A total of 105 clinical isolates of Acinetobacter spp. were collected from five hospitals in mainland China between January 2012 and December 2018 ( Supplementary Table 1 ). All strains were isolated from sputum samples of patients and cultured at 37°C in lysogeny broth (LB) media. To confirm the species level of these Acinetobacter isolates, a 305-bp partial rpoB gene was amplified, sequenced, and analyzed as described in Gundi et al. (2009). Furthermore, the antimicrobial susceptibility of A. pittii was determined using a Vitek 2.0 compact system (BioMérieux Clinical Diagnostics, Paris, France). Minimum inhibitory concentrations (MICs) were determined according to the breakpoints recommended by the Clinical and Laboratory Standards Institute. No humans or animals were involved in this study; therefore, ethical approval was not required.

Nucleic acids of the obtained A. pittii strains were extracted using a High-Pure Polymerase Chain Reaction (PCR) Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions and sequenced on an Illumina HiSeq platform (Illumina, San Diego, CA, USA). The obtained raw reads were assembled de novo using the Unicycler v0.4.8 pipeline (Wick et al., 2017). The CPS (KL or K types) of A. pittii was identified using the online tool Kaptive v2.0.0, which was updated in 2021 (Wyres et al., 2020). Multilocus sequence typing (MLST) was performed via the public databases for molecular typing and microbial genome diversity (https://pubmlst.org) using seven housekeeping genes (cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB) (Diancourt et al., 2010).

Phage IME-Ap7 was isolated following the procedure described by Liu et al. (2019b). Briefly, an A. pittii strain with different KL types was selected as the host bacterium for screening bacteriophages. The raw sewage collected from the Fifth Medical Center of the Chinese PLA General Hospital was centrifuged to obtain the supernatant, which was then mixed with the different A. pittii cultures in the exponential growth phase. The mixtures were cultured at 37°C in LB medium for 6 h. After 5-min centrifugation at 10,000 rpm, the supernatants were filtered with 0.22-μm membrane filters. The phage lytic ability was determined through a double-layer agar plate assay to observe the presence of phage plaques, as previously described (Kropinski et al., 2009).

Phage nucleic acids were extracted and purified using the High-Pure Viral RNA Kit (Roche Diagnostics). The obtained nucleic acids were sequenced and assembled, as described above. The phage genomic sequence was annotated using Rapid Annotation in Subsystem Technology (RAST; http://rast.nmpdr.org/), and the putative function of the coding sequences (CDSs) was predicted using NCBI BLASTP. The phage genome was visualized using the online software Proksee (Grant et al., 2023).

Based on the results of the phage genomic annotation, a gene encoding the tail fiber protein (ORF27, GenBank accession number: WRM43609.1) was predicted to have polysaccharide depolymerase activity. The sequence of ORF27 was amplified by using PCR with primers (forward: 5′-CAAATGGGTCGCGGATCCATGACAAACCCAACTTTAG-3′, reverse: 5′-GTGGTGGTGGTGCTCGAGTTATATCAACTTAACGTGA-3′) and cloned into the pET28a vector with restriction sites for BamHI and XhoI using the pEASY^® -Uni Seamless Cloning and Assembly Kit according to the manufacturer’s protocol (TransGen Biotech, Beijing, China). Clones containing inserts were selected using PCR and restriction enzyme digestion analysis and verified through DNA sequencing.

The depolymerase was expressed and purified as previously described (Liu et al., 2019b). Briefly, the recombinant plasmid containing a C-terminal hexahistidine-tag (6×His-tag) was transformed into the Escherichia coli BL21(DE3) and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich, MO, USA) at 16°C overnight. Cells were pelleted at 10,000 rpm for 5 min and lysed by sonication (20 min with a 3-s pulse and a 4-s pause) in lysis buffer (50 mM NaH₂PO₄ and 300 mM NaCl, pH 8.0). Bacterial lysates were centrifuged and passed through a 0.45-μm filter. The expressed protein was purified on a gravity column with Ni-NTA resin according to the manufacturer’s instructions (Sangon Biotech, Shanghai, China). The eluted protein was collected in an 8- to 14-kDa-molecular-mass-cutoff membrane (Viskase, IL, USA) and dialyzed against a 1,000-fold volume of lysis buffer for 24 h. The molecular weight of the purified protein was determined using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was measured using a fluorometer (Qubit 2.0; Thermo Fisher Scientific, Waltham, MA, USA).

The depolymerase activity of Dpo27 against host bacteria was semi-qualitatively determined using a modified single-spot assay as previously described (Liu et al., 2019b). Briefly, A. pittii 7 culture in the exponential growth phase was mixed with molten soft LB agar and poured onto the surface of an LB agar plate. After solidifying, the protein dilution (0.1–2 ng) in 5 μL of PBS was dropped onto the plate, with the same volume of PBS used as a negative control. The plates were observed for the formation of translucent spots while incubating overnight at 37°C.

The host range of IME-Ap7 was determined using a double-layer agar plate assay (Kropinski et al., 2009). Briefly, plates containing a 10-fold dilution of IME-Ap7 and A. pittii strains with different KL types were incubated overnight. The formation of singular phage plaques on bacterial lawns was used to evaluate the lytic activity and host range of isolated phage. Next, bacterial sensitivity to Dpo27 (2 ng) was determined using a modified single-spot assay, as described in the previous section.

Extraction and purification of bacterial exopolysaccharides (EPS) containing both CPS and liposaccharides (LPS) were conducted using a modified hot water–phenol method, as previously described (Hsieh et al., 2017). Briefly, 1 mL of A. pittii 7 cultured overnight in LB with 0.25% glucose was harvested and resuspended in 200 μL of double-distilled water (ddH₂O). An equal volume of hot-water-saturated phenol (pH 6.6; Thermo Fisher Scientific) was added and vortexed vigorously. After incubating at 65°C for 20 min, the mixture was extracted and purified using chloroform to remove bacterial debris. The obtained EPS was lyophilized and stored at −20°C.

The enzymatic activity of Dpo27 against bacterial surface polysaccharides was measured using the 3,5-dinitrosalicylic acid (DNS) method with minor modifications (Oliveira et al., 2017). Briefly, the EPS solution of A. pittii 7 (2 mg/mL) was mixed with Dpo27 or heat-inactivated Dpo27 (100°C for 15 min; 10 μg/mL) to a final volume of 1.0 mL and subsequently incubated at 37°C for 1 h; EPS or enzyme alone, respectively, served as the controls. Next, two volumes of DNS reagent (Solarbio, Beijing, China) were immediately added to each reaction mixture, and the mixtures were boiled for 5 min. The absorbance was measured at 540 nm using a Synergy HT Multi-Detection Microplate Reader (BioTek, VT, USA). The experiment was repeated at least three times.

The degradation of bacterial CPS was confirmed using Alcian blue staining, as previously described (Pan et al., 2013; Hsieh et al., 2017). Briefly, each of the mixtures described above was loaded and separated on 10% SDS-PAGE gel. After running, the gel was washed three times (5, 10, and 15 min) with the fix/wash solution (25% ethanol and 10% acetic acid in water) at room temperature and stained with 0.1% Alcian blue (Sigma-Aldrich) dissolved in the fix/wash solution for 15 min in the dark at 37°C. After the gel was destained overnight in the fixed/washed solution, CPS was visualized as a blue band.

The effects of pH and temperature on the enzymatic activity of Dpo27 were determined as previously described (Liu et al., 2019b). The lyophilized EPS powder of A. pittii 7 (described in a previous section) was resuspended in 50 mM sodium acetate buffer (pH 4.0–5.0), 50 mM Na₂HPO₄ buffer (pH 6.0–7.0), 50 mM Tris-HCl buffer (pH 8.0–9.0), and 50 mM sodium carbonate buffer (pH 10.0–11.0) to a final concentration of 2 mg/mL, and then mixed with Dpo27 (10 μg/mL) to obtain a final volume of 1.0 mL. The mixtures were then incubated for 1 h at 37°C. To test the thermal stability of the enzyme, the EPS powder was dissolved in 50 mM Na₂HPO₄ buffer (pH 6.0) and then incubated with the Dpo27 (10 μg/mL) at different temperatures (20–70°C) for 1 h. EPS solution or enzyme alone was used as a control. Enzymatic activity was determined using the DNS method, as described in the previous section. All experiments were performed in triplicate.

The ability of Dpo27 to enhance bacterial susceptibility to serum killing was determined as previously described with minor modifications (Lin et al., 2017; Pan et al., 2017). Briefly, the overnight A. pittii 7 culture (approximately 10⁷ CFU/mL) was treated with Dpo27 (10 μg/mL) for 1 h at 37°C. The human serum from healthy volunteers or its inactivation (heated at 56°C for 30 min) was then added to the enzyme-pretreated bacteria at a volumetric ratio of 1:3, and the overnight bacteria culture was incubated with enzyme or active serum as control. After incubation for 1 h at 37°C, the mixture was serially diluted and plated for bacterial enumeration. The experiment was performed independently three times.

The hemolytic effect of Dpo27 on erythrocytes was evaluated as described previously, with minor modifications (Wang et al., 2015). Briefly, blood samples from healthy donors were centrifuged (1,000 rpm for 10 min) to collect serum and erythrocytes. Next, the obtained erythrocytes were washed three times and diluted to a concentration of 5% (v/v) with PBS. The erythrocytes were incubated with Dpo27 (10 μg/mL) at 37°C for 1 h with gentle shaking. The erythrocytes treated with PBS or 0.1% Triton X-100 were included as negative or positive controls, respectively. Supernatant (100 μL) was transferred to a 96-well microplate after the sample was centrifuged at 1,000 rpm for 10 min, and another 100 μL of PBS was added to the wells. The absorbance of the hemoglobin was measured at 540 nm. All experiments were repeated in triplicate.

All experimental data are presented as means ± standard deviation (SD), and statistical analyses were performed using Prism 7 (GraphPad Software, CA, USA). One-way analysis of variance (ANOVA) was used to compare multiple groups, with p-values < 0.05 considered to be statistically significant.

To determine the prevalence of different Acinetobacter spp. in the collected isolates, a 305-bp partial rpoB gene of the 105 clinical strains was sequenced and analyzed. As shown in Supplementary Table 1 , the isolates were identified as A. baumannii (72.38%, 76/105), A. pittii (21.91%, 23/105), A. nosocomialis (3.81%, 4/105), and A. soil (1.90%, 2/105). The antimicrobial susceptibility of A. pittii was tested using the Vitek 2.0 compact system. The susceptibility rates of A. pittii to different classes of antibiotics are shown in Supplementary Table 2 . Among the 23 A. pittii strains, 16 (69.57%, 16/23) were non-susceptible to one or more agents in three or more antimicrobial categories (MDR). Moreover, nine strains (39.13%, 9/23) were resistant to imipenem, a type of carbapenem. Furthermore, the KL types and MLST of 23 A. pittii strains were identified. As presented in Table 1 , these isolates were assigned to KL14 (K14; 4.34%, 1/23), KL32 (K32; 4.34%, 1/23), KL38 (8.70%, 2/23), KL111 (8.70%, 2/23), KL163 (47.83%, 11/23), KL207 (17.39%, 4/23), and KL220 (8.70%, 2/23). According to the Pasteur MLST scheme, seven isolates (30.43%, 7/23) could not be typed, whereas other strains belonged to ST63 (52.17%, 12/23), ST119 (4.35%, 1/23), ST205 (8.70%, 2/23), and ST248(4.35%, 1/23).

Seven A. pittii strains (653, 1316, 1178, 1477, 910, 1483, and 7), representative of different KL types, were used as host bacteria to screen for phages. A lytic phage, IME-Ap7, was isolated using KL220 A. pittii 7 as an indicator bacterium. Following incubation overnight at 37°C, the phage formed clear plaques surrounded by translucent halos on the double-layer agar plate ( Figure 1A ). Remarkably, even at room temperature, the size of translucent halos continued to increase, suggesting that some depolymerases produced from phage virions might degrade bacterial surface polysaccharides.

The phage genome was assembled after high-throughput sequencing, and the obtained sequence was deposited in GenBank under the accession number OR791279. The linear genomic sequence of phage IME-Ap7 was 45,048 bp with a G+C content of 37.9%. According to the results of RAST and NCBI BLASTP, the phage genome contains 92 CDSs, the function of 21 of which has been predicted ( Figure 1B , Supplementary Table 3 ). ORF27 was predicted to encode a polysaccharide depolymerase with a length of 704 amino acids and a molecular weight of 77.60 kDa.

To determine whether the predicted depolymerase exhibited activity for polysaccharide degradation, a recombinant plasmid containing the ORF27 sequence was constructed and expressed. As depicted in Figure 1C , the protein Dpo27 migrated as a single band on 10% SDS-PAGE gel and had an estimated size of approximately 77.60 kDa. The concentration of purified Dpo27 was determined to be 0.5 mg/mL using a fluorometer. The polysaccharide-degrading activity of Dpo27 was evaluated using a modified single-spot assay with different enzyme dilutions (0.1–2 ng). As illustrated in Figure 1D , the size of the semi-clear circles decreased with a reduction in Dpo27 concentration, and the halo disappeared at an enzyme concentration of 0.25 ng.

To determine the lytic spectrum of phage IME-Ap7, 23 A. pittii strains representing seven KL types were tested using a double-layer agar plate assay ( Table 1 ). Phage IME-Ap7 lysed the KL220 type of A. pittii 7 and 1492, producing clear plaques on the bacterial lawn. The sensitivity of these isolates to Dpo27 was assessed using a modified single-spot assay. As described in Table 1 , Dpo27 formed a translucent halo on the KL220 type of A. pittii 7 and 1492, indicating that the protein had the same sensitivity range as its parent phage, IME-Ap7.

Enzymatic activity was evaluated by monitoring the amount of reducing sugars released from the enzyme-digested bacterial surface polysaccharides. As shown in Figure 2A , treatment with Dpo27 resulted in the release of sugars to an OD₅₄₀ value of 0.477 ± 0.007, which was significantly higher than that observed for treatment with heat-inactivated Dpo27 or the corresponding controls. Thus, EPS was degraded after incubation with Dpo27 (p < 0.0001, one-way ANOVA). The capsular polysaccharide-degrading capacity of Dpo27 was further verified using Alcian blue staining ( Figure 2B ). The results of gel electrophoresis showed that a smeared band was formed upon incubation of A. pittii CPS with Dpo27 when compared to the CPS alone or the CPS treated with the inactivated enzyme, indicating that the CPS of A. pittii 7 was degraded by Dpo27.

To determine the optimal pH for Dpo27 activity, the enzymatic activity of the protein at pH 4–11 was determined by measuring the production of reducing sugars. As shown in Figure 3A , Dpo27 remained active at pH 5.0–10.0. The thermal stability of Dpo27 was evaluated in 50 mM Na₂HPO₄ buffer (pH 6.0) using a previously described method. Dpo27 maintained enzymatic activity at temperatures ranging from 20–50°C, with an optimum temperature of 37°C ( Figure 3B ).

To verify the capacity of Dpo27 to enhance bacterial susceptibility to killing by incubation with serum, the bacterial counts of A. pittii 7 were determined across different treatment groups ( Figure 4A ). A. pittii 7 exhibited resistance to killing by incubation with serum, as indicated by a slight reduction in viable counts of the serum-treated bacteria. However, upon treatment with depolymerase Dpo27, A. pittii 7 became sensitive to serum, and a significant reduction in the viable bacterial count was observed (p < 0.0001, one-way ANOVA). Additionally, the acute toxicity of Dpo27 toward red blood cells was assessed to further evaluate the safety of depolymerase as an antimicrobial agent. As shown in Figure 4B , Dpo27 displayed no hemolytic activity against erythrocytes in vitro.