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Based on the literature (Ghajavand et al. 2017) the best source of A. baumannii phages is hospital sewage. However, procurement and amplification of these phages entail many difficulties. Therefore, it is extremely important to adjust and optimize these methods for each individual phage. The methods for phage isolation and use are presented in Fig. 1.
Multidrug resistance among A. baumannii strains poses serious difficulties for treating infections (Perez et al. 2007). However, phage therapy could be a new weapon in the fight against A. baumannii infection (Sulakvelidze et al. 2001). Bacteriophages have no harmful effects on patients' microbiome and are known to be specific and selective to pathogens. Phage therapy against A. baumannii infection has therefore high potential to be an effective, natural, and safe treatment for patients with serious chronic infections (Clark and March 2006; Międzybrodzki et al. 2012; Van Helvoort 1992). Basic information about bacteriophages against A. baumannii is presented in Table 1.
Phage symbol Family Morphology Source of isolation Number of tested strains Type of life cycle Animal model application References IsfAB78 Myoviridae Six-sided symmetry, 100 nm long Water sample 43 MDR strains (12 of those strains were sensitive to phage) Lytic N.A. Ghajavand et al. (2017) IsfAB39 Podoviridae Six-sided symmetry, short tail and 50 nm head Water sample 43 MDR strains (11 of those strains were sensitive to phage) Lytic N.A. Petty phage Podoviridae Short 15 nm long tail and 60 nm head Sewage 40 strains (25 of those strains were MDR; 4 strains were sensitive to phage) N.A. N.A. Hernandez-Morales et al. (2018) Acibel004 Myoviridae 105 nm long contractile tail and 70 nm icosahedral head and formed 1–2 mm plaques Wastewater sample 34 MDR strains (28 of those strains were sensitive to phage) Lytic N.A. Merabishvili et al. (2014) Acibel007 Podoviridae 10 nm long noncontractile tail, 60 nm head and forms 3–5 mm plaques Wastewater sample 34 MDR strains (28 of those strains were sensitive to phage) Lytic N.A. vB_AbaS_Loki Siphoviridae Isometric 57 nm capsid and non-contractile 176 nm long tail (and 10 nm diameter); short spikes at the tail terminus Sludge 34 strains (2 of those strains were sensitive to phage) Lytic N.A. Turner et al. (2017) SH-Ab 15599 Myoviridae Head with tail (both 88 nm long), formed round, clear plaques (2–3 mm diameter) with haloes Sewage 48 carbapenem resistant strains (13 of those strains were sensitive to phage) Lytic N.A. Hua et al. (2018) SH-Ab 15708 Myoviridae Head (88 nm long diameter) and tail (63 nm long) formed round, clear plaques (2–3 mm diameter) with haloes Sewage 48 carbapenem resistant strains (14 of those strains were sensitive to phage) Lytic N.A. SH-Ab 15497 Siphoviridae Tail (125 nm long and 4 nm wide) and head (55 nm diameter) formed small plaques (0.5 mm diameter) Sewage 48 carbapenem resistant strains (14 of those strains were sensitive to phage) Lytic N.A. SH-Ab 15519 Podoviridae Short, non-contractile, straight tail (18 nm long) and polyhedral, symmetrical head (55 nm diameter) formed round, clear plaques (8–9 mm diameter) with haloes Sewage 48 carbapenem resistant strains (8 of those strains were sensitive to phage) Lytic Mouse model- lung infection; intranasally administration of phage; the survival rate 90% vB-GEC_Ab M-G7was (phi G7) Myoviridae 120 nm contractile tail and 100 nm diameter icosahedral head Sewage water 200 strains (136 of those strains were sensitive to phage) Lytic Rats’ wund model; phage added on the wound; the survival rate 100% Kusradze et al. (2016) PBAB08 Myoviridae Head 180 nm in diameter and 360 nm long tail Bacteriophage Bank of Korea 14 MDR strains (5 of those strains were sensitive to phage) N.A. Mice model- lung infection; intranasal phage cocktail (PBAB08, PBAB25, PBAB68, PBAB80, PBAB93) injection; the survival rate 35% Cha et al. (2018) PBAB25 Myoviridae Head of 80 nm in diameter and 90 nm long tail Bacteriophage Bank of Korea 14 MDR strains (1 of those strains were sensitive to phage) N.A. Mice model- lung infection; intranasal phage cocktail (PBAB08, PBAB25, PBAB68, PBAB80, PBAB93) injection; the survival rate 35% PD-6A3 Podoviridae Short tail (9 nm in length), isometric head (50 nm in diameter); the phage created significant 2–3 mm diameter halos Sewage 552 MDR strains (179 of those strains were sensitive to phage) Lytic Sepsis mouse model; intraperitoneal administration; the survival rate of endolysin therapy group, endolysin + phage therapy group, phage therapy group and phage cocktail (14 phages) therapy group were 70, 70, 60, and 50%, respectively Wu et al. (2019) AB3P1 N.A. 0.8–1.5 mm oval plaques Different regions in Baghdad city including sewage, farm soil, feces of sheep, chicken litter, and swab from surgical lounge of several hospitals in Baghdad 23: 11 extensive- and 12 pan-drug resistant strains (18 of those strains were sensitive to phage) Lytic Mice model; intreperitoneally administration of AB3 phages; the survival rate 100% Jasim et al. (2018) AB3P2 N.A. 0.5 mm oval plaques Different regions in Baghdad city including sewage, farm soil, feces of sheep, chicken litter, and swab from surgical lounge of several hospitals in Baghdad 23: 11 extensive- and 12 pan-drug resistant strains (18 of those strains were sensitive to phage) Lytic Mice model; intreperitoneally administration of AB3 phages; the survival rate 100% AB3P3 N.A. 2.5–4.0 mm round plaques Different regions in Baghdad city including sewage, farm soil, feces of sheep, chicken litter, and swab from surgical lounge of several hospitals in Baghdad 23: 11 extensive- and 12 pan drug resistant strains (18 of those strains were sensitive to phage) Lytic Mice model; intreperitoneally administration of AB3 phages; the survival rate 100% AB3P4 N.A. 35 mm oval plaque Different regions in Baghdad city including sewage, farm soil, feces of sheep, chicken litter, and swab from surgical lounge of several hospitals in Baghdad 23: 11 extensive- and 12 pan drug resistant strains (18 of those strains were sensitive to phage) Lytic Mice model; intreperitoneally administration of AB3 phages; the survival rate 100% Table 1. Bacteriophages against A. baumannii.
Ghajavand et al. (2017) examined bacteriophages against A. baumannii isolated from intensive care units of Ishan Medical University hospitals. The study collected 350 samples including urine, catheter, wound, blood, eye swabs, sputum, and cerebrospinal fluid for A. baumannii isolation. Examination of multidrug resistance of 43 isolates showed that 100% were resistant to ciprofloxacin; 93% were resistant to meropenem, imipenem, ampicillinsulbactam, and cefepime; 91% were resistant to trimethoprim-sulfamethoxazole; 86% were resistant to ceftazidime; 84% were resistant to tetracycline; and 54% were resistant to amikacin. Bacteriophages in these studies were obtained from water samples (environmental samples and hospital waste). After incubation of waste samples from hospitals, clear plaques formed on lawns of AB78 and AB39 bacterial strains. Twelve A. baumannii isolates were sensitive to IsfAB78 phage and 11 were sensitive to IsfAB39 phage. Morphology of both lytic phages was examined with transmission electron microscopy, which indicated that both phages had six-sided symmetry. They significantly reduced the turbidity of the bacterial culture from OD600—2.8 in the control to OD600—0.4 in A. baumannii cultures treated with IsfAB78 or IsfAB39 phage, which suggested that the examined phages have potential for application in phage therapy. Moreover, studies by Hernandez-Morales et al. (2018) resulted in isolation and characterization of Petty phage obtained from sewage. The latent period for this phage was 25 min and burst size was estimated as 240 particles. Among 40 Acinetobacter strains tested, 4 were multidrug resistant (resistance to cefazolin, cefotaxime, chloramphenicol, and tetracycline) and susceptible to Petty phage.
Merabishvili et al. (2014) described two phages (Acibel004 and Acibel007) selected from wastewater samples. Two A. baumannii strains isolated from the nose of a Queen Astrid Military Hospital patient were used for phage amplification. A host range study showed that non-A. baumannii strains (A. nosocomialis and A. pittii) were resistant to both phages. Out of 28 chosen A. baumannii strains, 2 were resistant and 15 were sensitive to the phages. Adsorption and/or propagation of phages was possible on 11 strains. Acibel007 propagated on 61% and adsorbed on 71% of 28 A. baumannii strains, while Acibel004 propagated on 75% and adsorbed on 89% of strains. Based on efficiency of the plating method, phage Acibel007 had higher lytic activity than Acibel004. Moreover, A. baumannii 070517/0072 showed low frequency of mutation during phage application, which suggests low capacity for selection of phage-resistant mutants among bacteria. Maximum adsorption for Acibel007 and Acibel004 was 95% in 10 min and 85% in 15 min, respectively. The latent period and burst size for Acibel007 were 21 min and 145 virions per infected bacterium, respectively, and for Acibel004 they were 27 min and 125 virions per host cell, respectively. Bacteria were incubated either with a mixture of two phages or with a single phage. After 24 h of incubation, OD600 was 0.13, 0.18, and 0.07 for Acibel004, Acibel007, and simultaneous incubation, respectively, compared to 0.42 in an untreated control. OD600 measurements suggested that simultaneous phage application resulted in greater bacterial titer reduction than single phage application.
Turner et al. (2017) characterized another phage against A. baumannii, the bacteriophage Loki (vB_AbaS_Loki), isolated from sludge. The latent period for Loki phage was 40 min and burst size was 43 PFU per infective center. Mutation in A. baumannii ATCC 17978 gene lpxA, encoding a protein required for LPS synthesis, prevented phage adsorption. The authors suggested that an LPS component could be used by Loki as a receptor on the bacterial host. Loki formed turbid plaques (0.5 mm in diameter) on A. baumannii ATCC 17978 and Ca2+ was necessary for plaque formation. Thirty-six out of 38 examined A. baumannii strains were sensitive to Loki phage. Genome analysis showed that Loki has genes gp50 (a putative class I holin) with two transmembrane domains and gp51 containing an N-acetylmuraminidase and a domain that binds peptidoglycan at the C-terminus (Briers et al. 2007).
Hua et al. (2018) looked for phages against MDR A. baumannii clinical isolates. They found that 48 isolated strains were resistant to ciprofloxacin, cefepime, ceftazidime, and piperacillin-tazobactam. The study obtained 30 bacteriophages but only four of them were described: phage SH-Ab 15599, SH-Ab 15708, SH-Ab 15497, and SH-Ab 15519. The phage cocktail containing all these phages was effective against 88% of A. baumannii isolates but no synergic effect was described (i.e., strains resistant to single phage application were also resistant to the phage cocktail). Phages SH-Ab 15599, SH-Ab 15708, SH-Ab 15497, and SH-Ab 15519 were effective against 27%, 29%, 29%, and 17% of A. baumannii isolates, respectively. For phage SH-Ab 15519, an enzyme was suggested as cause of bacterial exopolysaccharide degradation. This phage demonstrated high absorption (90%) within 10 min. Subsequently, the phage was shown to be stable at pH 5 to 12 and temperatures from 4 to 50 ℃.
Kusradze et al. (2016) described another phage against A. baumannii, phage vB-GEC_Ab-M-G7 (phi G7), which was classified in the Myoviridae family (Matsuzaki et al. 2005). The phage proved to be active against 65% of the 200 A. baumannii strains tested (Kusradze et al. 2016). Phage activity was determined at different temperatures and it was observed that incubation at 37 ℃ had no effect on phage activity. After 24 h incubation at 50 ℃, 90% of phage particles were still active but total inactivation was obtained after 24 h incubation at 70 ℃. Moreover, phage phi G7 remained stable when incubated for 24 h in the presence of chloroform and for 5 h at pH from 5 to 11. Phage titer reduction to 103 was caused by 24 h incubation at pH 3.
Cha et al. (2018) examined the sensitivity of 14 MDR A. baumannii strains to nine bacteriophages: PBAB05, PBAB06, PBAB07, PBAB08, PBAB25, PBAB68, PBAB80, PBAB87, and PBAB93. Seven of the tested bacterial strains were sensitive to four or five phages. A. baumannii 28 was sensitive to five out of nine phages. These bacteriophages were used to prepare a phage cocktail, which was used in in vivo studies. Burst size for PBAB25 and PBAB08 was 630 and 215, respectively, and both phages were able to cause bacterial cell lysis 25 min after infection. PBAB08 and PBAB25 showed stability at pH 5–10 and temperatures from 4 to 55 ℃ for 1 h. Moreover, the authors sequenced whole genomes of phages PBAB08 and PBAB25 and they found that the genome of these phages was linear, with dsDNA of 42312 bp (PBAB08) and 40260 bp (PBAB25). Sequenced genomes were compared with reported genomes of phages against Acinetobacter. The results showed that phage PBAB08 had 57% sequence coverage and 99% similarity to AB1 phage (Yang et al. 2010), whereas phage PBAB25 had 78% sequence coverage and 97% similarity to IME_AB3 phage (Zhang et al. 2015).
Wu et al. (2019) presented the characterization of 14 lytic bacteriophages, which were tested on 552 A. baumannii strains. The phage cocktail combining the mentioned phages lysed 54% of 552 bacterial isolates. One of the lytic phages (phage PD-6A3) had a wider lytic spectrum (32% of 552 bacterial isolates) than the other 13 bacteriophages. PD-6A3 bacteriophage was stable at temperatures between 4 and 50 ℃ and at pH from 5 to 10. The burst size for this phage was 129 PFU per infected bacterial cell.
Liu et al. (2019) described another phage against A. baumannii. Phage IME200 formed 2 mm diameter clear plaques with halos. The halo was associated with depolymerase activity. Phage IME200 was able to infect 10 out of 41 A. baumannii isolates and it encoded an enzyme that might be useful in the treatment of biofilm formed by A. baumannii strains. This biofilm is resistant to disinfectants, so the use of phages with depolymerizing genes in their genomes creates a chance for treatment of infections caused by biofilm-forming resistant A. baumannii strains.
In another study, Jasim et al. (2018) isolated 23 A. baumannii strains from patients with septicemia, urinary tract infection, meningitis, wound infection, or pneumonia. Twelve out of 23 isolates were pan-drug resistant and 11 of them were extensive-drug resistant. Pan-drug-resistant clinical isolates are defined as strains causing difficulties in treatment and resistant to all antimicrobial drugs (Göttig et al. 2014). Analyses of bacterial sensitivity to bacteriophages indicated that 111 out of 136 A. baumannii-specific bacteriophages had high lytic properties and formed clear plaques with diameter greater than 3 mm (Jasim et al. 2018). For 10 randomly selected bacteriophages, the burst time ranged from 30 to 45 min and the minimum and maximum burst size was 130 and 245, respectively. Moreover, a phage cocktail (64 bacteriophages specific against A. baumannii isolates) was examined on 23 isolates (AB1-AB23). The results showed that 21 bacterial isolates (except AB2 and AB8) were infected by more than one bacteriophage, and AB3 was infected by six bacteriophages.
Wang et al. (2018) studied the use of phage ϕm18p against A. baumannii clinical isolate KM18. Murine RAW 264.7 macrophages were infected with KM18 and treated with the phage φm18p. After phage application [multiplicity of infection (MOI) 4, 0.4, and 0.04], the cell number was around two times higher than among cells infected with bacteria without treatment. A. baumannii titer in infected cells without treatment was around × log CFU/mL, but in cells treated with phage at MOI of 4, 0.4, and 0.04 it was around 4, 6, and 7 log CFU/mL, respectively.