extracts on bacterial growth:

As indicated by Obeidat et al. (21), the agar well diffusion method was used. The bacterial suspension was prepared by taking a number of bacterial colonies by means of a wire loop and placing them in tubes containing brain heart infusion broth for the purpose of bacterial activation. The tubes were incubated for 18 hours at 37°C, and the bacterial suspension was compared with the standard McFarland solution, which depends on the degree of turbidity of the bacterial suspension, close to (1.5×10⁸) cells/ml.

 

Then the bacterial suspension was spread on the plates containing the Muller-Hinton agar by sterilized swab and in several directions, and the plate was left for a while to dry. Wells measuring (5) mm diameter were made in the culture medium by using a cork borer. 100 µl of pre-prepared aqueous and alcoholic extracts (12.5, 25, 50, 100 and 200) mg/ml were added to the wells. Control wells were prepared by adding distilled water to the aqueous extracts, and buffer phosphate solution was used for the alcoholic extracts. Three replicates were done for each dish, then incubated at 37°C for 24 hours in the incubator. The effectiveness of each concentration of plant extracts was determined by measuring the diameter of the inhibition zone for each extract with a standard ruler in millimeters.

 

Determination of the Minimal Inhibitory Concentration (MIC) of Curcuma longa root extracts on the bacteria:

The plant agar dilution method was used as indicated by NCCLs (22). Two ml of each concentration of the above-mentioned plant extracts were mixed with 18 ml of the Muller-Hinton agar which is cooled to 50°C in addition to the control dish containing the culture medium, and then the dishes were inoculated with 100 µl of the bacterial suspension in the form of spot.

 

The dishes were left for half an hour to dry and incubated in the incubator at 37°C for 18-24 hours. The results were recorded on the basis of presence of growth (positive) or absence of growth (negative). The minimal concentration in which no bacterial growth appeared was considered as the minimal inhibitory concentration (MIC).

 

Determination of the Minimal Inhibitory Concentration (MIC) of silver nanoparticles against A.baumannii isolates:

The method adopted by Krishnan et al. (23) was followed. A fixed amount of bacteria (0.1) ml was cultured in sterile test tubes containing brain heart infusion broth based on the number of concentrations required to be prepared from the nanoparticle. Nanoparticles were added to the previous tubes with serial concentrations. The tubes were incubated at 37°C for 24 hours. The minimal inhibitory concentration (MIC)was considered as the first clear tube that comes after a series of turbid tubes and here the concentration of the substance is considered as (MIC) value.

 

Determination of the Minimal Inhibitory Concentration (MIC) of Azithromycin:

Stock solution with a final concentration of 10 mg / ml of Azithromycin was prepared according to the CLSI standards (17) by dissolving 1 gm of the antimicrobial agent in 90 ml of distilled water, then the volume was completed to 100 ml and the solutions were sterilized with 0.22 micrometer diameter micro filters, and kept in refrigerator at 4°C until use.

 

The minimal inhibitory concentration (MIC) of bacterial isolates was determined according to the standard criteria of CLSI (17). Duplicate serial concentrations of Azithromycin ranging from (2-4-8-16-32-64-128 – 256 - 512- 1024) μg / ml were prepared by addition of different concentrations of this antimicrobial agent from the prepared stock solution to the previously sterilizedand cooled to 45°C Muller-Hinton medium.The bacterial suspension of each isolate prepared with its turbidity compared with the turbidity of Macfarland standard solution. An amount of 5 μl of the above dilutions was pipetted by a micro-pipette, and inoculated by the spotted distillation method as a single drop on the antimicrobial solution media. The process was repeated for all cultures serially at a rate of two repeats for each single concentration, and the dishes were left at room temperature until the drops were dried before inverting the dishes. The culture media were incubated at 37°C for 24 hours. The minimal inhibitory concentration (MIC) was calculated as the minimal concentration of antibiotic which inhibition bacterial growth after 24 hours incubation at 37°C temperature.

 

 

 

 

The Co-Culture method for biofilm formation inhibition:

The Co-Culture method was conducted according to the method of Al-Marjani and Salman (24). This method involves detecting the inhibition of the biofilm formation by using Microtiter Plate (MTP) by filling the column wells of these media with 180μl of brain and heart broth containing 2% sucrose and 20µl of the A. baumannii bacterial culture to represent the control, while the rest of the wells were filled with 100µl of the concentration under the minimal inhibitory concentration of curcuma, silver nanoparticles and Azithromycin extracts (the substances whose effects to be tested on the biofilm) and 80 µl of the of the heart and brain broth containing 2% sucrose and 20µl of the bacterial culture.

 

The dish was covered and incubated at 37°C for 24 hours. The contents of the wells were then poured and washed with distilled water and left to dry at room temperature for 15 minutes (for drying). After that, 200 μl of 0.1% crystal violet dye was added to the wells and left for 20 minutes. Then the wells were washed many times with distilled water, and left to dry for 15 minutes. After their dryness was confirmed, 200µl of 95% ethyl alcohol was added per well. The absorbance of the wells was then measured at 630 nm wavelength using the ELISA reader. The inhibitory rate of biofilm formation is calculated according to the following equation:

 

                                                               (A-B)

The inhibitory rate biofilm formation =--------- X 100   

                                                                   (A)

 

A = Optical density of the control equation.

B = Optical density of the equation with the presence of inhibitory substance.

 

RESULTS AND DISCUSSION:

Isolation of Acinetobacter baumannii:

The results of morphologic and biochemical tests conducted on bacteria isolated from (140) pathogenic samples showed that 14 (9.3%) of them were Acinetobacter baumannii. The increasing existence of this bacteria in the hospital environment may be due to their ability to live on dry and wet surfaces, as well as to their resistance to many types of antibiotics and disinfectants and their production of many types of virulence factors (25). The results showed an increase in the number of male patients infected with A. baumannii 82 (54.7%) when compared with the infected females 68 (45.3%) as shown in table (2). The high incidence of male infection may be related to certain conditions such as smoking, alcoholism and Diabetes mellitus (26).

 

 

 

Table (2) Distribution of number and percentage of clinical specimens according to gender

 

Susceptibility of isolates to antibiotics:

The susceptibility of A.baumannii isolates was tested to 16 antibiotics from different groups depending on measuring the inhibition zone diameter around the antibiotic disc, and the results were compared with the CLSI criteria (17). All isolates showed 100% resistance to Cefepime, Ceftazidime, Piperacillin, Cefotaxime, Augmentin and Ceftriaxone, while the resistance rates of the isolates to Ciprofloxacin and Tetracycline showed (92.9%) for each of them, and the resistance rate to each of Gentamicin and Co-Trimoxazole was (85.7%), whereas the resistance rate to Tobramycin was (78.6%), Aztreonam and Amikacin (57.1%), Meropenem (50%), Imipenem (42.8%) and Colistin (14.3%) as observed in table (3).

 

Table (3): Resistance and susceptibility rates of A.baumannii to antibiotics

 

The antibiotic resistance of A.baumannii is due to the production of β-lactamase enzymes, the modification of target sites, the acquisition of efflux pumps and their production of aminoglycoside-modifying enzymes, as well as the possession of Cephalosporinase enzymes that attack cephalosporin antagonists and render them ineffective (27). The Imipenem and Meropenem belong to Carbapenem antagonist group. The cause of bacterial resistance to antagonists of this group is the ability of bacteria to produce two types of beta-lactamase enzymes: Metallo β- Lactamase and Carbapenem hydrolyzing class D β-Lactamase, which hydrolyze and destroy Carbapenem antagonists. A change in the proteins associated with the outer membrane (OMPs) leads to bacterial resistance to Carbapenem antagonists (28). One of the mechanisms associated with A. baumanni iresistance to Colistin antagonist is a mutation to the pathway of lipid (A) synthesis of lipopolysaccharide (LPS) pathway that causes its loss from the outer layer of the bacterial plasma membrane and thus removes the antibiotic-bacteria binding            site (29).

 

Testing the inhibitory effect of Curcuma longa extract:

The effect of different concentrations of aqueous extract (cold and hot) and alcoholic extract on Anti-imipenem-resistant A. baumannii by using the agar well diffusion method showed that the cold aqueous extract of curcuma roots was more effective at the higher concentration 200 mg/ml, as the mean inhibition zone diameter was 22 mm, followed by the concentrations (25, 50, 100) mg/ ml, with a mean inhibition zone diameter of (9, 14 and 17) mm respectively, while there was little effect at the concentration of 12.5 mg / ml as seen in table (4).

 

Table (4): Effect of cold aqueous extract of curcuma roots on A. baumannii

Type of extract	Concentration mg/ml	Mean ±St. Error
Cold aqueous extract of Curcuma longa	200	22.00 ±0.57
	100	17.00 ±0.57
	50	14.00 ±0.57
	25	9.00 ±0.57
	12.5	0.00 ±0.00

Mean= mean of inhibition zone, S.Error=standard error

 

In addition, the results revealed that the inhibition zone of hot aqueous extract of curcuma roots was 19 mm at the concentration of 200mg/ml, followed by concentrations (25, 50, 100) mg/ml and the mean inhibition diameters were (8, 12, 15) mm respectively, While there was no significant effect at the concentration 12.5 mg/ml. (Table 5).

 

Table (5): Effect of hot aqueous extract of curcuma roots on A. baumannii

Type of extract	Concentration mg/ml	Mean ±St. Error
Hot aqueous extract of Curcma longa	200	19.00 ±0.57
	100	15.00 ±0.93
	50	12.00 ±0.57
	25	8.00 ±0.57
	12.5	0.00 ±0.00

Mean= Mean of inhibition zone, S.Error=Standard error

 

Regarding alcoholic extract, the highest rate of inhibition was 15 mm at the concentration of 200 mg / ml, followed by concentrations (25,50, 100) mg/ml with a mean inhibition diameters (9,12 and 13) mm respectively, while there was little impact at the concentration 12.5 mg/ml as shown in table (6).

 

Table (6): Effect of alcoholic extract of curcuma roots on A. baumannii

Type of Extract	Concentration mg/ml	Mean ± St. Error
Alcoholic extract of Curcma longa	200	15.00 ±0.57
	100	13.00 ±0.57
	50	12.00 ±0.57
	25	9.00 ±0.57
	12.5	0.00 ±0.00

Mean= mean of inhibition zone, S.Error=standard error

 

The results of the present study agreed with the findings of Ashgar (30) which was conducted in Saudi Arabia, who confirmed that A. baumannii bacteria is sensitive to the aqueous and alcoholic extracts of curcuma plant.

 

Curcuma root extracts contain curcumin or diferuloymethan substance, a highly effective substance that binds to the cell membrane lipids, causing loss of its function, while its effect is different on the biofilm, causing changes in gene expression and inhibition of quorum sensing (31). It contains Tannins, which are polyphenols that are soluble in water and have the ability to precipitate bacterial cell proteins leading to their death (32).

 

The results of our study indicate that the cold aqueous extract has a significant effect on the bacteria than other extracts, indicating that the active substances in the plant extract are negatively affected by heat and alcoholic solvents.

 

Determination of MIC and sub-MIC values for curcuma extracts, silver nanoparticle and Azithromycin:

Table (7) involves the MIC and Sub MIC values for the three inhibitory substances. The agar dilution method was used to determine the MIC value for aqueous extracts (cold and hot) and alcoholic extract of the curcuma roots against A. bumannii isolates. This method is one of the most suitable to test the effectiveness of plant extracts on bacterial cultures, and it does not need to sterilize plant extracts (33).

 

 

The MIC value of the cold aqueous extract was 6.25mg /ml towards bacterial isolates and Sub-MIC value was 3.13mg/ml. While for hot aqueous and alcoholic extract, MIC value was 12.5 mg / ml and Sub MIC was 6.25mg/ ml for each of them. The Dilution method was used to determine the MIC value for silver nanoparticles (AgNPs) with a MIC value of 20000 μg / ml and a Sub-MIC value of 10000 μg / ml. The nanoparticles of modern times are of good importance, showing low toxicity and high effectiveness to solve the problem of bacterial multiple antibiotic resistance. Silver is always used against various diseases; it has been used in the past as a disinfectant and antimicrobial agent against Gram negative and Gram positive bacteria.

 

Studies have shown that silver nanoparticles have an anti-inflammatory activity that it contributes to wound healing, and these particles were found to be useful through speeding up the wound healing process, as well as increasing the surface tension of the skin in surgery and making it look similar to normal skin (34). Patra and Beak (35) indicated that silver nanoparticles release Ag + ions that interact with the Thiol group in bacterial proteins, and affects DNA function, therefor kills bacteria. It is thought that silver nanoparticles, after penetrating the bacterial cell membrane, may disrupt some of the cell's enzymes generating hydrogen peroxide (H₂O₂), which is a bacteria-killing substance.

 

The MIC of Azithromycin was determined by multiple serial concentrations on the solid Muller-Hinton medium, which is preferred over other culture media, because it contains a small amount of sodium chloride and a small amounts of calcium and magnesium ions, which does not affect calculated MIC values (36). The MIC value of Azithromycin was 512 μg / ml and the Sub-MIC value was 256 μg/ml. Azithromycin belongs to the Macrolides and its structure is similar to Erythromycin, which has a broad spectrum effect against Gram-negative bacteria. This antibiotic binds to the 50s ribosome unit in microorganisms susceptible to the antibiotic and interferes with microbial protein synthesis (37, 38).

 

 

 

Table (7): Determination of MIC and Sub-MIC values of curcuma extracts, silver nanoparticles and Azithromycin

 

Inhibition of Imipenem-resistant A. baumannii biofilm using Sub-MIC:

In the microtiter plate, Sub-MIC of aqueous (cold and hot) and alcoholic extracts,silver nanoparticles, and Azathromycin were used to inhibit the biofilm of Imipenem-resistant A. baumannii isolates. The results shown in table (8) demonstrated that there was no inhibitory effect of curcuma extracts used to inhibit the biofilm (negative result) through reading the ELISA results. While silver nanoparticles, used as 20 nm size, showed a clear effect (positive result) on biofilm inhibition. Silver nanoparticles have antibacterial characteristics, as they attach to the cell wall and penetrate the Gram negative bacterial cell membrane, therefore increase the cell permeability, and lead to uncontrolled transport through cytoplasmic membrane, and eventually to cell death. In addition, the biofilm matrix of A. baumannii bacteria contains extracellular DNA (eDNA), where a reaction occurs between the positive charge of silver nanoparticles and the negative charge of (eDNA), which plays a key role in the A. baumannii biofilm inhibition (39).

 

Bogdan et al., (40) in a study conducted in Croatia showed that the Sub-MIC for antibiotics had an effect on some of the different molecular mechanisms, resulting in morphological or physiological changes to the bacteria itself. In their study, they confirmed that MIC of Azithromycin could reduce the biofilm in the isolates of A.baumannii bacteria that form biofilm at an intermediate level.

 

 

Table (8): Biofilm inhibition of the Impemem-resistant A. baumannii by Sub-MIC for curcuma plant extracts, silver nanoparticles and Azithromycin.

Isolates of A. baumannii forming biofilm and imipenem resistant	Sub MIC of materials
	Silver nanoparticles  (AgNPs)	Azithromycin	Cold aqueous extract	Hot aqueous extract	Al-coholic extract
Isolate 1	+	-	-	-	-
Isolate 2	+	-	-	-	-
Isolate 3	+	-	-	-	-
Isolate 4	+	-	-	-	-
Isolate 5	+	-	-	-	-

(+)Represents presence of inhibitory effect,(-) represents absence of inhibitory effect

 

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Accepted on 02.04.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4463-4470.