In vivo efficacy of phage cocktails against carbapenem resistance Acinetobacter baumannii in the rat pneumonia model

Shibin Li; Bingdong Wei; Le Xu; Cong Cong; Bilal Murtaza; Lili Wang; Xiaoyu Li; Jibin Li; Mu Xu; Jiajun Yin; Yongping Xu

doi:10.1128/jvi.00467-24

. 2024 Jun 12;98(7):e00467-24. doi: 10.1128/jvi.00467-24

In vivo efficacy of phage cocktails against carbapenem resistance Acinetobacter baumannii in the rat pneumonia model

Shibin Li ¹, Bingdong Wei ², Le Xu ¹, Cong Cong ¹, Bilal Murtaza ¹, Lili Wang ¹, Xiaoyu Li ¹, Jibin Li ³, Mu Xu ⁴, Jiajun Yin ^5,^✉, Yongping Xu ^1,^4,^✉

Editor: Kristin N Parent⁶

PMCID: PMC11265278 PMID: 38864621

ABSTRACT

Acinetobacter baumannii, an opportunistic pathogen, poses a significant threat in intensive care units, leading to severe nosocomial infections. The rise of multi-drug-resistant strains, particularly carbapenem-resistant A. baumannii, has created formidable challenges for effective treatment. Given the prolonged development cycle and high costs associated with antibiotics, phages have garnered clinical attention as an alternative for combating infections caused by drug-resistant bacteria. However, the utilization of phage therapy encounters notable challenges, including the narrow host spectrum, where each phage targets a limited subset of bacteria, increasing the risk of phage resistance development. Additionally, uncertainties in immune system dynamics during treatment hinder tailoring symptomatic interventions based on patient-specific states. In this study, we isolated two A. baumannii phages from wastewater and conducted a comprehensive assessment of their potential applications. This evaluation included sequencing analysis, genome classification, pH and temperature stability assessments, and in vitro bacterial inhibition assays. Further investigations involved analyzing histological and cytokine alterations in rats undergoing phage cocktail treatment for pneumonia. The therapeutic efficacy of the phages was validated, and transcriptomic studies of rat lung tissue during phage treatment revealed crucial changes in the immune system. The findings from our study underscore the potential of phages for future development as a treatment strategy and offer compelling evidence regarding immune system dynamics throughout the treatment process.

IMPORTANCE

Due to the growing problem of multi-drug-resistant bacteria, the use of phages is being considered as an alternative to antibiotics, and the genetic safety and application stability of phages determine the potential of phage application. The absence of drug resistance genes and virulence genes in the phage genome can ensure the safety of phage application, and the fact that phage can remain active in a wide range of temperatures and pH is also necessary for application. In addition, the effect evaluation of preclinical studies is especially important for clinical application. By simulating the immune response situation during the treatment process through mammalian models, the changes in animal immunity can be observed, and the effect of phage therapy can be further evaluated. Our study provides compelling evidence that phages hold promise for further development as therapeutic agents for Acinetobacter baumannii infections.

KEYWORDS: bacteriophage, Acinetobacter baumannii, multi-drug resistant, pneumonia, immune processes

INTRODUCTION

Acinetobacter species are recognized as prominent hospital pathogens, with Acinetobacter baumannii emerging as a paramount concern in nosocomial infections (1). The World Health Organization underscored the severity of antibiotic-resistant pathogens, listing A. baumannii as the most menacing in 2017 (2). Multidrug-resistant (MDR) A. baumannii, an opportunistic pathogen, poses a substantial threat to human health, giving rise to various untreatable infectious diseases, including pneumonia, bacteremia, and meningitis (3 –5). The China Antimicrobial Resistance Surveillance System (CHINET) reported that in 2023, A. baumannii ranked as the fourth most frequently isolated pathogen in clinical settings and the second most common pathogen isolated from respiratory tract specimens (http://www.chinets.com). This highlights the critical role of A. baumannii in clinical infections and the urgent need for effective intervention strategies to mitigate its impact on public health.

Carbapenem antibiotics, such as imipenem and meropenem, serve as the last line of defense against severe Gram-negative bacterial infections (6). However, 2023 CHINET reported the alarming finding that clinical isolates of A. baumannii from tertiary hospitals in China had significantly higher rates of resistance to imipenem (78.6%) and meropenem (79.5%) compared to other multidrug-resistant bacteria (http://www.chinets.com). This heightened resistance underscores the critical and urgent necessity for effective therapeutic agents to combat infections caused by multidrug-resistant A. baumannii. In response to the growing antibiotic resistance crisis, bacteriophages (phages) are re-emerging as a promising alternative to antibiotics, leveraging their capacity to effectively eliminate MDR bacteria (7, 8). There have been many studies reported on the use of phages for the control of A. baumannii infections (9 –12).

Currently, a primary approach to assess the potential of phage application involves analyzing novel phage genomes. The evaluation of lytic phages includes considerations of pH and temperature stability (13, 14) and in vitro bacterial inhibition. These assessments contribute to determining the potential for phage applications, and their characteristics extend the possibility of in vivo applications. Although numerous preclinical animal studies have demonstrated the feasibility of phage therapies for various diseases, including MDR bacterial lung infections (15 –17), current research on phage therapy for lung infections caused by A. baumannii has predominantly focused on its efficacy as a treatment. Rigorous in vivo studies, especially those addressing critical questions about the impact on the immune system, are limited. Therefore, additional comprehensive studies are imperative before considering the application of phages for the treatment of A. baumannii pneumonia.

In this investigation, we isolated two lytic phages specific to A. baumannii from wastewater. Subsequently, the safety of phage application was assessed by excluding the presence of antibiotic-resistance genes and virulence genes in their genomes. We determined phage pH, temperature stability, and in vitro capabilities to lay the foundation for phage applications. A rat model of A. baumannii pneumonia was established, and phage cocktails were administered to evaluate the efficacy of phage therapy. Significantly, our study employed a combined approach of RNA-seq and qPCR to monitor alterations in the immune system of rats throughout the treatment. As anticipated, the phage cocktail exhibited efficient bacterial eradication and contributed to the alleviation of stress on the immune system. The outcomes of our study furnish a more comprehensive basis for advocating the application of phages in the treatment of A. baumannii lung infections.

RESULTS

Description of the phage genome

The whole genome sequence of vB_AbaM_P1 contains 107,537 bp with 37.68% G + C content. A total of 197 gene regions were predicted [183 open reading frames (ORFs) plus 14 tRNAs]; 36 of them were leftward-directed, and 162 were rightward-directed. Among them, 58 of the predicted ORFs have designated functions (Table S1). The whole genome of phage vB_AbaM_DP45 contains 164,031 bp with a G + C content of 36.77%. A total of 256 gene regions were predicted (248 ORFs plus 8 tRNAs), of which 216 were leftward-directed and 40 were rightward-directed (Table S2). Of these, 123 putative ORFs predict function. Both PHaTYP and Bacphlip predictions showed vB_AbaM_P1 and vB_AbaM_DP45 as lytic phages. As predicted by Pharokka’s results, the genomes of both phages did not contain integrase genes, virulence factors, or antibiotic resistance genes, suggesting that the two phages were lytic phages eligible for application.

Taxonomic relationships of the two phages

The taxonomic relationships of the two A. baumannii phage strains were evaluated within a broader context of phages using vContact2 (Fig. 1). Within the network, the Acinetobacter phages were classified into distinct groups, and the relationships between these groups reflected their nucleic acid similarity and shared protein content. Specifically, Phage vB_AbaM_P1 was assigned to the Saclayvirus category. Phages in this group typically have genomes ranging from 99.7 to 113 kb and contain 153–184 ORFs. According to the classification by the International Committee on Taxonomy of Viruses (ICTV), Saclayvirus phages have three representative phages, and the prediction of their lifestyles showed that all of them are lytic phages. vB_AbaM_DP45 belongs to the Lazarusvirus. Lazarusvirus phages have genomes ranging from 164 to 168.2 kb and contain 244–253 ORFs. All eight Lazarusvirus representative phages in ICTV were predicted to be lytic phages.

Temperature and pH stability of phages

The phage could be considered stable when the free phage titer after 1 h of incubation decreases within 1 log₁₀PFU/mL (plaque forming unit) compared to the initial phage titer. Phage P1 was stable in the range of pH 5–9 (Fig. 2a), beyond which the titer decreased significantly to 4.7 log₁₀ PFU/mL and 6.8 log₁₀ PFU/mL at pH 4 and 10, respectively. DP45 was stable in the pH 6–9 range (Fig. 2c) with a drop in titer at pH 5 and 10 to 6.6 log₁₀ PFU/mL and 5.6 log₁₀ PFU/mL, respectively, and inactivated at pH 1 and 12. P1 was stable within the range of 30°C–70°C (Fig. 2b), with a drop in titer of more than 2 log₁₀ from 8 log₁₀ PFU/mL to 5.5 log₁₀ PFU/mL when the temperature was higher than 80°C and complete inactivation at 100°C. DP45 was stable in the range of 30°C–60°C (Fig. 2d), with a drop in titer from 8 log₁₀ PFU/mL to 4.7 log₁₀ PFU/mL when the temperature was higher than 70°C and complete inactivation at 100°C.

Fig 2 — pH and thermal stability of phage P1 and DP45. P1 was incubated at different pH (a) and temperatures (b), while DP45 was incubated at different pH (c) and temperatures (d) for 1 h. The surviving phage titers were determined by the double-layer plate method. Error bars represent the SD of three independent replicate experiments, and different letters represent significant differences.

Bacteriostatic effect of phage

Both phage P1 and DP45 were able to inhibit bacterial growth for more than 6 h at all multiplicity of infections (MOIs; 100, 10, 1, and 0.1). This was a significant difference from what occurred when phosphate-buffered saline (PBS) was added to the bacteria. P1 was able to inhibit bacterial growth within 12 h of the assay when the MOI was 0.1 (Fig. 3a), and DP45 could also inhibit bacterial growth for 8 h when the MOI was 1 (Fig. 3b). After combining the two phages, both could inhibit bacterial growth for more than 12 h at all MOIs (Fig. 3c).

Fig 3 — Phage inhibition time at different MOIs. Inoculated with different MOIs, the OD600 values were measured every hour to observe the production of resistant bacteria. Equal amounts of PBS-inoculated bacteria were used as controls. (a) Inhibition curve of P1. (b) Inhibition curve of DP45. (c) Inhibition curve after combination of two phages. Error bars indicate the SD of three replicate counts.

Endotoxin content

Endotoxin, also known as lipopolysaccharide (LPS), constitutes a component of the cell wall in Gram-negative bacteria and is commonly found in phage lysates. In our investigation, we quantified the endotoxin content in the purified phage lysate. The determined amount of LPS was 62.957 EU/10⁹ PFU, significantly lower than the pre-purification level of 4132.655 EU/10⁹ PFU. Consequently, the presence of LPS carried by the phages did not result in any notable adverse effects.

Phage therapy improves survival of A. baumannii pneumonia

For the EG group, A. baumannii pneumonia was fatal, and all rats died within 72 h after infection (Fig. 4a). Phage treatments significantly increased the survival rate of the rats infected with A. baumannii (90%, P < 0.001).

Bacterial load

The bacteria grew rapidly in the lungs after being infected with A. baumannii, reaching 9.22 ± 1.11 log₁₀ CFU/g at 12 h post-infection and growing to 11.60 ± 1.19 log₁₀ CFU/g at 48 h (Fig. 4b). When compared to the infection group, the bacteria in the lungs were effectively eradicated after the administration of the phage treatment. Bacterial counts after administering the phage therapy decreased by 4.59, 6.43, and 8.48 log₁₀ at 12 h, 24 h, and 48 h after infection, respectively, and the mean colony count was 4.63 ± 0.94 log₁₀, 3.81 ± 0.75 log₁₀, and 3.12 ± 0.35 log₁₀ CFU/g, respectively.

Histology

The results of the hematoxylin-eosin (HE) staining of lung tissue collected at each time point are shown in Fig. 5a through h. At 0 h of infection, HE staining results of lung tissues were similar in the EG (Fig. 5a) and DG (Fig. 5e) groups. At 12 h post-infection (Fig. 5b), the lung tissue showed obvious alveolar wall thickening and inflammatory cell infiltration. Large numbers of alveolar structures disappeared, and a small amount of light pink edema fluid was visible in the alveolar cavity. As the infection time increased, the alveolar structure continued to gradually decrease. The inflammatory cell infiltration was aggravating, and the edema fluid in the alveolar cavity increased (Fig. 5c). At 48 h post-infection (Fig. 5d), the alveolar structures were largely lost, and the lung tissue showed a large number of substantial lesions of inflammatory cell infiltration. Compared with the infected group, the lung tissue from rats treated with the phage cocktail showed a lower degree of pathological changes and alveolar fusion. At 12 h post-infection (Fig. 5f), the inflammatory cell infiltration and the general inflammatory conditions showed a decrease with time (Fig. 5g and h).

As shown in (Fig. 5i), phage treatment can significantly reduce pulmonary mean linear intercept (MLI), with an increasing disparity between the two groups as time progresses.

Cytokine levels

The expression of all three inflammatory factors increased to varying degrees. As shown in Fig. 6, for the EG group, TNF-α and IL-1β had the highest expression at 12 h, followed by a gradual decrease. The expression of IL-6 was highest at 24 h, followed by a decrease in expression. Unlike the EG group, the DG group showed a lower degree of change in cytokines. The expression of TNF-α showed a significant increase at 12 h, followed by a gradual decrease. The expression of IL-1β was significantly different from both the CG and EG groups at 12 h and then gradually decreased. IL-6 expression rose at 12 h and then fell back rapidly. All three cytokines were similar in the CG group at 48 h.

Transcriptome changes

Immunological alterations in the processes of A. baumannii pneumonia were observed using transcriptome analysis. A large number of differentially expressed genes (DEGs) were observed after infection. To validate the results of our RNA-seq analysis, several highly expressed DEGs were selected for validation using the qRT-PCR method (primer sequences for qRT-PCR are listed in Table S3). The results showed that the expression trends of the selected DEGs during A. baumannii pneumonia were consistent with RNA-seq (Fig. 7a), indicating that the RNA-seq results reliably reflected the gene expression trends.

The results of the clustering analysis of the expression of 50 immune-related genes are shown in Fig. 7b. Compared with the CG group, the expression of immune genes was significantly upregulated in the EG-12 h and EG-24 h groups and then decreased in the EG-48 h groups.

Effect of phage treatment on immune gene expression

DEGs in all groups after infection were analyzed, and a total of 3,195 DEGs were found (Fig. 8a). To investigate the gene functions of the DEGs, we performed KEGG (Kyoto Encyclopedia of Genes and Genomes） pathway annotation and enrichment analysis on 3,195 DEGs. The results of the KEGG analysis showed that there were 41 pathways with q < 0.01 (Table S4). Of these, seven pathways are immune-related: cytokine-cytokine receptor interactions, chemokine signaling pathways, tumor necrosis factor (TNF) signaling pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Platelet activation. Based on the enrichment results of KEGG, the protein-protein interaction (PPI) analysis of the selected DEGs in immune-related pathways is shown in Fig. 8b, in which the genes with the highest association and expression were TNF-α, IL-6, IL-1β, Myd88, and Ccl2. For the expression results of these hub genes after being treated with phages, our qRT-PCR result is shown in Fig. 8c, which showed a continuous decrease in the expression of the highly associated genes.

DISCUSSION

In this study, both phages composing the phage cocktail showed reliable performance for application. The two phages remained stably active after 1 h of incubation at pH 6–9 (Fig. 2a and c), and they also remained stable when the temperature was lower than 60°C (Fig. 2b and d). Interestingly, both phages we used could survive at 70°C, but the stability decreased as the temperature increased, similar to the results of previous studies (18). The optimal MOI for phage vB_AbaM_P1 was 0.1, and the host did not develop phage resistance within 12 h (Fig. 3a). Phage vB_AbaM_DP45 also inhibited the growth of host bacteria for 8 h at an MOI of 1 (Fig. 3b). The phage cocktail they formed showed a cooperative effect (Fig. 3c), which could better kill the pathogenic host bacteria and reduced the possibility of the generation of resistant bacteria. Therefore, the cocktail formed by the combination of two phages can be more effective in killing pathogenic bacteria.

In clinical studies of lethal pneumonia caused by MDR A. baumannii, the main concerns include bacterial infection load, tissue damage, and immune system status. However, these factors are almost impossible to rapidly and precisely determine in actual clinical situations. Therefore, studying changes in various factors in mammals after phage application is important for the wide application of phages and the pharmacodynamic study of phages. In the actual clinical setting, it is almost impossible to accurately determine the bacterial load. Therefore, we used a high dose of A. baumannii for modeling to ensure the stability of the pneumonia model. In addition, since phage concentration in vivo is dose-dependent and high phage/bacteria ratio conditions have better therapeutic efficacy (19 –21), we used a high MOI for treatment. When phages enter the animal body, they are phagocytosed by mononuclear macrophages, filtered and cleared by the immune organs, and gradually reduced over time. In previous studies, phages have been shown to achieve therapeutic efficacy by killing the target bacteria before they are cleared (22).

As bacteria are more likely to develop resistance mutations against phages when treated with a single phage (23 –25), we used a cocktail of two phages. Our results showed that treatment using two phages was effective in improving the survival rates in rats infected with lethal A. baumannii pneumonia (Fig. 4a), indicating the potential use of phages to treat lethal pneumonia. After treatment, the number of bacteria in the lung tissue rapidly decreased to low levels. Interestingly, the bacteria were not completely cleared within 48 h (Fig. 4b). Since the bacteria were not uniformly distributed throughout the lungs, some of the bacteria were hidden in the crevices of the tissue and, therefore, were not infected by phages.

In the immune system, immune cells are the first line of defense against pathogens, and the recruitment of neutrophils and monocytes/macrophages plays a crucial role in the response to bacterial infections. The depletion of neutrophils, monocytes, and macrophages increases the bacterial burden of the organism, and the recruitment of a large number of immune cells can cause unrecoverable damage to infected tissues. As shown in the histological results (Fig. 5a and e), the lung tissue in the DG and EG groups was similar in the initial phases of infection (12 h). Unsurprisingly, recruitment of inflammatory cells also occurred in the lung tissue after phage treatment. This is a normal immune response caused by the entry of bacteria and the lipopolysaccharide released by phage lysis of bacteria, which can lead to the aggregation of immune cells (26 –28). After 24 h, the DG group normalized due to a significant reduction in bacterial numbers and diminished immune cell recruitment. This indicates that a small number of bacteria cannot induce a strong immune response. In contrast, the increasing number of bacteria in the EG group caused the immune system to overreact, large numbers of inflammatory cells infiltrated, and the alveolar structures gradually disappeared, which is similar to the results of previous studies (19, 22).

Immunity is a complex process in which cytokines, as signaling molecules in the immune system, play an important role. Most immune responses caused by severe bacterial infections are systemic, and the levels of cytokines in the serum better reflect the immune status. In the EG group, measurements of serum levels for three important cytokines showed a rapid increase in cytokine release after infection (Fig. 6), and the results were consistent with our expectations. After phage treatment, the release of cytokines decreased rapidly after the initial upregulation of release. Similar to immune cell recruitment in the lungs, cytokines in the blood vary with the degree of the infection. Notably, cytokine release in the EG group suddenly dropped to lower values at 48 h, similar to the DG and CG groups, which we analyzed as a result of infected rats reaching a near-death state and the organism becoming dysfunctional and unable to respond normally to the infection.

A sustained state of high immune activation may cause immune exhaustion (29 –31). In our transcriptomic studies, we focused on immune-related genes and investigated immune system changes at the genetic level during A. baumannii pneumonia. Clustering analysis of immune-related genes showed (Fig. 7b) that the expression profile of immune-related genes showed a rapid increase followed by a rapid decrease, suggesting that the expression of immune-related genes was impaired after severe infection. Interestingly, several genes related to signal transduction and metabolism were expressed abnormally in the late stages of infection, indicating a severe dysregulation of the gene expression system in infected animals, which further led to a reduction in cytokine release in the late stages of infection.

Throughout the immunization process, different genes contribute to immunity to different degrees. The genes that have been differentially expressed across the different samples (Fig. 8a) were enriched by KEGG. We conducted a PPI analysis of genes enriched for immune-related pathways and screened five key genes that play important roles in the immune pathways involved in A. baumannii pneumonia (Fig. 8b). The effect of phages on the expression profile of the immune system can be derived by measuring the expression of these key genes after phage treatment. Based on previous studies, the levels of IL-6, TNF-α, and IL-1β were strongly correlated with the activity of macrophages (32, 33). Ccl2 is an important chemokine that regulates the migration and infiltration of monocytes/macrophages (34). Myd88 is a key regulator that plays an important role in immunity (35). The qPCR results (Fig. 8c) showed that the expression of these genes was reduced to different degrees after phage treatment, indicating that phage treatment reduced the burden on the immune system and avoided high loads and exhaustion of the immune system.

In summary, our study demonstrates that severe and uncontrolled inflammatory responses lead to lung injury and immune system exhaustion. In comparison, the use of phages killed A. baumannii and prevented lethal immune changes. Up until now, no mammalian immune changes have been reported during phage treatment of A. baumannii pneumoniae. These data provide a deeper understanding of immune changes in mammals during phage treatments.

MATERIALS AND METHODS

Animal maintenance

Male Wistar rats, aged 6 weeks and weighing 180–200 g, were acquired from Liaoning Changsheng Biotechnology Co., Ltd. Throughout the entire experiment, rats were placed in specific pathogen-free, light-controlled, and temperature-controlled conditions and supplemented with standard feed and water.

Bacteria and bacteriophages

The strains used in this experiment are listed in Table S5, and the strains were grown at 37°C in Luria-Bertani (LB) with shaking or on LB plates containing 1.5% agar (wt/vol). The multidrug-resistant strain AB-5 was isolated from the sputum of patients with A. baumannii pneumonia. Two phages used for phage therapies, named vB_AbaM_P1 (OL960030) and vB_AbaM_DP45 (OP585103), were isolated from medical sewage. The host spectrum of two phages is listed in Table S5.

Phage identification and analysis

The genomes of two phage strains were sequenced using the Illumina Hiseq 2500 platform, and phage lifestyles were predicted using PhaTYP (36) and Bacphlip (37). Gene function prediction of phages was performed using Pharokka (38), a phage rapid annotation tool, and ORF function analysis of phages was performed combined with RASTtk (https://rast.nmpdr.org/) (39). Functional validation of the annotated ORFs was performed using BLASTP, HHpred (40) (https://toolkit.tuebingen.mpg.de/tools/hhpred), and InterPro (41) (https://www.ebi.ac.uk/interpro/). The presence of transfer RNA (tRNA) was detected using tRNAscan-SE (42) (http://lowelab.ucsc.edu/tRNAscan-SE/). Based on Pharokka predictions, the Comprehensive Antibiotic Resistance Database (CARD) (43) and the Virulence Factor Database for Pathogenic Bacteria (VFDB) (44) were used to determine that the genome did not contain antibiotic resistance genes and virulence factors.

Taxonomic relationships of phages

To explore the association between Acinetobacter phages and the broader phage population, a gene-sharing network comprising 199 Acinetobacter phage genomes from GenBank was established using vContact2 (45). Subsequently, the generated network was visualized and examined utilizing Cytoscape (46).

Effects of temperature and pH on phage activity

The temperature and pH stability of phages were determined as described previously (47). Briefly, 10⁸ PFU/mL of phages were incubated at temperatures ranging from 30°C to 100°C for 1 h, and the number of surviving phages was then counted by the double-layer plate method. Using 1 M HCl and 1 M NaOH to prepare SM buffers with different pH values (1–12), 10⁸ PFU of phages were added to each pH solution, and the surviving phages were counted after 1 h of incubation.

Bactericidal assays

To assess the inhibitory potential of the phages, a 200 µL suspension of mid-log phase A. baumannii strain CGMCC 1.90331 was introduced into 96-well plates along with 20 µL of phage at various MOI levels. A blank control was included with an equivalent volume of PBS. All cultures were incubated at 37°C and 160 rpm, and OD600 was measured at 1 h intervals using a microtiter plate reader. Based on the results of the single phage assay, a 1:1 mixture of the two phages was used to determine the bacteriostatic effect of the phage cocktail under the same conditions. The experiments lasted for 12 h, and all experiments were performed in triplicate.

Endotoxin assay

Endotoxin removal from the phage lysate was achieved through the dialysis method. The endotoxin level in the phage lysates was measured using a dynamic colorimetric assay in accordance with the manufacturer’s guidelines (abs50045, absin Biotechnology Co. LTD, Shanghai, China). The result was expressed as endotoxin units per phage titer (EU/PFU).

Phage treatment of pneumonia

Rats were assigned to A. baumannii infection and phage therapy groups (n = 10) and were anesthetized with sodium pentobarbital (50 mg/kg). MDR A. baumannii was administered by non-invasive intratracheal drops of 2 × 10⁹ CFU under direct vision, while the phage therapy group was treated 1 h after infection in the same manner but using the phage cocktail (2 × 10¹⁰ PFU). The survival rate of the rats was observed 7 days post-infection.

Based on 7-day survival, rats were partitioned into three groups (n = 15): the control group (CG), the A. baumannii infection group (EG), and the phage therapy group (DG). At different time points after infection (12 h, 24 h, and 48 h), rats (n = 3) were euthanized, and samples of lung tissue and serum were collected. After sampling, the lung tissue was immediately homogenized and prepared for bacterial enumeration.

Histological tests

Lung tissues from rats were collected at different time points and fixed with 4% paraformaldehyde for 48 h. The fixed tissues were embedded using paraffin, sectioned into 5 µm slices, and then stained using HE to observe any histomorphological changes. The extent of lung injury was assessed using Image Pro Plus 6.0 software to quantify the MLI of the lung.

Cytokines quantification

The concentrations of inflammatory factors were measured from the collected rat serum at different time points. Factors including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) from each group were determined by ELISA.

Transcriptome analysis

For the lung tissues of the CG and EG groups collected at different times, total RNA was extracted, followed by isolating and purifying the mRNA to create cDNA libraries. Transcriptome sequencing was performed using an Illumina NovaSeq 6000 sequencing system. Gene expression with paired-end clean reads was aligned to the reference genome of Rattus norvegicus (Rnor_6.0_release95). Genes with significantly different expression (|FC|≥ 2, FDR < 0.01) were selected for analysis, and those DEGs with high expression were selected for qPCR to verify the quality of the transcriptome results.

Variation in immune gene expression

DEGs at all three time points were enriched in the KEGG pathway. Immune-related pathways were selected from the enriched pathways, and PPI were performed on DEGs using the Search Tool for Retrieval of Interacting Genes and Proteins (STRING) database, with the highest confidence interaction score of 0.900, and Cytoscape software was used to determine the ranking of gene associations in these immune-related pathways.

The expression of the most strongly associated genes in the EG group and DG group was quantified using the ABI7500 system. We used β-actin as a housekeeping gene, and we quantified gene expression using the 2^-ΔΔCT method. Gene expressions were compared between the groups. Primer sequences are detailed in Table S3.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software. The survival rate was calculated by Log-rank tests, and all experimental data were expressed as mean ± SD. A t test and one-way ANOVA were used for comparison between groups, followed by Tukey’s multiple comparison test. Differences were considered significant when P-values were less than 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (****).

ACKNOWLEDGMENTS

This study was funded by the third batch of “the open competition mechanism to select the best candidate” science and technology research project of Liaoning Province (No.2022JH1/10900013); A project admitted in the Dalian Deng Feng Program: key medical specialties in construction funded by the People’s Government of Dalian Municipality (No. 243, 2021); Dalian Key R&D Program (2021YF16SN012).

S.L. designed and coordinated the trial, as well as wrote and revised the manuscript. B.W. contributed to the trial’s design and organization and revised the manuscript. L.X. participated in writing and revising the manuscript. C.C. directed the trial. B.M. contributed to writing and revising the manuscript. L.W. designed and organized the trial. X.L. designed and organized the trial. Y.X., J.L., M.X., and J.Y. designed, organized, and directed the manuscript. All authors have reviewed and approved the final manuscript.

Contributor Information

Jiajun Yin, Email: yinjiajun@dlu.edu.cn.

Yongping Xu, Email: xyping@dlut.edu.cn.

Kristin N. Parent, Michigan State University, East Lansing, Michigan, USA

DATA AVAILABILITY

The transcriptome sequencing results have been uploaded to the NCBI database, and the BioProject ID is PRJNA1009251. Phages used in this study can be found in GenBank with accession numbers OL960030 and OP585103; pathogenic bacteria used here were deposited in the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 1.90331.

ETHICS APPROVAL

All animal experiments followed the ARRIVE guidelines. All procedures were approved by the Ethics Committee of Dalian University of Technology. The ethical approval number is DUTSBE220624_01.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.00467-24.

Supplemental tables. jvi.00467-24-s0001.xlsx.

Tables S1 to S5.

jvi.00467-24-s0001.xlsx^{(50.2KB, xlsx)}

DOI: 10.1128/jvi.00467-24.SuF1

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental tables. jvi.00467-24-s0001.xlsx.

Tables S1 to S5.

jvi.00467-24-s0001.xlsx^{(50.2KB, xlsx)}

DOI: 10.1128/jvi.00467-24.SuF1

Data Availability Statement

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[B32] 32. Lv J, Chen J, Wang MJ, Yan F. 2020. Klotho alleviates indoxyl sulfate-induced heart failure and kidney damage by promoting M2 macrophage polarization. Aging (Albany NY) 12:9139–9150. doi: 10.18632/aging.103183 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Gou XM, Xu WC, Liu YS, Peng Y, Xu WL, Yin YB, Zhang XM. 2022. IL-6 prevents lung macrophage death and lung inflammation injury by inhibiting GSDME- and GSDMD-mediated pyroptosis during pneumococcal pneumosepsis. Microbiol Spectr 10:e0204921. doi: 10.1128/spectrum.02049-21 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34. Aikawa S, Deng WB, Liang XH, Yuan J, Bartos A, Sun XF, Dey SK. 2020. Uterine deficiency of high-mobility group box-1 (HMGB1) protein causes implantation defects and adverse pregnancy outcomes. Cell Death Differ 27:1489–1504. doi: 10.1038/s41418-019-0429-z [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

In vivo efficacy of phage cocktails against carbapenem resistance Acinetobacter baumannii in the rat pneumonia model

Shibin Li

Bingdong Wei

Le Xu

Cong Cong

Bilal Murtaza

Lili Wang

Xiaoyu Li

Jibin Li

Mu Xu

Jiajun Yin

Yongping Xu

Roles

ABSTRACT

IMPORTANCE

INTRODUCTION

RESULTS

Description of the phage genome

Taxonomic relationships of the two phages

Fig 1.

Temperature and pH stability of phages

Fig 2.

Bacteriostatic effect of phage

Fig 3.

Endotoxin content

Phage therapy improves survival of A. baumannii pneumonia

Fig 4.

Bacterial load

Histology

Fig 5.

Cytokine levels

Fig 6.

Transcriptome changes

Fig 7.

Effect of phage treatment on immune gene expression

Fig 8.

DISCUSSION

MATERIALS AND METHODS

Animal maintenance

Bacteria and bacteriophages

Phage identification and analysis

Taxonomic relationships of phages

Effects of temperature and pH on phage activity

Bactericidal assays

Endotoxin assay

Phage treatment of pneumonia

Histological tests

Cytokines quantification

Transcriptome analysis

Variation in immune gene expression

Statistical analysis

ACKNOWLEDGMENTS

Contributor Information

DATA AVAILABILITY

ETHICS APPROVAL

SUPPLEMENTAL MATERIAL

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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