Keywords: multidrug-resistance,Acinetobacter baumannii, bacteriophage therapy, mouse model, genome analysis

Ethics statement

All animal studies were approved by and followed the guidelines and regulations of the Ethical Committee for Animal Experiments of Hankuk University of Foreign Studies (approval number 2017-0001).

Bacterial strains and phages used

Fourteen clinicalA. baumannii strains were obtained from patients in Samsung Medical Center, Sungkyukwan University School of Medicine. Antibiotic resistance profile of the strains is shown in Table1. To determine the genotypes of theA. baumannii isolates, Oxford scheme multilocus sequence typing (MLST) was performed as described previously (Bartual et al.,2005;http://pubmlst.org/abaumannii/info/primers_Oxford.shtml). One of the strains (strain 28) was selected and further used for mice experiment. It was resistant to both kanamycin (3.5 mg/ml, Sigma, USA) and ampicillin (5 mg/ml, Sigma, USA), and this fact was used to enumerate recovered bacteria from mice lung. The 9 bacteriophages tested against the 14 strains were PBAB05, PBAB06, PBAB07, PBAB08, PBAB25, PBAB68, PBAB80, PBAB87, and PBAB93. Bacteriophages used for making the therapeutic cocktail were PBAB08, PBAB25, PBAB68, PBAB80, PBAB93 (Bacteriophage Bank of Korea).

Phage isolation and purification

Bacteriophage isolation and characterization were described thoroughly (Clokie and Kropinski,2009a,b; Clokie et al.,2018). The isolation and characterization of phages have been described previously (Kim et al.,2013). The phages were purified using a glycerol gradient centrifugation method (Sambrook and Russell,2006). A single plaque was used to inoculate 5 ml of a mid-exponential-phase culture of the bacterium, followed by incubation at 37°C for 3 h. A phage lysate was obtained by centrifugation at 11,000 xg for 10 min and discarding the supernatant. Five ml of the lysate was used to inoculate 100 ml of a mid-exponential-phase culture of the bacterium, and the mixture was then incubated until lysis was completed. NaCl was added to the lysate at a final concentration of 1 M, and it was incubated at 4°C for 1 h. After centrifugation at 11,000x g for 10 min, 10% (wt/vol) polyethylene glycol 8000 (PEG 8000) was added, and the mixture was then incubated at 4°C for 1 h. The supernatant was discarded after centrifugation at 11,000x g for 10 min. Then the pellet was resuspended in 750 μl of SM buffer [100 mM NaCl, 8 mM MgSO4·7H₂O, 50 mM Tris-Cl (pH 7.5)], and chloroform was added at a ratio of 1:1 (vol/vol), followed by vortexing and then centrifugation at 3,000x g for 15 min. The upper phase was isolated and was added to a polycarbonate centrifuge tube containing 3 ml of 40% glycerol in the lower layer and 4 ml of 5% glycerol in the upper layer. After centrifugation at 151,000x g for 1 h, the supernatant was discarded, and the pellet was resuspended in 400 μl of SM buffer. For animal experiments, any contaminating lipopolysaccharide (LPS) was removed before treatment. Triton X-114 (Sigma, USA) was added to phage solution at a final concentration of 1% (vol/vol) and mixed using vortex for 10 s. After incubation on ice for 10 min, the mixture was transferred to 37°C water bath and incubated for 1 min. After a centrifugation at 15,000x g for 5 min, supernatant was collected and used as the final phage solution. Purified phages were stored at 4°C until use. Standard double agar overlay plaque technique was used for phage enumeration (Kropinski et al.,2009).

Transmission electron microscopy (TEM) of phage particles

Purified phage sample was loaded onto a copper grid for 1 min followed by negative staining with 2% (vol/vol) uranyl acetate (pH 6.7) and drying. The phage morphology was observed using a Carl Zeiss LIBRA 120 EF-TEM (Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 120 kV.

Genome sequencing, annotation, and analysis

Whole genome sequencing of phage DNA was carried out using PacBio RS II system in Macrogen, Korea. Open reading frames (ORFs) were searched using NCBI ORF Finder. Genomic sequence similarity comparison was done using MAUVE. ORF map was drawn using CLC Genomics Workbench 10. Genomic tree was drawn using Mega 7.

One step multiplication

One step growth experiment was described previously (Hyman and Abedon,2009). Briefly, phage PBAB08 or PBAB25 was added to a fresh culture ofA. baumannii at an MOI 0.001 and allowed to adsorb for 5 min. The mixture was then centrifuged at 1,738 xg for 10 min. The supernatant was discarded to remove any free phages and the pellet was resuspended in 3 ml of fresh LB broth incubated at 37°C with shaking. One hundred micro liters of sample was removed from the culture every 5 min and subjected to titration using double-layer agar plate methods (Kropinski et al.,2009). This assay was performed at least in triplicate.

Phage stability

For temperature stability test, phage titer was measured after incubation of phage lysates for 1 h at different temperatures (4, 37, 45, 55, 60, 65, or 80°C) using a double-layer agar plate method (Kropinski et al.,2009). For pH stability test, phage titer was measured after 1 h's incubation of phage lysates at room temperature by mixing with equal volume of pH buffer solutions of different pH values (pH 3, 4, 5, 6, 7, 8, 9, 10, and 11). pH buffer solutions were made by adding HCl drop by drop to 1 M NaOH solution. The resulting pH was measured using S220 seven compact™ pH/Ion (Mettler Toledo, Columbus, USA).

Phage protection studies using a mice model

Six week-old female Balb/c mice were obtained from Young Bio, Korea. Mice were divided into 4 groups; Group 1 was treated with SM buffer only. Group 2 was infected with MDRA. baumannii (strain 28) only. Group 3 was treated with the phage cocktail only. Group 4 was infected withA. baumannii and treated with the phage cocktail. Each group contained 20 mice. Mice were intraperitoneally injected with cyclophosphamide (Sigma, USA) at the concentration of 150 mg/kg at −1 and −3 days of bacterial infection. Before intranasal bacterial or phage injection, mice were anesthetized with 125 mg/kg of avertin (Sigma, USA) by intraperitoneal injection. 1 × 10⁸ CFU ofA. baumannii was intranasally injected to each mouse at 0 and +1 days. SM buffer was injected to the control group mice instead. 1 × 10⁹ PFU of phage cocktail was intranasally injected to experimental mice every day from −1 to +7 days. At days 0 and +1, phages were injected 4 h after bacterial injection. Survival of mice was observed until 7 days after bacterial infection. Bacterial load in mice lung was counted as follows; lung was isolated at +3 and +4 days and weight was measured. After homogenization, 500 μl of SM buffer was added and was subjected to centrifugation at 2,000x g. The supernatant was used for countingA. baumannii in a medium containing both kanamycin (3.5 mg/ml) and ampicillin (5 mg/ml).

Cytokine, IgE, and histamine assays

To see immune reactions against phages, the cocktail was introduced every day for 7 days to mice in three different routes; intraperitoneal, intranasal, and oral. Three mice were used for each route. Control group was treated with SM buffer. One day after the last treatment of a phage cocktail, mice were sacrificed and serum was obtained for analysis of cytokine profile using Multi-Analyte ELISArray kits (Qiagen, USA), of IgE profile using RayBio^®; Mouse IgE ELISA Kit (RayBiotech, USA), and of histamine using Histamine Enzyme Immunoassay Kit (SPI Bio, France).

Statistical analyses

Student'st-test (Bailey,1959) was used to calculate the significance of the difference among test groups. For each assay, all determinations were carried out at least in triplicate. Statistically significant values were defined asP < 0.05 orP < 0.01.

Antibiotic resistance of clinically isolatedA. baumannii strains

Fourteen bacterial isolates were selected and their antibiotic resistance and phage susceptibility were observed (Table1). All the isolates were multidrug-resistant (MDR) strains. Many of them were resistant even to colistin or tigecycline. The 14A. baumannii isolates showed five sequence types (STs) based on MLST (Table1), and they belonged to the same clonal complex (CC), global complex II. NineAcinetobacter bacteriophages, PBAB05, PBAB06, PBAB07, PBAB08, PBAB25, PBAB68, PBAB80, PBAB87, and PBAB93, were selected from the Bacteriophage Bank of Korea and tested for infection to the clinicalA. baumannii strains. Seven out of 14 isolates could be infected by four or five phages. The phage susceptibility was not related to antibiotic resistance profile. Isolate 28 was susceptible to all five phages and thus selected for mice infection experiments later. We further characterized two of the phages PBAB08 and PBAB25 from which whole genomic DNA sequences were successfully obtained as single contigs after next generation sequencing.

Bacteriophage characterization

Two phages, PBAB08 and PBAB25, belonged to family Myoviridae of order Caudovirales (Figures1A,B). Their virions were composed of a spherical head and a long, rigid tail. Phage PBAB08 had a head of 180 nm in diameter and a tail of 360 nm in length. The smaller phage PBAB25 had a head of 80 nm in diameter and a tail of 90 nm in length. A complete lysis of host bacteria infected with PBAB08 or PBAB25 occurred in 25 min post infection (Figure1C). Burst size for each phage was 215 and 630, respectively.

The infectivity of PBAB08 and PBAB25 remained intact when exposed to pHs ranging from 5 to 10 for 1 h (Figures2A,B). It decreased rapidly at pHs above 10. The infectivity of both phages remained intact when exposed to temperatures ranging from 4 to 55°C for 1 h and dropped rapidly above 65°C (Figures2C,D).

Genomic sequence analysis

The whole genomes of phages PBAB08 and PBAB25 were sequenced (GenBank accession numbersMG366114 andMG366115, respectively). Their genomes were double stranded linear DNAs of 42,312 base pairs and 40,260 base pairs, respectively. The genome sequences were compared to reportedAcinetobacter phages using BLASTN. PBAB08 showed a 99% similarity with 57% sequence coverage to phage AB1 (Yang et al.,2010), and PBAB25 showed a 97% similarity with 78% sequence coverage to phage IME_AB3 (Zhang et al.,2015). BLASTN significant alignment coverage of all other known phages were <30%. A mosaic structure between the genomes of PBAB08 and AB1, and that of PBAB25 and IME_AB3 were observed (Figure3). An extensive rearrangement occurred between genomes of PBAB08 and AB1, while genomic structure was conserved between PBAB25 and IME_AB3.

Seventy six and seventy genes encoding phage proteins were found in the genomes of PBAB08 and PBAB25, respectively (Figure4). Functional annotation of putative ORFs of each phage is shown in Tables2,3. They are divided into four categories according to functions; structural proteins, those involved in DNA packaging, those involved in replication and regulation, and those involved in lysis. It was evident that sequence similarities for both pairs were more prominent in virion proteins than in proteins involved in replication and regulation. It is not fair to compare virion proteins of the two pairs in parallel since the extent of annotations were different. Nevertheless, it is notable that PBAB25 had a wide array of tail proteins suggesting a complicated tail structure. Since both phages had their own putative DNA polymerases, a phylogenetic tree of closely relatedAcinetobacter phages in GenBank was drawn based on the sequence comparison of their DNA polymerase genes (Figure5). The phages could be grouped as two, in which PBAB08 belonged to one group (upper 10 phages in Figure5), while PBAB25 belonged to the other group (lower 10 phages). No DNA polymerase was found in functional annotation of ORFs of phage AB1, thus it was not included in the tree.

In vivo efficacy of phage cocktail

Only 15% of mice infected withA. baumannii intranasally survived at 7 days post infection (Figure6A). On the contrary, 35% of mice infected with the bacteria followed by treatment with the phage cocktail survived. A better survival of phage-treated mice was observed at day 4 post infection. Sixty percentage of mice survived with phage treatment while 20% of mice survived with only bacterial infection. Mice treated with phage only remained healthy for the entire experimental period.

Reduction of bacterial load in infected lung of mice was observed (Figure6B). At 3 and 4 days post bacterial infection, bacteria resistant to both kanamycin and ampicillin were counted. Double-resistant bacterial count was reduced more than 100-fold for both days. Nevertheless, it was clear that a complete elimination ofA. baumannii was not achieved with this phage treatment.

Immune reactions against the phage cocktail

Phages are foreign substances to mice and there are chances they could elicit immune responses. We checked changes in serum IgE level indicating any allergic responses for three different routes of phage injection (Figure7A). A 20% increase in serum IgE was observed for intraperitoneal injection route, while no significant change was observed for either intranasal or oral administration. For cytokines, a slight increase of serum GM-CSF was observed for all three routes of phage administration (Figure7B). In intraperitoneal route, slight increases of IL2, IL10, and IL17A were also observed. Thus, inflammatory response against the phage cocktail was minimal. No significant change in histamine level was observed when phage cocktail was administered (Supplementary Figure 1).

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Supplementary Materials