Efficacy of Isolated Bacteriophage Against Biofilm Embedded Colistin-Resistant Acinetobacter baumannii

Objective: Acinetobacter baumannii is responsible for most nosocomial infections in hospitals. It has the ability to form biofilms and has a high degree of antibiotic resistance. Colistin is one of the last therapeutic options for the treatment of Multi Drug Resistance infections. Recently, strains of this pathogen resistance to the colistin were reported increasingly. Therefore, alternative antibacterial methods such as phage therapy are being researched. Results: From 15 MDR A . baumannii clinical isolates, 26.6% were resistant to colistin, 80% were able to produce strong biofilm, and 20% produce weak biofilm. The isolated lytic phage (IsfAB78) was able to reduce the biofilm by up to 87%. Since most of the MDR colistin-resistant strains produce biofilm, and MDR A . baumannii infections are difficult to treat, development of phage therapy could be an alternative in the future. Phage IsfAB78 is a good candidate for this purpose.

resistant to all known antibiotics, which has urged to find an alternative approach to treat MDR bacteria [11]. In recent decades, increasing evidence has shown the possibility of phage therapy to treat drug-resistant bacterial infections [12]. Bacteriophages are present in all the environments containing their host and play an important role in biological activities [13]. Phages have bacteriolytic performance and can kill bacterial cells in biofilms [14]. The most important characteristics of phages include their specificity and proliferation within the host and at the site of infection, with no side effects [15].
In this study, we found out the biofilm formation of clinical isolates of colistin-resistant. Acinetobacter baumannii, and assessed their sensitivity to the isolated bacteriophage.

Main Text Collection and identification of isolates
In this study, all 15 A. baumannii clinical samples, were collected from patients with burn wound infections at Isfahan Medical University hospitals during the years 2017-2018. All A. baumannii isolates were confirmed by conventional phenotypic and molecular methods [16].

Determining the Minimum Inhibitory Concentration
Minimum Inhibitory Concentration (MIC) for Colistin (Sigma-Aldrich, USA) was performed using the standard microdilution broth technique according to CLSI guidelines (Clinical and Laboratory Standards Institute). The MIC was defined as the lowest concentration of antibiotic that inhibits the growth of bacteria after overnight incubation. The test was repeated three times for each isolate. E. coli (ATCC 25922) was used as a positive control [11].

Biofilm formation
Acinetobacter baumannii biofilm formation was carried out in 96-well flat-bottomed polystyrene tissue culture plate (SPL, Korea). Isolates were cultured for 48 hours in 5 ml tryptic soy broth (TSB) supplemented with 1% glucose. The cultures were diluted to 1: 1000 (equivalent to 10 8 CFU), and 200μL of each aliquot were added to each well and incubated at 37° C. After 24 h of incubation, the plates were rinsed three times thoroughly with normal saline (NS) and allowed to dry at room temperature for 1 hour. Finally, they were stained with crystal violet (1%) and their optical density was measured at 570 nm. The test was repeated three times [17,18]. Biofilm formation was classified into 4 categories according to the amount of optical density: (1) strong (4OD control < OD test ); (2) medium biofilm producer (2OD c < OD t 4OD c ); (3) weak biofilm producer (OD c < OD t ≥2 OD c ); and (4) non-biofilm producer (OD t ≥OD c ) (22). Finally, three colistin-resistant MDR-AB isolates, with strong biofilms, were selected and used in experiments.

Phage Isolation
IsfAB78 is a lytic phage isolated from hospital wastewater. The characteristics of this phage are described in our previous study (23). Briefly, 50 ml of water samples from hospital wastewater were centrifuged at 13,000 rpm for 10 min. The supernatant was filtered through a 0.45μm pore size membrane and mixed with 50 ml of 2x nutrient broth (containing MgSO 4 , 1mM CaCl 2 ,1 mM) and 1 ml (10 8 CFU) of a 24-hour culture of the indicator bacteria. After 24 h incubation at 35 ° C and 160 rpm, a few drops of chloroform were added, and 15 minutes later the mixture was centrifuged for 10 minutes at 4000 g. The supernatant was filtered through a 0.45 pore filter membrane. The doublelayer agar and spot test methods are used to detect phage plaque formation [19].

Double-Layer Agar
Melted nutrient agar medium (1.15%) was poured on a plate, and after solidification, 1 ml of the filtered phages were mixed with 1 ml of 24-hour bacterial culture (10 8 CFU) and added to liquid nutrient agar medium (0.7%) with the temperature of 45 ° C, and the mixture was placed on the plate containing nutrient agar. The solidified plate was incubated for 24 hours at 37 ° C. The clear plaques were picked by sterile loops and inoculated into 5 ml of nutrient broth supplemented with fresh cultured bacterial suspension. After 24 h at 35°C and 160 rpm, the mixture was centrifuged at 4000 g for 15 min and the supernatant was filtered through a 0.2 µm syringe filter. The filtrate was processed through double-layer assay as described above and again an individual plaque was picked. This step was repeated several times in order to increase the purity of the phage [19,20]. The titer of the isolated phage was determined by standard plaque assay and stored at 4°C for further use [21].

Spot Test
Briefly, 1 ml of bacterial culture at the concentration of 10 8 pfu/ml was inoculated in 2.5 ml of the melted nutrient agar medium (0.07%) and the mixture was poured onto a plate containing 1.5% nutrient agar, where two-layer plates were created. When the top layer was solidified, 10 µl of the filtered phage were spotted in the plate and incubated at 37 ° C for 24 hours. The created plaque indicates bacterial susceptibility to the phage [22].

Optimum phage eradication concentration
The antibacterial activity of the isolated phage in the biofilm was measured using different concentrations of the phage. Optimum phage eradication concentration indicates the best concentration of the phage leading to clear wells in 96-well culture plates. For this experiment biofilm was grown according to the described method. After the biofilm formation, the planktonic cells were discarded and each well was washed 3 times with normal saline (NS). A total of 3× 10 9 PFU, 3× 10 8 PFU, 3× 10 7 PFU, 3× 10 6 PFU, 3× 10 5 PFU equal to MOI of 10, 1, 0.1, 0.01, 0.001 in a total volume of 200μl of the diluted phage were added to each well. The plate was incubated for 48 hours at 37 ℃.
The well-containing biofilms and not added phage were used as a negative control, and wells containing the medium were used as sterility control.
Evaluation of antibacterial of the phage activity against biofilm Finally, MTT assay (3-[4.5-dimethylthiazol-2-yl] -2.5-diphenyltetrazolium bromide) was used to determine the biofilm formation [23]. For this purpose, bacterial suspension was removed and 50μl of MTT (0.3%) in 150μl of PBS was added for 2 hours at 37°C. Afterward, the MTT solution was removed and 150μl DMSO lysis solution was added into each well and kept at room temperature and in a dark environment for 30 minutes. The absorbance of the solution was measured at a wavelength of 550 nm using a Microplate Reader Model 550 (BioRad). In order to measure the individual biofilm formation of each isolate, the ratio of the biofilm OD of the isolate that was incubated with the phage was calculated in relation to the biofilm OD of the same isolate without phage (native biofilm).

Minimum inhibitory concentration
The MIC of the isolates against the colistin antibiotic was done according to CLSI.  Biofilm formation The results of biofilm formation in the microtiter plate showed that 20% of isolates formed weak biofilm, 40% isolates created intermediate biofilm, and 40% produced strong biofilm (Fig. 1).

Phage Identification
Phage IsfAB78 was isolated from hospital wastewater (23). MDR-AB biofilm used as a host indicator for the isolation of lytic phages. As shown in Fig. 2

Effect of Phage on Biofilm of Different Isolates
Three colistin-resistant MDR A. baumannii isolates were selected to assess the potential of the phage to disintegrate biofilms. The established biofilm was treated with different concentrations of phages.
The results were represented as a percentage of the biofilm biomass in control samples that were left untreated. The lytic phage seemed to be able to reduce the biofilm (19-87%) but the percentage of biofilm reduction was dependent on the concentration of added phages. At low concentration (10 6 pfu/ml), the reduction in biofilm was significant (up to over 87%) (Fig. 3 (supplementary)).

Discussion
Treatment of bacterial infections involving biofilm formation is very difficult, especially for multidrugresistant pathogens. Therefore, there is an urgent need to develop new alternative treatments to combat such increasing infections. As a nosocomial pathogen, the multi-drug resistant Acinetobacter baumannii (MDR-AB) illustrates an increasing global health threat [24]. Colistin is now considered as the last resort treatment for gram-negative bacilli including MDR-A. baumannii. Unfortunately, the increasing use of colistin has resulted in the emergence of resistance as well [22][23][24][25]. Therefore, looking for an alternative therapeutic method is imperative. Bacteriophage therapy may provide new treatment strategies to combat drug-resistant bacterial infections associated with biofilm. Phages have demonstrated to damage biofilm by disintegrating its structural components [26].
In this study, a specific lytic phage was isolated from hospital wastewater. In similar studies, lytic bacteriophages are isolated on different bacteria, and also investigate their effects and properties on the considered bacteria [27,28]. of the isolates produce biofilm, and the rest were weak biofilm producers. In another study, it was shown that 60% and 62% of their isolates produced biofilm [31,32]. The percentages of the biofilmforming bacteria obtained in our study were slightly more than other researchers because of lower number of the experimented samples. Although the number of samples is effective in the experiment, the increase in pathogenicity of the bacteria cannot be denied. The effect of the isolated phage was evaluated on three biofilm embedded colistin-resistant MDR-AB isolates using the MTT assay. The analysis of the data showed that the isolated lytic phage could reduce the biofilm content of these isolates up to 87%. Bacteriophages have significant applications to degrade biofilm on medical devices' surface. P. mirabilis and E. coli could reduce about a 90% reduction in biofilm when treated with phage as compared to untreated [33]. For all isolates the results were not the same; The phage did not result in total removal of some isolates biofilms. It seems for more effective and complete removal of biofilms, a combination of more than one lytic phage or combination of phage with antibiotics may be useful. The results also showed that different phage dilutions affect the removal of 8 biofilm. The best dilution in all three isolates was MOI 0.01(0 6 pfu/ml). Similar to other studies, this study showed that the isolated lytic phage had an impact on the biofilm of Acinetobacter baumannii [20,22,23,34]. The use of phage has been proposed as an alternative method due to the high drug resistance of Acinetobacter baumannii and bacterial adaptation at hospital levels. Due to the appropriate performance of phage in reducing the biofilm, the phage is suggested as a therapeutic agent for Acinetobacter baumannii. However, for the required therapeutic usage, further studies are needed on animal models, genome identification and its properties, and also on the extent of phage co-administration with antibiotics.

Limitations
The limitations that we confront with that in this study were the lack of several phages to investigate the effect of them on isolates, and also in-vivo and cell culture analysis should do in further investigation. Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request  Spot assay for identification phage on the nutrient agar plate after 24h Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementary.pdf