Bacterial strains
Two hundred and fifty (250) bacterial strains including Gram-negative (GN) and Gram-positive (GP) strains were provided by Suez General Hospital, Egypt. All strains were immediately transported in the sterilized box into the bacteriology laboratory.

Culture condition and maintenance media
The provided bacterial strains were propagated and subcultured at 37°C for 24 h on different media including Muller-Hinton broth, Nutrient agar medium, Trypticase soy agar, Mannitol salt agar, Blood agar base medium and Muller-Hinton agar then the pure colony was kept on agar slant before carrying out the tests for antibiotic sensitivity profile, identification and antimicrobial assays.

Antibiotic sensitivity test of bacterial isolates
Seventeen commercially prepared (6 mm in diameter) antibiotic disks, obtained from Oxoid, UK, belonging to a variety of groups, were selected for their potency against bacterial isolates. Properties of the target antibiotics included: Ampicillin (10 mcg), Flucloxacillin (5 mcg), Clindamycin (2 mcg), Levofloxacin (5 mcg), Erythromycin (15 mcg), Kanamycin (30 mcg), Tobramycin (10 mcg), Ofloxacin (5 mcg), Rifamycin (30 mcg), Aztreonam (1 mcg), Gentamicin (10 mcg), Norfloxacin (10 mcg), Gatifloxacin (5 mcg), Cephradine (30 mcg), Tetracycline (30 mcg), Ciprofloxacin (5 mcg) and Oxacillin (1 mcg). The test was performed according to the standard Kirby-Bauer disk diffusion method¹⁰. The findings were presented according to standardized protocols for the assay of antibiotic compounds as directed by National Committee for Clinical Laboratory Standards “NCCLS”, and were classified as sensitive (S), intermediate sensitive (I) and resistant (R)¹¹.

Primary identification of bacterial isolates
According to Bergey’s manual, almost all microscopically examinations and biochemical testing used for identification were carried out^12,13.

Secondary identification
It was by automated identification using the Biomerieux Vitek 2 System. The procedure of the identification was used according to the recommendations of the manufacturer. The databases of the VITEK 2 identification based on established biochemical methods and newly developed substrates, according to Funke and Funke-Kissling^14,15.

Plant materials
Different parts of 5 medicinal plants, including Nigella sativa (seeds), Ziziphus spina-christi (leaves), Rosmarinus officinalis (leaves and stem), Origanum majorana (leaves) and Allium sativum (fruit). Plant parts were obtained from Ain Shams Uni., where the collected plant parts were cleaned, washed, and shade dried, exposure to sunlight was avoided to prevent the loss of active components. Dried plant parts were homogenized to a fine powder and stored in airtight bottles¹⁶.

Aqueous extract preparation
The fine powder of different plants parts weighing 100 (g) was extracted with one liter of distilled water (1:10 w/v) in a conical flask, plugged with cotton wool, and then kept for 7 days, by means of cold extraction. Then the extract was filtered with 8 layers of muslin cloth. The extract of each plant was concentrated in vacuo using a rotary evaporator at 40°C. The water remaining in the extract was finally removed by placing the extract in porcelain dishes in the temperature-controlled oven to give a residue weighing about 6.3-9.2 g for different extracts. The residues were stored at 4°C for further use^17,18.

Acid-Base fractionation of most potent plant extract
About (100g) of Z. spina-christi plant powder was extracted with one liter of distilled water. The water extract of the plant was concentrated under reduced pressure until dryness at a temperature not exceeding 60°C, acidified with HCl 3% until pH=1.5 and then filtered. The filtrate obtained was transferred to separated funnel, and the chloroform (v/v) was added to the filtrate and left for 10-15min after proper mixing, two layers were formed (upper layer was chloroform layer “acid-alkaloid fraction”) and (lower layer was aqueous layer), the previous step repeated about three times until the upper layer become colorless. The aqueous layer was transferred to separated funnel and ammonia was added to adjust the medium until pH=10.5, then chloroform in (v/v) was added and two layers formed, the upper chloroform layer (“Base alkaloid fraction”) and the aqueous lower layer (“rich polyphenol fraction”). The previous step was repeated about three times until the upper layer become colorless. The fractions were filtered and evaporated under reduced pressure until dryness then weighed to calculate the percent (w/w)¹⁹.

Investigation of antibacterial activity of plant extracts
Test organisms
The most Multidrug-resistant bacterial strains namely, Klebsiella pneumoniae (Gram-negative), Escherichia coli (Gram-negative), Pseudomonas aeruginosa (Gram-negative), Staphylococcus aureus (Gram-positive) and Methicillin-resistant Staphylococcus aureus (Gram-positive) which isolated from the clinical specimens were selected for the study of anti-bacterial and antibiofilm assays of used extracts.

Seeding method for the bacterial strains under study
In a 150 ml conical flask containing 25 ml of Muller Hinton Broth, a loop full of each strain was inoculated, followed by incubation in a rotary shaker at room temperature for 24 hours to activate the bacteria with a final cellular concentration of 1 × 10⁸ cfu/ml. The molten Mueller Hinton Agar was inoculated with an inoculum of 200 μl and poured into the sterilized petri dishes where precaution was taken to ensure full homogenization²⁰.

Preparation of the extracts for antibacterial assay
Plant extracts were dissolved in sterile distilled water for antibacterial study. The concentration of the extracts was evaluated and labeled on the specified tube. The antibacterial study of different plant extracts was assayed by two methods as follows:

Disc diffusion method
The disc diffusion method was performed for the determination of antimicrobial of the five tested extracts against previous mentioned MDR-strains. A standard blank paper disk (7 mm in diameter) were separately soaked in high concentrations of extracts (100 mg/ml) and transferred onto the surface of growth media previously seeded with the test organisms in petri plates using a sterile pair of forceps. The Petri dishes were closed with parafilm and left in the refrigerator for 1 hour to allow the active compounds of the extracts diffuse. Sterile distilled water was used as a negative control instead of active compounds. The plates were then incubated at 37°C for 24 hours. By calculating the diameter of the inhibition zones in millimeters around the disc using a caliper or ruler, the sensitivity of the bacteria to each extract was determined when the values were reported as an average of three replicates²¹.

Broth microdilution method [Mean growth inhibition percentage (%)]
The bacterial suspension equivalent to the turbidity of 0.5 McFarland standard (10⁸cfu/ml) prepared from a fresh subculture of tested bacteria in Mueller Hinton Broth (MHB), then the suspension was diluted to (10⁶ cfu/ml) using MHB. The adjusted microbial inoculums (100 µL) were added to each well of a sterile 96-well flat-bottomed microtiter plate containing the tested concentration 100 (mg/ml) of each plant extract 100 (µL/well). As a result, the last inoculum concentration of (5×10⁵ CFU/ml) was obtained in each well. This plate included two wells containing only the media used as background control and three wells containing microbial suspension without the extract tested used as growth control. The optical density was assessed at 620 nm 37°C after 24 hours using ELISA microplate reader (Sunrise™-TECAN, Switzerland) at the Department of Botany and, Faculty of Science, Al-Azhar University, Cairo, Egypt. Ampicillin and Gentamicin were used as standard antibiotics for Gram-positive and Gram-negative bacteria, respectively. Finally, cell concentrations were transformed into a percentage of bacterial inhibition. The percentage of bacterial growth reduction (GR %) was calculated using GR%= as reference the control treatment (without extract). Since T is the concentration of cells under the extract treatment, and C is the concentration of cells under the control treatment. The results were reported as means ±SE of the triplicate experiment¹¹.

Determination of (MICs) and (MBCs) of the most potent plant extract and its polyphenol fraction
Using the broth microdilution method, the MICs of total extract and polyphenol fraction of Z. spina-christi was performed in 96 polystyrene wells according to Andrews²² and Sampaio, et al.²³. For each bacterial strain, an inoculum suspension contains a cell concentration of 10⁸ CFU/mL was prepared in Mueller-Hinton broth. In the test each wells of the plate 20 μl of tested plant extract was added to a 180 μl of bacterial suspension. For tested extracts which, diluted to yield concentrations 50, 25, 12.5, 6.25, 3.125 and 1.562 mg/ml for total extract and 5, 2.5, 1.25, 0.626, 0.312, 0.156 and 0.078 mg/ml for polyphenol portion of Z. spina-christi. In plate setup both control positive wells (Media + bacteria) and control negative wells (media + tested extract) as well as a background wells (media + sterile distilled water) were contained. The highest dilution of the tested extract (lowest conc.) inhibit the visible bacterial growth defined as the MIC¹¹. After determination of MICs of Z. spina-christi total extract and its polyphenol fraction against each bacterial strain, the wells of MICs were poured on the surface of Muller- Hinton agar plates and then incubated at 37°C for 24 h. After incubation the developed bacterial colonies were counted and the lowest concentration of the tested extract showed bactericidal effect (no growth) was defined as minimum bactericidal concentration (MBC).

Biofilm Formation of MDR-bacterial isolates
Quantitative assessment was carried out using the Microplate method (MP) suggested by Christensen, et al.²⁴ using 96 flat bottom well tissue culture plates. For determination of biofilm formation activity of S. aureus, MRSA and P. aeruginosa the wells of microtiter plates were loaded by 0.2 ml of bacterial inoculum (10⁵ CFU/ml) in Trypticase soya broth (TSB) medium containing 2% glucose (Glc), the plates were aerobically incubated for 24 h at 37°C and then twice washed by  PBS (pH 7.2). After washing 0.1% crystal violet (CV) was added onto the wells to staining for 2 min. The plates were measured at 570 nm using ELISA microplate reader (Sunrise™-TECAN, Switzerland). Negative control wells (sterile media) also contained in the plates. The vales of the different optical density (OD) were calculated to detect the slime producing strains (biofilm formers) in comparison with O.D. of negative control or cut off. The interpretation of the production of biofilm was made in accordance with the criteria set out by Stepanovic, et al.²⁵.

Antibiofilm formation activity of most potent plant extract and its polyphenol fraction
Sub-MICs of total extract and polyphenol fraction of Z. spina-christi at concentrations 2, 1, 0.5 and 0.25 mg/ml and 200, 100, 50, and 25 μg/ml, respectively were checked for their ability to inhibit biofilm formation in biofilm-producing strains. At the time of inoculation, they were added to the growth medium and the cells were allowed to form a biofilm. Two-fold serial dilutions of aliquot (100 μL) were prepared in a 96-well microtiter TSBGlc-containing plate containing different concentrations of extracts tested under the MIC values. Bacterial suspensions (100 μL; 5 × 10⁵ CFU/mL, final concentration) were then transferred into the plate. The wells of TSBGlc medium with 0.2% DMSO was used as a negative control, while the wells of the medium without tested extracts serve as positive control and wells of different concentrations of tested extracts acts as a blank.  The plates were placed in incubated shaker in aerobic condition for 24h at 37^◦C. After the incubation period the effect of each extract concentration was measured at O.D. 570 nm using the microplate reader (Sunrise™-TECAN, Switzerland) against the biofilm activity of S. aureus, MRSA and P. aeruginosa according to the previously mentioned method²⁶. In addition, the growth of the tested strains without the extracts was also determined at O.D. of 620 nm. The experiment was performed in triplicate.