Characteristic and Potential Therapeutic Effect of Isolated MDR-Acinetobacter Baumannii Lytic Phage

Background: Acinetobacter baumannii is a major pathogen in the hospital, especially in Intensive Care Units (ICU) and the resistance to multiple drugs as a major contributor to hospital infection. Bacteriophages are viruses that attack bacteria and kill them that could be used for clinical treatment. The aim of the study is in evaluating the function of bacteriophage specicity of multi-drug resistant Acinetobacter baumannii, to be used as a useful method for treating of Acinetobacter Infections. Methods: Cross-sectional study during the year 2017, from patients admitted to the ICU, First, 48 isolates of Acinetobacter baumannii were identied by phenotypic method and amplied with blaOXA-51 gene. Then, the sensitivity of phages to pathogens namely ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp) evaluated. DNA of the phage was extracted using the Viral Nucleic Extraction Kit II (Geneaid, Taipei, Taiwan) according to the manufacturer's instructions. Then for protein analysis, PEG-precipitated puried phages were subjected directly to SDS-PAGE, and protein bands were visualized by coomassie Blue G-250 staining method. Finally for cell survival assay we investigated the toxicity of the isolated phage to Hela cells. Results: In the bacterial resistance pattern, the highest resistance belongs to ciprooxacin. In optimal phage test, at dilution of 1 (MOI 1) it produced the best effect on bacteria in 30 minutes. Phage sensitivity to different hosts performed by double layer agar method, the phage was treated with ESKAPE bacteria and after 24 hours’ incubation at 37°C, only for Acinetobacter baumannii Plaque created. The genome analysis indicated that phage pIsf-AB2 has a double-stranded DNA genome. In bacterial control, all cells were killed by A. baumannii, and no live-cell was seen. The cells remained in control of the phage, and the phage did

Isolation, puri cation, and titration of lytic phages For isolating phages, sewage samples were collected from various water sources in Alzahra General Hospital (Isfahan, Iran). The phages were isolated by enrichment method [13]. Brie y, 50 ml of centrifuged sewage supernatant was ltered through a 0.45 µm pore size membrane and mixed with an equal volume of 2x nutrient broth containing 1 ml exponential phase MDR A. baumannii. After incubation at 35 ° C for 24 hours with shaking at 160 rpm, the mixture was centrifuged for 10 min at 13000 rpm, and the supernatant was ltered through 0.45 µm membrane size lter. Subsequently, 200 µl of the ltrate was mixed with 2.5 ml of 0.7% soft nutrient agar in the presence of 100 µl of the log-phase bacterial indicator. Then, the mixture was poured onto a solid medium of 1.5% nutrient agar and incubated for 24 hours at 37 ° C. Double-layer agar method was used to detect plaque formation by phage. Single plaques were picked. A double-layer agar method was performed To obtain puri ed phage strains, several times and plaque picking was repeated until single-plaque morphology was observed [14]. The titer of phage was determined by standard plaque assay [15].

Phage concentration and storage
Each single puri ed plaque was added into 5 ml of nutrient broth containing the MDR-AB indicator (OD600 = 0.6) and cultured at 37 °C for 24 h. Then, the suspension was transferred into 500 ml of nutrient broth and shaken overnight at 35 °C. Chloroform was then added to the to a nal concentration 0.1% mixed gently and allowed to stand at room temperature for 15 min to kill the bacteria. Solid NaCl was added to the culture to a nal concentration of 1 M, mixed and dissolved, and the culture was incubated in an ice bath for one hour. After centrifugation at 10,000 × g for 10 min, and solid PEG6000 was added to the supernatant to a nal concentration of 10% (w/v) mixed and dissolved at room temperature. The solution was incubated overnight on ice to precipitate the phage particles. The pellet was obtained by centrifugation (10,000 × g) for 10 min at 4 °C, and suspended in 5 ml of SM buffer (50 mM Tris-Cl, 100 mM NaCl, 8 mM MgSO4, pH 7.5) [45]. An equal volume of chloroform was then added to separate the phage particles from PEG6000. Following centrifugation at 3,000 × g for 10 min, the aqueous phase was collected and ltered through a 0.22 µm pore-size membrane lter and stored at 4 °C [16].

Examination phage morphology by transmission electron microscopy
Negative staining and TEM electron microscopy were used to observe bacteriophages. A drop of concentrated phage was placed onto a copper mesh grid surface and negatively stained with 2% phosphotungstic acid (PTA). The grid was examined by transmission electron microscopy (Zeiss-EM10c, Germany) at an operating voltage of 100 kV.

pH, thermal and chloroform stability
For pH stability test, 10 10 PFU/ml of the phage was incubated at different pH buffers (3, 5, 7.9, and 11) at 37 °C in SM buffer for 1 hour. The phage titer was determined by the double-layer agar method, as described above. For thermal stability, the phage aliquots were collected after 5 min, 15 min, 45 min, 1hour intervals at different temperatures (37, 50, and 70 °). To determine chloroform stability, 1 ml (1 × 10 10 PFU) of the phage was mixed with 0.4 ml chloroform, and phage was collected and titered after 1hour incubation at room temperature [17].
Determination of MOI MDR A. baumannii cultures in OD 600 = 0.1 (10 8 CFU/ml) were divided into identical aliquots and infected with a serial dilution of bacteriophage stocks and let to adsorb for 15 min. then, free phages were removed by centrifugation at 5000 g for 10 min. Precipitants were resuspended in NB medium, and bacteriophages were tittered after 4 hours of incubation at 30 °C by double-layer method [18].

One-step growth curve
For the one-step growth curve experiment, one milliliter of the MDR A. baumannii (OD600 = 0.1) in the exponential phase was mixed with the phage at an MOI 0.01 and let to adsorb for 10 min. The unadsorbed phages were removed by brief centrifugation (6000 g, 10 min) and 50 µl of the pellet was transferred to 50 ml of NB medium and placed at 37 ° C on a shaker (160 rpm). Samples were collected every 10 min over a time period of 120 min, and the number of phages was immediately assessed by the double-layer agar method [18]. This experiment was done in triplicate.
Phage genome isolation and analysis by restriction enzymes DNA of the phage was extracted using the Viral Nucleic Extraction Kit II (Geneaid, Taipei, Taiwan) according to the manufacturer's instructions. DNA of the phage was digested with the HindIII restriction enzyme (sigma aldrich) according to the manufacturer's protocol. The digested DNA was analyzed by 0.8% agarose gel electrophoresis with 0.5% TBE (Tris-Borate EDTA) running buffer [18].

Phage protein analysis
For protein analysis, PEG-precipitated puri ed phages were subjected directly to SDS-PAGE, and protein bands were visualized by coomassie Blue G-250 staining method [19]. In order to determine the protein of the phage with a lytic activity, we used SDS-PAGE under non-denaturing conditions [20]. Brie y, phage lysates were centrifuged at 14000 × g at 4 °C for 30 minutes. Then, the supernatant was ltered through a 0.22 µm lter and concentrated through centrifugation in 10000 g. The specimen was mixed with protein loading buffer without β-mercaptoethanol. The samples were then loaded on an SDS-PAGE without boiling. After electrophoresis (80 V), the resolved gel was placed onto an agar coated plate, in which soft agar mixed with the MDR AB was previously poured onto the gel [21].

Bacteriophage host range
For determining the phage host range, 3 ml of 0.6% melted agar (50 °C) was mixed with 200 µl of 10 8 CFU/ml of the phage and poured onto a plate which was previously coated with 1.5% solid agar. After agar was solidi ed, 10 µl of the ltered phage was spotted on each plate with A.baumannii clinical isolates, P. aeruginosa (ATCC 27853 ), E. coli (ATCC 25922 ), K. pneumonia (ATCC 10031). The appearance of lysis plaques was investigated after 12 h [22].

Bacterial reduction assay
For this assay, we used a previously described method [23]. One milliliter OD600 = 0.1 culture of the bacterial indicator was inoculated to two separated asks containing 100 mL nutrient agar. One vial was inoculated the isolated phage, and the other one was taken without phage as a negative control. The cultures were incubated at 35 °C at 160 rpm. The optical density (OD 600) of samples was measured at 20 min intervals for 4 hours [24].

Determination of Phage Titer
Bacteriophage dilutions of 10 serial dilutions were prepared, and then each dilution was equally mixed with bacteria and placed in an incubator at 35 ° C and 160 rpm for absorbance. Then centrifugation was performed for 10 minutes, and the precipitate was remixed in the Nutrient Broth medium. After 8 hours of incubation at 37 ° C, the bacteriophage titer was determined [15].

Cells survival assay
We investigated the toxicity of the isolated phage to Hela cells. HeLa cell line (ATCC CCL-2) was obtained from the National Cell Bank of Iran, Pasteur Institute of Iran (Tehran, I.R. Iran). The cells were plated (1.5 × 10 4 well/plate) in 96-well plate in the presence of 100 µl Dulbecco's modi ed eagle's medium (DMEM, Gibco, USA) supplemented with 5% fetal calf serum (FCS; Gibco, USA), at 37 °C in 5% CO2 (15). When cells reached 70-80% con uent, 10 6 CFU of A. baumannii (AB-2) was added to each well, followed by the addition of the phage at different MOI (0, 0.01, 0.1, 1, 10). As a control, Hela cells were treated with 10 8 PFU of phage without the addition of A. baumannii. In a separate experiment, the cells were rst infected with 10 6 CFU of AB-2. After 2 hours, the phage was added to the infected wells. The cells were incubated for an additional 24 hours, and the number of living cells was counted using trypan blue [18].

Results
Bacterial identi cation and antibiotic susceptibility pattern All 48 isolates, which were phenotypically identi ed as A. baumannii, harbored the bla OXA−51 gene ( Fig. 1).
Based on the pattern of A. baumannii (Fig. 2 ), 82% showed resistance to amikacin, 97% to cefepime, 96% to ceftazidime, 99% to cipro oxacin, and 82% to rifampin. Few samples had the intermediate resistance pattern, while susceptibility was not found among them.
Isolation of lytic phages MDR A. baumannii strain, MDR-AB2, isolated from catheter of a patient with pneumonia at Alzahra hospital, was resistant to several antibiotics (table 1). The bacteria were used as an indicator to screen bacteriophages in sewage samples of the same hospital. The isolated phage named pIsf-AB2 forms clear, round, 2-3 mm plaques in the double-layer agar (Fig. 3). The morphology of pIsf-AB2 was examined by negative staining of the phage and observation by electron microscopy. The phage had an icosahedral head of 70 ± 10 nm and a tail of about 60 nm (Fig. 4). The phage belongs to the order Caudovirales and family Myoviridae following the current guidelines of the ICTV (International Committee on Taxonomy of Viruses, http://ictv.global/taxonomyRelease.s.asp).

Phage Growth Curve
One-step growth experiment showed that the latent period of pIsf-AB2 was about 30 min and was followed by the lysis phase, which lasted for 70 min. The burst size was 120 PFU per infected cell (Fig. 5 ).
The optimal phage titer For determining the optimal MOI of pIsf-AB2, different MOIs of the phage were inoculated into AB-2 (10 8 CFU/ml). As shown in the Fig. 6 The pIsf-AB2 with MOI of 1 reduces MDR AB-2 from 10 8 CFU/ml to The results indicate that although higher MOI reduced A. baumannii more quickly, but is not necessary for lysis.

Reduction bacterial assay
Infections of A. baumannii with high titer of lysate was monitored for 7.5 h. Phage infection signi cantly decreased the A. baumannii culture turbidity in comparison to control. However, an increase of the turbidity (OD600) was observed after about 4 hour of culture incubation. This increase of turbidity was most probably due to the growth of phage-resistant bacteria (Fig. 7).

Phage stability
The stability of the pIsf-AB2 to different pH, chloroform, and the temperature was tested. The phage pIsf-AB2 lost its infectivity at pH 3 and 11, while pH 7 is the most suitable condition to maintain the phage. The phage was stable at different pH ranging from − 20 to 25 °C. However, the phage titer was slightly dropped at 50 °C and reduced dramatically at 70 °C. The activity of the virus was not affected by chloroform treatment.

Bacteriophage host range
Host range spectrum surveyed on forty eight A. baumannii strains and showed that the pIsf-AB2 phage was able to infect 56.3% of the A. baumannii strains. The results demonstared that phage was speci c for the A. baumannii and didn't has any effect on Klebsiella, Psodomonas, E .coli.

Genome size and protein pro les
The genome analysis indicated that phage pIsf-AB2 has a double-stranded DNA genome ( approximately 12.6 kb). The genome of pIsf-AB2 could be digested by HindIII endonuclease ( g.8). It was found that HindIII has three cutting site. Although, endonucleases , HincII have no cutting site. The puri ed phages were denatured in loading buffer (containing SDS and 1% β-Mercaptoethanol) and heated in boiling water for 5 min. The phage proteins were separated in SDS-PAGE. The results pIsf-AB2 has nine structural protein bands in 10% SDS-PAGE, with a molecular weight ranging from 14.5 to 150 kDa. The most abundant protein band in the gel above 35 kDa, which was assumed to be the phage putative capsid protein ( g.9 ).

Phage endolysin activity
The proteins of pIsf-AB2 were loaded on SDS-PAGE without boiling as describes in the materials and methods. The AB-2 overlay on SDS-PAGE showed a clear band at 15 kDa (Fig. 10).

Cell survival assay under bacterial infection
The safety of phage was examined in the Vero cells line in the presence of MDR-AB-2. In 12 primary wells, we added phage in different dilutions MOI = 0, 100, 1, 0.1, 0.01, separately to each well, and the bacterium was combined with a concentration of half Mac Farland. A well was considered as a cell control, a phage control well, and a well was considered as control of bacteria.
The plate was incubated for 24 hours, then counted by neobar cell count; in all dilutions, the living cells were observed, and by decreasing the dilution of the phage, the number of live cells decreased, so the lowest cell viability was found at dilution of 0.01. In bacterial control, all cells were killed by A. baumannii, and no live-cell was seen. The cells remained in control of the phage, and the phage did not affect the cells (diagrams 1 and 2).

Discussion
The aim of our study was to demonstrate a new approach for the usage of the bacteriophage for treating a life-threatening MDR A. baumannii infection. Growth under challenging conditions and prolonged shelflife in dry conditions, as well as resistance to most commonly used antibiotics, A. baumannii has become an essential and effective pathogen in the hospital [25]. Similarly, this bacterium is found as the cause of the spread of several diseases worldwide [26]. The importance of this bacterium and the increasing resistance to antimicrobial agents necessitates more veri cations. In this study, 48 isolates were tested with an antibiogram and tested for resistance patterns using the CLSI guidelines. They had multidrug resistance (MDR), with the highest resistance to 100% ampicillin, 99% cipro oxacin, 97% cefepime, and Ceftazidime being 96%; resistance to amikacin was 82%, and rifampin was 86%. According to a study by Sadiqifard et al. in 2006 in Tehran, resistance to A. baumannii has been 100% to ceftazidime and 52% to amikacin [27]. Another study by Henwood et al. in the United States, of 649 isolates from different hospitals; 595 isolates of A. baumannii were diagnosed and cipro oxacin and ceftazidime resistance, was 30% and 70% respectively [28]. In our study, the resistance to these antibiotics was 99% and 96%, respectively. The results of the antibiotic resistance pattern obtained in this study are in contrast to other studies, which can be due to the diverse clinical samples, therapeutic methods in different regions, and the time of the survey.
In similar studies in different locations, the MDR rate has increased in all countries, and resistance of A. baumannii to antibiotics has been rising, exhibiting that resistance levels vary across different geographic areas. In the study of Simhon et al., A. baumannii resistance to cipro oxacin from 49% in 1990 increased to 87% in 2000, which shows elevating the antibiotic resistance [29].
Bacteriophage, commonly known as phage, is de ned as viruses that infect bacteria. Phages are present everywhere and require a bacterial host for this presence [30]. Phage therapies were used for human beings for the rst time in 1915 by d'Herelle, which received signi cant results from early phlebotomy experiments [31]. The use of bacteriophages for phage therapies is the use of lytic phages that infect bacterial cells and eliminate them by lysis [32]. Unlike antibiotics that may affect body ora, phages target the host bacteria and do not affect other bacterial species, which present the speci city of this [33].
Most categorized Acinetobacter phages are tailed viruses with double-stranded DNA genomes. They are arranged into three families, including Myoviridae, Podoviridae, and Siphoviridae that are in the order of Caudovirales [18,23].
Tailed bacteriophages make their progeny to protrude the cells and infect other cells with endolysins which cause to breaking down the bacterial cell wall. In a study by Regeimbal JM et al. in the United States, they were able to separate the A. baumannii particular phage from the sewage and, by examining the electron microscope, stated that the phage had a shaft-shaped head and a contractile tail, and was in Myoviridae group [34]. In the study we performed, we were able to isolate the phage from sewage by hosting A. baumannii. Placing a phage in a plate on an A. baumannii, a zone of the plaque was formed and examined by morphology with an electron microscope, a 6-cupsid, fractured, and constricted phage of the myoviriridae family in which morphological results overlapped with Regeimbal JM studies. Another study by Merabishvili M in Belgium in 2014, the phage of A. baumannii was isolated from the river. By microscopic examination, a 6-cupsid head and taut phage were introduced from the myoviridae family [10].
A study by Kusradze in 2016, the bacteriophage separated from A. baumannii from sewage, was introduced by microscopic examination as the myoviridae family. They also investigated the phage secretion stability at different temperatures, pH, and chloroform, which stated that after 24 hours, the phage incubation was maintained at 37 ° C, 100% of the phages. At 50, 90% of the phage was retained, and at 70 ° C, the phage was not observed. Also, it was stable in exposure to chloroform and had no phages at pH 5 and 11 (19).
Broadly speaking, high pH stability and high thermal resistance made phage remarkably pledged for practical usage in deracination of A. baumannii contaminations and or treatment of A.baumannii infections. In our study, we investigated phage at different temperatures, pH and chloroform stability, and after 24 hours of inoculation, the phage exhibited a steady-state for chloroform; and at 37 ° C temperature, the best effect was on bacteria which the phage titer was not diminished; in different pH analyzes, phages at pH 5 and 11 disappeared. These results are consistent with the results of the Kusradze study. Thermal resistant phages were usually isolated from extreme thermal habits [31,32], but they could also be found in other environments. Recently, thermal resistant phages have been isolated and characterized from various dairy products [33,34]. There are several possible explanations for thermal an pH resistance of phages that one of the main reason is lysin (endolysin). Most doublestranded DNA phages accomplish host cell lysis through the holin-endolysin system. The similarity of bacteriophage endolysin genes is essential for structural analysis, which contributes to the potential of using endolysin as an antimicrobial agent.
The endolysins antibacterial activity is generally attribute to their enzymatic function, which make, rupturing of the covalent bonds in peptidoglycan. However, some endolysins, especially those from phages of Gram-negative bacteria, are capable of affecting bacterial cells by means of a mechanism completely independent of their enzymatic activity (During et al. 1999; Orito et al. 2004). in our study the protein that caused lysis estimated with SDS PAGE about 15 kDa that corroborate with previous results. The lysin is utterly stable and constant over a wide range of pHs. A number of concerns have been raised about the potential toxicities and the practicality of bacteriophage therapy for MDR bacterial infections. However, in this particular case, we overcame these hurdles and did not observe any discernible adverse clinical events.

Conclusion
The results of this study highlighted that A. baumannii resistance has been increased, and treatment of infections caused by it was painful and should be considered as an appropriate alternative to antibiotics. Considering the results of the effect of the phage on A. baumannii, which caused the bacterial lysis in the plate as well as the results of cell culture, the phage treated the bacteria infected with the cells, as well as other experiments including optimal phage titers for reducing the concentration of the bacteria, during 30 minutes, signi cantly reduced the level of bacteria. According to these results, bacteriophages can be used as a useful therapeutic alternative to antibiotics to combat MDR bacterial infections.

Declarations
Ethics approval and consent to participate 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.

Competing interests
The authors declare that they have no competing interests Funding This study was nancially supported by Isfahan University of Medical Sciences, Isfahan, I.R. Iran through the Grant No 396188.
Authors' contributions BS was a signi cant contributor to doing and writing the manuscript. AM , MS , NH and VK collaborated in doing the thesis that results in the paper.SM Designed and supervised the manuscript. All authors read and approved the nal manuscript.