Biosynthesis of TiO2 nanoparticles using prodigiosin and evaluating its antibacterial activity against biofilm producing MDR- Acinetobacter baumannii

Keywards: A. baumannii, Antimicrobial activity, Biofilm, Prodigiosin, TiO2 nanoparticles A rising number of hospital infections were caused by multi drug resistant A.baumannii. This microorganism has become a big global concern for clinicians. This study aimed to evaluate the antimicrobial activity of biosynthesized TiO2 nanoparticles against biofilm producing multi drug resistant A. baumannii. Bacteria were isolated from burn wounds. The selected isolate was identified using the routine biochemical assays, viteck 2, and confirmed by PCR technique, targeting the 16S rRNA and blaOXA-51 genes. Antimicrobial susceptibility tests were performed using Viteck 2 system and the biofilm production was tested by using microtiter plate method. S marcescens was used for production of the prodigiosin which characterized later by UV-visible spectroscopy and then was used for biosynthesis of titanium dioxide nanoparticles (TiO2) NPs. Atomic force microscopy, X-ray diffractometer and field emission scanning electron microscopy were used for characterization of TiO2 NPs. Antimicrobial activity of TiO2 NPs was examined by well diffusion assay using concentration of 0.40.006 mg/ml. The studied isolate was beta-lactamase producer and showed resistance to aminoglycosides, quinolones, furanes and trimethoprim/ sulphonamide, PCR amplification of 16S rRNA and blaOXA-51 genes was used for detection of A baumannii. The selected isolate was a strong biofilm producer with 5.9 times more than the OD values of the control. Atomic force microscopy images showed that the synthesized TiO2 NPs were in spherical shape with an average diameter of 67.49 nm. The TiO2 NPs inhibited the bacterial growth at concentrations of ≥ 0.1mg/ ml and a maximum zone of inhibition recorded was 22 mm at concentration of 0.4 mg/ ml. Biosynthesis of TiO2 NPs using prodigiosin was showed a promising antibacterial activity against strong biofilm producing MDRA. baumannii. Biosynthesis of TiO2 nanoparticles using prodigiosin and evaluating its antibacterial activity against biofilm producing MDRAcinetobacter baumannii


‫بكتريا‬ ‫تعد‬
. A red pigment, Prodigiosin, was discovred in S marcescens, it is an important secondary metabolite (6). The generation of prodigiosin in different strains, in particular through a quorum sensing mechanism, seems to be induced by cell density at later stages of bacterial growth (7,8). Prodigiosin plays a crucial role in nanoparticles biosynthesis, in addition to its unique anticancer properties, it also found to have antibacterial, antiprotozoal, and antiinflammatory activity (9). Also, it is used or nanoparticular biosynthesis of gold and silver (10,11). In recent years Acinetobacter baumannii has become primarily a multi-drug resistant bacterium and a major concern (12). WHO reported it among major antibiotic resistant "pathogen priorities" highlighting its serious threats to public health (13,14). Advanced molecular methods have been employed today to identify A. baumannii, such as 16S rRNA, blaOXA 51 and RNA ß subunit (rpo) genes amplification and sequencing (15,16,17,18). According to a National Health Institutes report, over 80 % of all bacterial diseases contain biofilm. Biofilms are associated with various medical conditions which of, upper respiratory tract infections, urogenital infections, dental plaque and indwelling medical devices infections. (19) These biofilms are extremely difficult to eradicate as the antibiotic resistance can be increase 1,000 times (20). There are different techniques for biofilm det ection, like, tissue culture or microtiter plate (TCP), tube method (TM), congo red agar method (CRA), bioluminescent tests, and fluorescent microscopic examinations (21). The aims of this study are to synthesize, characterize and evaluate the antibacterial activity of biosynthesized TiO2 NPs using prodigiosin pigment against biofilm producing multidrug resistant A. baumannii using well diffusion method.

Bacterial isolation and Identification
Twenty bacterial isolates were collected from Burn, Reconstructive and plastic Surgery Hospital in Sulaimani -KRG-Iraq during the period of 01/7/2019 to 20/12/2019. All Bacterial isolates were identified on the basis of routine biochemical assays and confirmed by VITEK 2 system (Biomérieux, France) using VITEK® 2 GN ID Card and VITEK® 2 GP ID Card (according to the manufacturer's instructions).
One isolate of multidrug-resistant A. baumannii was selected for further experiments. A. baumannii ATCC (19606) and Serratia marcescens strains supplied from biology department college of science, University of Sulaimani, KRG-Iraq, and used as a standard strain for molecular detection and also for antibacterial activity and biosynthesis of TiO2 NPs.

Molecular detection of A. baumannii
A-Extraction of DNA Genomic DNA was extracted from an overnight culture in tryptic soy broth using Bacterial Genomic DNA Kit (Geneaid, Taiwan) according to the manufacturer's protocol.

B-Detection of 16S ribosomal RNA gene
Specific 16S rRNA primers were used for identification of the isolate as described by (22). In brief, a 1500-bp fragment of the ribosomal RNA gene was amplified using the following primers F (5′TGGCTCAGATTGAACGCTGGCGGC-3′) and R(5′-TACCTTGTTACGACTT CACCCCA-3′). The final reaction volume was 25 μl, and the cycling conditions were as follows: an initial denaturation at 95°C for 4 minutes, followed by 30 cycles of denaturation at 95°C for 40 seconds, annealing at 64°C for 40 seconds, extension at 72°C for 40 seconds, and a final extension at 72°C for 5 minutes. The PCRamplified products were stained with ethidium bromide and analyzed by 1% agarose gel electrophoresis. Images were acquired using a Bio-Rad Gel Doc XR + imaging system.

C-Detection of blaOXA-51 gene
To confirm the identity of the isolates at the level of species, OXA-51 gene was amplified with expected amplicon size of 353bp (23, 18) using primers: F (5′-TAATGCTTTGATCGGCCTTG-3′) and R (5′-TGGATTGCACTTCATCTTGG-3′). The final volume was 25 μl, and the cycling conditions were as follows: an initial denaturation at 94°C for 5 minutes, followed by 30 cycles at 94°C for 25 seconds, 56.5°C for 40 seconds, 72°C for 50 seconds and a final extension at 72°C for 6 minutes. The PCR-amplified products were stained and analyzed as mentioned above.

Detection of Biofilm
A. baumannii was inoculated in a 5ml Trypticase soya broth (TSB) and grown to stationary phase then culture was diluted at 1:100. Subsequent, 200μl of diluted culture was pipetted into wells in 96-well flat bottom polystyrene microtiter plates. Incubation was carried out at 37º C for 24 hours. Cultures were then aspirated and the wells were washed 3 times with phosphate buffer saline (Ph 7.2). The plate was then air dried for 15 min at room temperature then stained by adding 200 μl of 0.1% crystal violet for 15 min. After removing the stain, 200μl of 95% ethanol were added to each stained well and left for 10 to 15min at room temperature. The optical density of the wells was measured at 590 nm using micro ELISA auto reader. Sterile TSB was used as a negative control. To compensate for background absorbance, the optical density (OD) reading value of control (C) was deducted from the test (T) values (24,25). The intensity of biofilm was measured as below: 1. ODT = ODc= Non biofilm producer. 2. ODc < ODT ≤ 2ODc = Weak biofilm producer. 3. 2ODc < ODT ≤ 4ODc =Moderate biofilm producer. 4. 4ODc < ODT = Strong biofilm producer.

Antibiotic susceptibility test
Susceptibility tests were performed using VITEK 2 system with AST-GN48 TEST KIT susceptibility card for Gram negative bacteria (Biomérieux, France) according to the manufacturer's instructions.

Prodigiosin production
Vol. 13 Issue:2, (2020) ISSN: P-1999:6527 E-2707:0603 Production of the prodigiosin was executed by inoculating the S. marcescens to the batch fermentation media. The batch fermentation media was prepared according to (26

Extraction and purification of prodigiosin
The extraction of Prodigiosin the red pigment was carried out after 30 hours of incubation from the cell-free broth culture of S. marcescens. Subsequently the amount of 250 ml of methanol was added to the harvested cells after centrifugation of the culture medium for 15 minutes at 8000 rpm and stirred completely for 3 hours at room temperature. Thereafter, a second centrifugation was done for the remaining mixture for 20 minutes at 8000 rpm, the supernatant was collected and filtrated through 0.2 μm, milipore filter (Gema medical, Spain). The process of concentration of methanol filtrate at 70˚C was accomplished by Rotary evaporator.
The extraction of the crude prodigiosin was done by adding double volume of chloroform (organic phase), the red elutes was collected and dried at 45 ˚C to get the red powder of the prodigiosin, finally, the obtained red powder was dissolved in methanol for the purpose of storage. The process of extraction of prodigiosin was carried out according to (26).

Synthesis of titanium dioxide nanoparticles
Process of the synthesis of titanium dioxide nanoparticles was done by utilizing tanium chloride (TiCl4, 99%). Ten mg of red powder of prodigiosin was dissolved in 1ml deionized water in a sterilized flask and dispersed by ultrasonic bath for 60 minutes. On the other hand, 5 ml of TiCl4 was dissolved in 50 ml deionized distilled water and was dispersed for 30 minutes by ultra-sonic bath. After the dispersion time completed, the two separate solutions (the prodigiosin solution and the TiCl4 solution) both were mixed and stirred thoroughly by using magnetic stirrer at pH 7 for 30 minutes and thereafter the mixture was left in a dark room overnight. The solution that include the precipitate of the titanium dioxide nanoparticles, centrifuged at 6000 rpm for 30 minutes, washed two times by DDW, then was centrifuged for the second time at 6000 rpm to concentrate the precipitate. The obtained nanoparticles precipitation was dried at 60 ˚C for 30 minutes in an oven. Finally, the resulting white powder was preserved in a dark bottle for further characterization and applications, the process of synthesizing TiO2 nanoparticle was done according to (27,28).

Antibacterial Activity of TiO2 Nanoparticles
Bacterial susceptibility to TiO2 NPs was det ermined by using agar well diffusion method. Bacterial isolates were cultivated overnight at37 ° C on Mueller Hinton medium. After incubation, standard inoculum for each bac terial isolate at a concentration of 1.5 X 10 8  incubation at 37 °C. After incubation, bacteri al susceptibility diameters in the inhibition zones were reported. Wells containing sterile distilled water alone were used as a negative control and well was contained gentamycin as a positive control (25).

Results and Discussion Bacterial identification
Vitek 2 system was identified the isolate as follows: A. baumannii (99% probability) using VITEK® 2 GN ID Card which is able to identify more than 150 fermentative & non-fermentative Gram-negative bacilli (Table1). VITEK2 compact device incorporates many advantages that may be of clinical significance for routine bacterial identification such as easy technique rapid detection and high degree of automation (29). Funke et al (1998) showed that within 3 h, the VITEK 2 system identified correctly 84.7% of selected species belong to enterobacteriaceae family and 70 different species of non-enteric bacilli, while 0.8% being misidentified strains and 1.2% being non-identified strains (30). In the present study, A. baumannii was identified by VITEK 2 system and also was confirmed by molecular technique targeting two specific genes (16S rRNA and blaoxa-51 genes) as shown below.

Antibiotic susceptibility assay
Minimum inhibitory concentration of the tested bacteria were obtained by using automated system (Vitek 2) with specific cards representing different classes, In a current study a substantial increase to most antibiotic Classes was observed, particularly antibiotics chosen for treating A. baumannii infections, it was beta-lactamase positive and showed resistance to aminoglycosides, quinolones, furanes and trimethoprim/ sulphonamide, while it showed an intermediate resistance to levofloxacin (Table2). Many of the antimicrobial agents can be reliably tested through VITEK 2 system. Vitek 2 is a closed system that can avoid cross-contamination or contamination of the environment (31). It can manage number of specimens at the same time automatically, preparation and processing of samples is simple to do, reduce processing and operating times drama tically, increase the performance of standard clinical laboratories (30,31) In China, a large study which included 851 hospitals for evaluating the performance of five commonly used automated susceptibility testing systems (Vitek 2, Phoenix, Microscan, TDR, and DL) against ESBL-producing Escherichia coli and KPC-producing Klebsiella pneumonia, except for cefepime and meropenem, Vitek 2 system seemed to have provided a reasonably reliable and conservative evaluation of MIC (32).

Molecular Detection of MDR-A baumannii
A. baumannii was identified by targeting two genes, 16S rRNA and blaOXA-51 gene by two separated PCR reaction. Primers targeted 16S rRNA gene were able to detect A baumannii in both clinical isolate and A. baumannii ATCC 9606 (Fig. 3). Our results were in agreement with previous studies (17,18,29,33 and 34)  baumannii, it was suggested that the identifica tion of this species may be based solely on the detection of an blaOXA-51 gene (38).

Production of prodigiosin
The spectra of absorption of the prodigiosin pigment which has been extracted from our sample of Serratia marcescens (ATCC 15365) was showed maximum production of the red pigment and demonstrated an absorption peak at 526 nm wavelength (39), the characterization of the prodigiosin was done by using UV-visible spectroscopy (Shimadzu, Japan), so as to reveal the ultimate absorption of this red pigment, as shown in fig.4.

Characterization of green synthesis TiO2 NPs Atomic force microscopy (AFM) AFM images show that the synthesized TiO2
NPs are in spherical shape and the size of an average diameter was 67.49 nm (table 3 and fig.5). The surface morphology of the TiO2 NPs was studied by atomic force microscopy ,the 2D and 3D of TiO2 NPs were given topology (40).
The interlacing parameters of titanium oxide nanoparticels were a = 0.3785Å, these parameters have been compatible with the reference of face-centered cubic (fcc) crystal lattice of metallic titanium. According to the Debye-Schereer, and XRD patterns all titanium oxide nanoparticles were in form of crystalline as shown in the equation below (41). D = [Kλ / βcosθ] Å Where: D: is the average crystallite size (Å) K: is the shape factor (0.9) Λ: is the wavelength of X-ray (1.5406 Å) Cu Kα radiation θ : is the Bragg angle β ; is the corrected line broadening of the nanoparticles.
Field emission scanning electron microscopy FESEM images measured the topography of the synthesized nanoparticles, the images were magnified at 50kx based on ( fig. 7), the whole nanoparticle samples demonstrate smooth planes and orderly organized in a shape of titanium oxide nanoparticles bunches, researches were revealed that increasing in the rate of calcination temperature will have an important effect on the size of the nano particles changing them from small to large particles by means of agglomeration, however, better small size nano particles were formed by low temperatures leading to formation of spherical shape of nano particles (42).

Detection of biofilm production
After incubation for 24 hr, the tested isolate produced biofilm in the TSB medium at 37 o C.The mean OD of biofilm production was (0.674), which was 5.9 times more than the control (Mean OD=0.114), which indicate that A. baumannii was a strong biofilm producer (Table 4 and Fig 8). Previous studies were evaluated three screening methods for biofilm production, tissue culture or 96 wells microtiter plate (TCP), tube method (TM) and congo red agar method (CRA), they were reported that the TCP method as superior to the TM and CRA methods (43,44 Albaiaty, 2019 also recorded that TiO2 NPs were able to inhibit bacterial growth at 500 μg / ml with maximum zone of inhibition of 14 mm against strong biofilm-producing MRSA isolates (49,50). Abdullah (2016) reported that, TiO2 NP exhibited an antibacterial activity against some biofilm producing gram negative bacteria, but when they were combined with antibiotics the effect was greater (47). Another work from Iraq (48) was concluded that the particle size may has an effect on the antibacterial activity of the NP, They have used three different particle sizes (10, 50 and 100 nm) of TiO2 against A. baumannii , they were found that 50 nm particle size was exhibited the best effect at concentration (25,000 μg/ml ), while a higher concentration (50,000 μg/ml) was needed when they were used 10 nm and 100 nm NP to reach the same antibacterial activity. TiO2 nanoparticles due to their small size and high surface to volume ratio undergo a higher level of interaction with the bacterial cells surface than the larger particles, resulting a high antibacterial activity (51). According to several studies, it was believed that the metal oxides carry the positive charge while the microorganisms carry negative charges; this causes electromagnetic attraction between microorganisms and the metal oxides which leads to oxidization and finally death of microorganisms, they are cause pits or holes of bacterial cell wall could be associated with internalized particles, leading to increase of permeability and cell death (52,53). One of the principal mechanisms of TiO2 action is the production of reactive oxygen species (ROS), when exposed to light at the pro per wavelength during the photo catalysis process on its surface (54, 55). The search for new antimicrobial substances has been directed on metal oxide nanoparticles. Specifically, titanium dioxide (TiO2) as an attractive antimicrobial compound; because it is non-toxic (widely recognized as safe substance), chemically stable, inexpensive and also due to its photocatalytic nature (56). The biosynthesis approaches are often ref erred to as "green synthesis," which were based on divers biologically available natural resources, including live plants (57), plant extracts, algae, fungi, yeasts (58) and bacteria for nanoparticles synthesis. These biological processes were regarded as safe, cost effective, biocompatible, un toxic, stable and environmentally friendly (56).

Conclusions
According to our knowledge this is a first study in KRG-IRAQ used Prodigiosin for Biosynthesis of TiO2 nanoparticles which showed a promising antibacterial activity against strong biofilm producing MDR-A. baumannii.